LG5, a Novel Allele of EUI1, Regulates Grain Size and Flag Leaf Angle in Rice

Grain size and flag leaf angle are two important traits that determining grain yield in rice. However, the mechanisms regulating these two traits remain largely unknown. In this study, a rice long grain 5 (lg5) mutant with a large flag leaf angle was identified, and map-based cloning revealed that a single base substitution followed by a 2 bp insertion in the LOC_Os05g40384 gene resulted in larger grains, a larger flag leaf angle, and higher plant height than the wild type. Sequence analysis revealed that lg5 is a novel allele of elongated uppermost internode-1 (EUI1), which encodes a cytochrome P450 protein. Functional complementation and overexpression tests showed that LG5 can rescue the bigger grain size and larger flag leaf angle in the Xiushui11 (XS) background. Knockdown of the LG5 transcription level by RNA interference resulted in elevated grain size and flag leaf angle in the Nipponbare (NIP) background. Morphological and cellular analyses suggested that LG5 regulated grain size and flag leaf angle by promoting cell expansion and cell proliferation. Our results provided new insight into the functions of EUI1 in rice, especially in regulating grain size and flag leaf angle, indicating a potential target for the improvement of rice breeding.


Introduction
Grain size is a major target of rice breeding that is not only a component of yield but also a quality trait. Rice grain yield mainly depends on four major components: grain weight, number of panicles (or tillers) per plant, number of grains per panicle, and the ratio of filled grains [1]. The grain size of rice directly determines the grain weight, which, in turn, affects the yield [2]. Rice grain size consists of grain length, grain width, grain thickness, and grain length-to-width ratio, which is a complex trait controlled by multiple quantitative trait loci (QTLs)/genes. More than 500 QTLs related to rice grain size have been identified on 12 chromosomes. To date, several QTLs/genes controlling grain size have been cloned in rice, such as GS3, GW2, qSW5/GW5, GS5, GL3.1/qGL3, GW8, TGW6, BG2, GW6a, GL7/GW7, GLW7, GS9, SMG1/OsMKK4, and SMG2/ OsMKKK10; these genes play a very important role in the regulation of grain size in rice [3]. Recent advances have identified several signaling pathways involved in determining grain size, including transcriptional regulatory factors, the ubiquitin-proteasome pathway, G-protein signaling, mitogen-activated protein kinase (MAPK) signaling, and phytohormones [3]. For instance, GS2 encodes the transcription factor OsGRF4, which controls grain size by promoting cell expansion and cell proliferation. GW2 and OsOTUB15 are two major genes controlling grain width, both of which regulate grain size through the ubiquitin pathway; GW2 encodes a

The lg5 Mutant Increase Grain Size and Elevate Flag Leaf Angle
To understand the mechanisms governing rice grain size and flag leaf angle, we previously identified a mutant with long grains and a large flag leaf angle in a mutant bank ( Figure 1A,F). The lg5 mutant was isolated from the cobalt 60 radiation-induced M 2 populations of the Xiushui (XS) mutant bank. The lg5 mutant showed larger grains compared to XS ( Figure 1A,C). The length and width of lg5 grains were significantly increased compared with those of XS grains ( Figure 1B,D). The average length of XS and lg5 grains was 7.51 ± 0.10 mm and 8.23 ± 0.06 mm, respectively. The average width of XS and lg5 grains was 3.43 ± 0.06 mm and 3.80 ± 0.10 mm, respectively. In addition, the thousand-grain weight of lg5 was increased compared with that of XS ( Figure 1E). The average thousand-grain weight of XS was 26.97 ± 0.44 g, whereas that of the lg5 mutant was 29.63 ± 0.26 g. The lg5 mutant also showed a larger flag leaf angle and higher plant height than XS ( Figure 1F,H). The average flag leaf angle of XS and lg5 was 22.67 ± 2.52 • and 60.33 ± 2.52 • , respectively ( Figure 1G). The average plant height of XS and lg5 was 85.33 cm and 118.33 cm, respectively ( Figure 1I). Furthermore, the lengths of the panicle and internode of every part of lg5 was increased significantly relative those of XS. The length of the panicle, first internode, second internode, third internode, fourth internode, and fifth internode of lg5 were increased by 5.30%, 62.78%, 60.93%, 59.76%, 73.27%, and 244.45%, respectively, relative to those of XS ( Figure S1). Together, these results indicated that LG5 influenced grain size and plant architecture in rice.
To understand the mechanisms governing rice grain size and flag leaf angle, we previously identified a mutant with long grains and a large flag leaf angle in a mutant bank ( Figure 1A,F). The lg5 mutant was isolated from the cobalt 60 radiation-induced M2 populations of the Xiushui (XS) mutant bank. The lg5 mutant showed larger grains compared to XS ( Figure 1A,C). The length and width of lg5 grains were significantly increased compared with those of XS grains ( Figure 1B,D). The average length of XS and lg5 grains was 7.51 ± 0.10 mm and 8.23 ± 0.06 mm, respectively. The average width of XS and lg5 grains was 3.43 ± 0.06 mm and 3.80 ± 0.10 mm, respectively. In addition, the thousand-grain weight of lg5 was increased compared with that of XS ( Figure 1E). The average thousand-grain weight of XS was 26.97 ± 0.44 g, whereas that of the lg5 mutant was 29.63 ± 0.26 g. The lg5 mutant also showed a larger flag leaf angle and higher plant height than XS ( Figure 1F,H). The average flag leaf angle of XS and lg5 was 22.67 ± 2.52 ° and 60.33 ± 2.52 °, respectively ( Figure 1G). The average plant height of XS and lg5 was 85.33 cm and 118.33 cm, respectively ( Figure 1I). Furthermore, the lengths of the panicle and internode of every part of lg5 was increased significantly relative those of XS. The length of the panicle, first internode, second internode, third internode, fourth internode, and fifth internode of lg5 were increased by 5.30%, 62.78%, 60.93%, 59.76%, 73.27%, and 244.45%, respectively, relative to those of XS ( Figure S1). Together, these results indicated that LG5 influenced grain size and plant architecture in rice.  Data are presented as means ± SD. * and ** indicate p < 0.05 and p < 0.01, respectively, according to Student's t-test.

Map-Based Cloning of LG5
We isolated the LG5 gene via map-based cloning using an F 2 population derived from a cross between the lg5 mutant line and the indica rice variety Huasizhan. The F 1 progeny showed the same phenotype as the XS plants, and 642 of 2554 individual plants in the F 2 population showed the lg5 phenotype, in line with a mendelian 3:1 (wild type: mutant) ratio, indicating that the mutant phenotype was controlled by a single recessive gene. We used 171 simple sequence repeats (SSRs) covering 12 chromosomes to locate the LG5 gene, which was initially mapped to an 847 kb interval of chromosome 5 (Chr. 5) between the SSR markers RM18847 and RM18903 ( Figure 2A) and further fine-mapped to a 150 kb interval between the makers RM18883 and RM18893. There are 33 predicted open reading frames. We failed to develop more polymorphic markers to further limit the candidate region, so we sequenced 33 open reading frames and associated them with the known genes between makers RM18883 and RM18893. Comparing the genomic sequences of the candidate region revealed one substitution of A to GT in the LOC_Os05g40384 gene ( Figure 2B) and resulted in premature transcription termination after a 345 bp translation ( Figure 2C). These results suggested that the LOC_Os05g40384 gene was responsible for the lg5 mutant.
We isolated the LG5 gene via map-based cloning using an F2 population derived from a cross between the lg5 mutant line and the indica rice variety Huasizhan. The F1 progeny showed the same phenotype as the XS plants, and 642 of 2554 individual plants in the F2 population showed the lg5 phenotype, in line with a mendelian 3:1 (wild type: mutant) ratio, indicating that the mutant phenotype was controlled by a single recessive gene. We used 171 simple sequence repeats (SSRs) covering 12 chromosomes to locate the LG5 gene, which was initially mapped to an 847 kb interval of chromosome 5 (Chr. 5) between the SSR markers RM18847 and RM18903 ( Figure 2A) and further fine-mapped to a 150 kb interval between the makers RM18883 and RM18893. There are 33 predicted open reading frames. We failed to develop more polymorphic markers to further limit the candidate region, so we sequenced 33 open reading frames and associated them with the known genes between makers RM18883 and RM18893. Comparing the genomic sequences of the candidate region revealed one substitution of A to GT in the LOC_Os05g40384 gene (Figure 2B) and resulted in premature transcription termination after a 345 bp translation (Figure 2C). These results suggested that the LOC_Os05g40384 gene was responsible for the lg5 mutant.
LG5-OE plants exhibited a decrease in plant height ( Figure 3H). We also examined the LG5 relative mRNA levels in the culm of XS and LG5-OE plants ( Figure 3I), and the expression levels of LG5 were found to be positively correlated with the plant height. To confirm that the phenotype resulted from the LG5 gene, the RNA interference plants of LG5 (LG5-RNAi) were generated in the Nipponbare (NIP) background. Transgenic plants exhibited increased phenotypes of grain length, grain width, and thousand-grain weight relative to NIP in the mature stage ( Figure 4A-E). In addition, transgenic plants produced higher plant height and larger flag leaf angle than NIP ( Figure 4F-H). The results reported above are consistent with previous research indicating that dwarfism is caused by elevated expression of EUI1 [30].    Our results indicated that the lg5 mutant phenotype was caused by the mutant of the LOC_Os05g40384 gene, which functioned as a negative regulator of grain size, grain weight, flag leaf angle, and plant height. These findings showed that the LOC_Os05g40384 was a valuable gene for rice genetics research and breeding, providing new potential for the improvement of seed size and plant architecture.

LG5 and its Homologs Are Conserved in Cereal Crops
To explore the evolutionary relationship between LG5 genes and their homologs among six cereal crop species, we conducted an online search (https://phytozomenext.jgi.doe.gov/blast-search) accessed on 6 December 2021 among Oryza sativa, Zea mays, Triticum aestivum, Sorghum bicolor, Hordeum vulgare, and Glycine max. Then, a total of 33 proteins from Oryza sativa (1), Zea mays (4), Triticum aestivum (12), Sorghum bicolor (5), Hordeum vulgare (5), and Glycine max (6) were selected because they had more than 75% identical proteins in common with LG5 protein, and a neighbor-joining phylogenetic tree was constructed using Mega 6. According to the homologous degree of the Our results indicated that the lg5 mutant phenotype was caused by the mutant of the LOC_Os05g40384 gene, which functioned as a negative regulator of grain size, grain weight, flag leaf angle, and plant height. These findings showed that the LOC_Os05g40384 was a valuable gene for rice genetics research and breeding, providing new potential for the improvement of seed size and plant architecture.

LG5 and Its Homologs Are Conserved in Cereal Crops
To explore the evolutionary relationship between LG5 genes and their homologs among six cereal crop species, we conducted an online search (https://phytozome-next. jgi.doe.gov/blast-search) accessed on 6 December 2021 among Oryza sativa, Zea mays, Triticum aestivum, Sorghum bicolor, Hordeum vulgare, and Glycine max. Then, a total of 33 proteins from Oryza sativa (1), Zea mays (4), Triticum aestivum (12), Sorghum bicolor (5), Hordeum vulgare (5), and Glycine max (6) were selected because they had more than 75% identical proteins in common with LG5 protein, and a neighbor-joining phylogenetic tree was Plants 2023, 12, 675 7 of 15 constructed using Mega 6. According to the homologous degree of the LOC_Os05g40384 protein, the 33 proteins could be divided into three major groups-modern, intermediate, and ancient-according to the homologous degree relative to the LG5 protein from highest to lowest ( Figure S2).
LG5 was conserved in cereal crops.

LG5 Regulates the Grain Size and Flag Leaf Angle by Controlling Cell Expansion and Proliferation
In rice, grain size is restricted by the size of the spikelet hull [31], which is determined by both cell expansion and proliferation. Therefore, we examined cell size and numbers in the cross sections of the central parts of the spikelet hulls just before heading between the lg5 mutant and XS ( Figure 5A), larger inner parenchyma cells were found in the lg5 mutant than in XS ( Figure 5B,C). The inner parenchyma cells of spikelet hulls in the lg5 mutant were longer (by 4.80%) and contained more cells (by 6.32%) than those in XS, with an increase in total cell length (11.49%) relative to XS ( Figure 5D-F). These results indicated that the expansive grain size of lg5 contributed to increases in both cell number and cell size. Scanning electron microscopy of the inner and outer surfaces of glumes also showed that lg5 had a larger cell size than XS ( Figure 6A,D) and that the inner spikelet hull surfaces in the lg5 mutant were longer (by 30.29%) and wider (by 16.11%) than those of XS ( Figure 6B,C), whereas the outer spikelet hull surfaces in lg5 mutant were longer (by 104.56%) and wider (by 9.02%) than those in XS ( Figure 6E,F). These results suggested that LG5 controlled grain size by regulating cell expansion and proliferation in rice glumes. LOC_Os05g40384 protein, the 33 proteins could be divided into three major groups-modern, intermediate, and ancient-according to the homologous degree relative to the LG5 protein from highest to lowest ( Figure S2).
LG5 was conserved in cereal crops.

LG5 Regulates the Grain Size and Flag Leaf Angle by Controlling Cell Expansion and Proliferation
In rice, grain size is restricted by the size of the spikelet hull [31], which is determined by both cell expansion and proliferation. Therefore, we examined cell size and numbers in the cross sections of the central parts of the spikelet hulls just before heading between the lg5 mutant and XS ( Figure 5A), larger inner parenchyma cells were found in the lg5 mutant than in XS ( Figure 5B,C). The inner parenchyma cells of spikelet hulls in the lg5 mutant were longer (by 4.80%) and contained more cells (by 6.32%) than those in XS, with an increase in total cell length (11.49%) relative to XS ( Figure 5D-F). These results indicated that the expansive grain size of lg5 contributed to increases in both cell number and cell size. Scanning electron microscopy of the inner and outer surfaces of glumes also showed that lg5 had a larger cell size than XS ( Figure 6A,D) and that the inner spikelet hull surfaces in the lg5 mutant were longer (by 30.29%) and wider (by 16.11%) than those of XS ( Figure 6B,C), whereas the outer spikelet hull surfaces in lg5 mutant were longer (by 104.56%) and wider (by 9.02%) than those in XS ( Figure 6E,F). These results suggested that LG5 controlled grain size by regulating cell expansion and proliferation in rice glumes.  The leaf lamina joint, which connects the leaf blade and sheath, is the most important tissue governing the leaf angle. It has been found that most of the identified rice mutants with altered leaf inclination arecaused by abnormal expansion and division of changed cells in the leaf lamina joint [20,22,32]. Therefore, we examined leaf lamina joint cells through paraffin sections between XS and lg5 mutants ( Figure 5G). The lg5 mutant displayed larger cell expansion than XS in the lamina joint ( Figure 5H). Paraffin sections showed compact and regular cells in lg5 compared with XS, resulting in longer (186.58%) and wider (100.85%) cells in lg5 than XS ( Figure 5I,J).
Several genes were found to control grain size by influencing cell proliferation and cell expansion processes, such as GS2; GL7 was found to be involved in the regulation of cell expansion; and GW2, GL3.1, TGW6, and GS5 were found to control grain size by regulating cell proliferation. To reveal how LG5 regulates cell expansion and cell proliferation in spikelet hulls, we detected its expression levels in the XS and lg5 young panicles. Compared with XS, GS2 and GS5 expression was significantly increased in the lg5 mutant (Figure 6G), suggesting that the increase in cell size and number of lg5 might result from the elevated expression of GS2 and GS5 because they are positive regulators of grain size [33,34]. The expression levels of TGW6, GL7, GW2, and GL3.1 were similar between XS and lg5 mutants according to reverse transcription polymerase chain reaction (RT-PCR) results.
We also detected the expression of genes involved in the cell cycle, such as CYCA1;1, CYCA3;2, CYCB1;1, CYCB2;2, CDKA2, and CDKB2;1. Compared with XS, the six cell-cyclerelated genes were all upregulated in the lg5 mutant ( Figure 6H). These results indicated that LG5 may regulate grain size by promoting cell proliferation through enhanced expression of several cell-cycle-related genes. The leaf lamina joint, which connects the leaf blade and sheath, is the most important tissue governing the leaf angle. It has been found that most of the identified rice mutants with altered leaf inclination arecaused by abnormal expansion and division of changed cells in the leaf lamina joint [20,22,32]. Therefore, we examined leaf lamina joint cells through paraffin sections between XS and lg5 mutants ( Figure 5G). The lg5 mutant displayed larger cell expansion than XS in the lamina joint ( Figure 5H). Paraffin sections showed compact and regular cells in lg5 compared with XS, resulting in longer (186.58%) and wider (100.85%) cells in lg5 than XS ( Figure 5I,J).
Several genes were found to control grain size by influencing cell proliferation and cell expansion processes, such as GS2; GL7 was found to be involved in the regulation of cell expansion; and GW2, GL3.1, TGW6, and GS5 were found to control grain size by regulating cell proliferation. To reveal how LG5 regulates cell expansion and cell proliferation in spikelet hulls, we detected its expression levels in the XS and lg5 young panicles. Compared with XS, GS2 and GS5 expression was significantly increased in the lg5 mutant ( Figure 6G), suggesting that the increase in cell size and number of lg5 might result from the elevated expression of GS2 and GS5 because they are positive regulators of grain size [33,34]. The expression levels of TGW6, GL7, GW2, and GL3.1 were similar between XS and lg5 mutants according to reverse transcription polymerase chain reaction (RT-PCR) results.
We also detected the expression of genes involved in the cell cycle, such as CYCA1;1, CYCA3;2, CYCB1;1, CYCB2;2, CDKA2, and CDKB2;1. Compared with XS, the six cell-cyclerelated genes were all upregulated in the lg5 mutant ( Figure 6H). These results indicated that LG5 may regulate grain size by promoting cell proliferation through enhanced expression of several cell-cycle-related genes.

The Expression Pattern and Subcellular Localization of LG5
To examine the temporal and spatial expression pattern of LG5, total RNA from eight organs from XS plants, including root, tiller bud, stem, node, pulvinus, leaf sheath, flag leaf, and young panicle, were extracted. We examined the expression of LG5 using quantitative real-time RT-PCR analysis. The results revealed that LG5 was constitutively expressed in various rice organs and especially highly expressed in the flag leaf, young panicle, and leaf sheath ( Figure 7A). A similar study also reported that the highest LG5 expression occurred in young panicles and internodes [29]. To further determine the involvement of LG5 in different stages of young panicles, we examined the expression of LG5 in four stages of spikelet (young 2 cm, 7 cm, 13 cm, and 20 cm panicles). We found that LG5 was most highly expressed in the young 2 cm panicle ( Figure 7B). This result was consistent with a previous study of EUI1-GUS, which was detected mainly in rapidly elongating or dividing tissues [29]. Those results showed that LG5 played an important role in regulating grain size, especially in the young panicles.

The Expression Pattern and Subcellular Localization of LG5
To examine the temporal and spatial expression pattern of LG5, total RNA from eight organs from XS plants, including root, tiller bud, stem, node, pulvinus, leaf sheath, flag leaf, and young panicle, were extracted. We examined the expression of LG5 using quantitative real-time RT-PCR analysis. The results revealed that LG5 was constitutively expressed in various rice organs and especially highly expressed in the flag leaf, young panicle, and leaf sheath ( Figure 7A). A similar study also reported that the highest LG5 expression occurred in young panicles and internodes [29]. To further determine the involvement of LG5 in different stages of young panicles, we examined the expression of LG5 in four stages of spikelet (young 2 cm, 7 cm, 13 cm, and 20 cm panicles). We found that LG5 was most highly expressed in the young 2 cm panicle ( Figure 7B). This result was consistent with a previous study of EUI1-GUS, which was detected mainly in rapidly elongating or dividing tissues [29]. Those results showed that LG5 played an important role in regulating grain size, especially in the young panicles. To examine the subcellular localization of LG5, we constructed LG5::GFP and HDEL-mCherry fusions driven by the 35S promoter. The two fusion protein constructs were transferred together into tobacco and rice protoplasts. We observed that the LG5::GFP and HDEL-mCherry fusion proteins were localized in the endoplasmic reticulum both in tobacco and rice protoplasts (Figures 7C,D). These results indicated that LG5 encoded an endoplasmic reticulum protein, consistent with previous evidence [29].

Discussions
Grain size and flag leaf angle are two important agronomic traits that influence grain yield in rice. Many genes associated with these two traits have been identified and characterized, but the molecular and genetic mechanisms underlying grain size and flag leaf angle remain largely unknown [3,11,35]. In the present study, a mutant with a large grain and flag leaf angle, named lg5, was identified and characterized ( Figure 1). Comparison To examine the subcellular localization of LG5, we constructed LG5::GFP and HDEL-mCherry fusions driven by the 35S promoter. The two fusion protein constructs were transferred together into tobacco and rice protoplasts. We observed that the LG5::GFP and HDEL-mCherry fusion proteins were localized in the endoplasmic reticulum both in tobacco and rice protoplasts ( Figure 7C,D). These results indicated that LG5 encoded an endoplasmic reticulum protein, consistent with previous evidence [29].

Discussions
Grain size and flag leaf angle are two important agronomic traits that influence grain yield in rice. Many genes associated with these two traits have been identified and characterized, but the molecular and genetic mechanisms underlying grain size and flag leaf angle remain largely unknown [3,11,35]. In the present study, a mutant with a large grain and flag leaf angle, named lg5, was identified and characterized ( Figure 1). Comparison of the genomic sequences of the candidate region indicated that LOC_Os05g40384/EUI1 is the most likely candidate gene for lg5 (Figure 2). A complementation test restored the grain size and flag leaf angle to the wild-type phenotype. Overexpression of LG5 sharply reduced plant height to a dwarf size and eliminated the ability to bear grains in the XS background ( Figure 3). These results confirmed our assumption that the LOC_Os05g40384 gene was the target gene. In our study, LG5 was found to be involved in regulating important agronomic traits including grain size, flag leaf angle, and plant height. To investigate the function and application of the LG5 gene, we generated LG5-RNAi lines in the NIP background. Some agronomic traits such as grain size, flag leaf angle, and plant height were enhanced in the LG5-RNAi lines (Figure 4). Our results indicated the possibility of applying the LG5 gene to modify seed size and plant architecture. EUI1 (elongated uppermost internode) produced a near doubling in the length of the uppermost internode [36]. To date, some evidence has been found that the EUI1 gene could affect multiple traits in rice; it was found to regulate disease resistance to bacterial blight and blast resistance in rice [37] and to play a role in root gravity responses in rice [38]. Interestingly, in our study, we found that EUI1 also acted as a negative regulator of grain size and flag leaf angle, providing new insights into the functions of EUI and supplying valuable germplasm resources for rice breeding.
Cell expansion and cell proliferation determined organ size [39][40][41]. In our study, cell length and total cell numbers were significantly increased in lg5 compared with those in XS in the cross sections of spikelet hulls ( Figure 5A-F), and the length and width of epidermal cells of the inner and outer glumes were also increased in lg5 compared with those in XS ( Figure 6A-F). To determine the possible regulatory relationship between LG5 and other identified genes that control grain size by regulating cell expansion, such as GS2, and GL7, we examined the transcript level of these genes and found that the expression level of the GS2 gene was significantly increased in the lg5 mutant. In addition, TGW6, GW2, GL3.1, and GS5 were found to control grain size by regulating cell proliferation, and the transcript level of GS5 was significantly increased in the lg5 mutant ( Figure 6G). These results suggested that LG5 may control grain size by regulating the expression of the cell expansion gene and cell proliferation gene. Furthermore, cell cycle genes played functional roles in the cell cycle regulation of seed size and development in plants [42,43]. In this study, several cell cycle genes, including two A-type cyclins (CYCA1;1 and CYCA3;2), two B-type cyclins (CYCB1;1 and CYCB2;2), and two cyclin-dependent kinases (CDKA2 and CDKB2;1), were upregulated in the lg5 mutant ( Figure 6H). The upregulation of these cell-cycle-related genes may contribute to cell division in the spikelet and lead to expanding grain size. These observations suggested that LG5 may control grain size by regulating the expression of cell cycle genes. Histological analysis of the leaf lamina joints, which exhibited significant increases in cell size in lg5 relative to those in XS ( Figure 5G-I), indicated that LG5 may control flag leaf angle by regulating cell expansion in leaf lamina joints. This finding was consistent with literature published during past decade study, indicating the EUI1 gene has a positive effect on parenchyma cell division and elongation [44].
Grain size and leaf angle are critical agronomic traits that determine final yields [27], and both are regulated by phytohormones [3,23]. In rice, BR played important role in the regulation of grain size, leaf angle, plant height, and tiller number [45,46]. For example, overexpression of OsBIM1, which was involved in BR signaling, could significantly increase rice leaf angles but decrease grain size and reduced yield [47]; knockout of OsBUL1, which was involved in BR signaling through regulation of HLH proteins, produced erect leaves with small grains [48]. GA was another predominant hormone regulating plant cell elongation and always exhibits crosstalk with BR in complex mechanisms [49]. GA signalingrelated genes simultaneously controlled grain size and leaf angle and were evident in the case of GW6/OsGSR1, which was a positive regulator of GA signaling. GW6/OsGSR1 could activate BR synthesis through direct interaction with the BR biosynthesis enzyme, DWF1, and the GW6/OsGSR1-RNAi lines showed erect leaves and decreasing seed size [50]. In our study, an lg5 mutant frameshift site occurred in the P450 binding domain region, which resulted in the loss-of-function mutant allele of Os05g40384. Additionally, EUI1 encoded a cytochrome P450 monooxygenase CYP714D1, which was associated with modulating GA responses [29]. Therefore, LG5 might be involved in GAs to coregulate grain size and flag leaf angle. A previous study found that EUI1 was associated with the loss of SLR1 during GA signaling and played a negative regulatory role in GA-mediated cell elongation [51,52]. EUI1 was involved in GA homeostasis not only in the internodes during the heading stage but also in the roots and seeds [38]. Our findings illustrated that LG5 might regulate grain size and flag leaf angle by participating in the phytohormone pathway. In summary, we characterized a mutant lg5, which probably functioned in the phytohormone pathway and contributed to increased grain size, thousand-grain weight, flag leaf angle, and plant height in rice.

Plant Materials and Growth Conditions
The japonica rice XS was radiated with 60 Co-γ. We selected the mutant lg5 with a long grain and flag leaf angle in the M 2 generation and further confirmed the phenotype in M 3 and M 4 progenies. Stable inheritance M 3 and M 4 were used in this research. The lg5 mutant and XS were planted in Hangzhou and Hainan in four generations over two years, and the characters of lg5 were stable in different environments. The lg5 mutant was crossed with the indica rice Huasizhan to generate an F 2 segregation population for gene mapping. All the parents, F 1 plants, and F 2 individuals used for morphological and genetic analyses were grown in paddy fields under natural conditions at the China National Rice Research Institute (Hangzhou, Zhejiang Province).

Map-Based Clone of LG5
The mutant individuals from the F 2 generation of a cross between the lg5 mutant and Huasizhan were selected for mapping. Bulked segregant analysis was used to rapidly locate the mutation as follows: equal amounts of flag leaf from each of 20 XS plants and 20 lg5 mutant plants were sampled for DNA extraction to form an XS pool and an lg5 pool. The parents and the two DNA pools were subjected to preliminary linkage analysis by genotyping 171 polymorphic SSR markers covering 12 chromosomes. Subsequently, 642 lg5 mutant individual plants from the F 2 population were genotyped to determine the physical location of LG5. Sequences of primers for mapping are listed in Table S1.

Phenotyping
The seeds were individually harvested for phenotypic investigation during the mature stage. Fully filled grains were used to measure grain length, grain width, and thousandgrain weight. The grain length, grain width, and thousand-grain weight were evaluated using an automatic seed counting and analyzing instrument (Model SC-G, Wanshen Ltd., Hangzhou, China). Each measurement was repeated three times, and the mean value was recorded. Flag leaf angles were measured by a protractor. Plant heights were measured by a ruler during the mature stage.

Vector Construction and Plant Transformation
We sequenced the entire genomic DNA region of LG5 in XS and NIP. XS and NIP had the same sequence of the LG5 gene. To construct a complementary vector, the LG5 gene containing the 1758 bp promoter region, entire genomic DNA region (9904-bp), and 783 bp downstream of LG5 was cloned into the vector PCAMBIA1300 (PC1300) by seamless cloning. To generate the overexpression lines, the 1734 bp CDS of LG5 was cloned into the vector PCAMBIA1300-CaMV35S (PC1300S), and PC1300 and PC1300s were digested with Kpn1 and Xba1 by double-enzyme digestion. To generate the RNAi lines, the vector pTCK303 was used as described by Wang et al. [53]. To avoid disturbing other homologous genes, 500 bp CDS of LG5 was used as the LG5-RNAi fragment, which was cloned to pTCK303 by BamH1 and Kpn1 for the sense strand and Spe1 and Sac1 for the antisense strand. Genetic transformations were conducted using rice embryogenic calli through agrobacterium tumefacien-mediated transformation [54]. The primers mentioned above are listed in Table S1.

Subcellular Localization of LG5
To determine the subcellular localization of LG5, the 1734 bp CDS of LG5 was cloned into the vector pYBA1132 containing the GFP gene. The Pro35S::LG5-GFP plasmid was introduced into GV3101 (Weidi, AC1003) and injected into young tobacco, whereas the Pro35S::LG5-GFP plasmid was introduced into rice protoplasts. The endoplasmic reticulum protein HDEL (LOC_Os05g45310) was cloned into the vector pYBA1138 fused with the mCherry gene, which was used as an endoplasmic reticulum marker. GFP and mCherry fluorescence signals were observed under a Zeiss LSM710 confocal laser-scanning microscope (Carl Zeiss AG, Jena, Germany). The primers mentioned above are listed in Table S1.

Phylogenetic Tree Analysis
To create a meaningful phylogenetic tree comparing homogenous LG5 in Oryza sativa, Zea mays, Triticum aestivum, Sorghum bicolor, Hordeum vulgare, and Glycine max, the proteins of the 33 varieties were compared. A FASTA file containing a list of proteins was downloaded from the phytozome database for each protein. Phylogenetic evolutionary tree analysis was performed by Mega 6 using the neighbor-joining method and modified online (https://www.evolgenius.info/evolview) accessed on 6 December 2021.

Paraffin Section Analysis
Cross sections of the rice glumes and flag leaf lamina joint areas were analyzed by paraffin sectioning. They were fixed in FAA (50% ethanol, 5% formaldehyde, and 5% glacial acetic acid) for more than 48 h. The fixed samples were dehydrated in a graded ethanol series (30,50,75,95, 100, 100, and 100%), cleared in a xylene series (50, 70, 90, 100%, and 100%), and embedded in paraffin. Cross sections were stained with toluidine blue, stored in paraffin, cut with the YD-335B microtome (Shanghai Zhisun Equipment Co. Ltd, Shanghai, China), and observed by the S-3000N scanning electron microscopy (Hitachi, Tokyo, Japan). They were operated as previously described by Ruan et al. [55].

Transmission Electron Microscopy Analysis
For rice glume cell observation, the spikelets of the lg5 mutant and XS were collected as samples during the maturity stage. The samples were fixed in FAA solution (formalin: glacial acetic acid: ethanol in a 1:1:18 ratio by volume) at 4 • C for 24 h, and dehydrated and dried as described by Feng et al. [56]. The inner and outer surfaces of the spikelet glumes were observed under the S-3400 scanning electron microscope (Hitachi, Tokyo, Japan) at Zhejiang University.

RNA Extraction and qRT-PCR Technology
Total RNA was extracted from various rice tissues using a Mini-BEST plant RNA extraction kit (TAKARA, Tokyo, Japan). First-strand cDNAs were synthesized using Prime Script RT Master Mix (TAKARA, Tokyo, Japan). Quantitative RT-PCR analysis was performed on an Applied Biosystems 7500 real-time PCR system (Invitrogen, Carlsbad, CA, USA) with a 2 × SYBR Green PCR Master Mix (Applied Biosystems, Foster, FL, USA). The rice ACTIN or UBIQUITIN gene was used as an internal control. Each measurement was replicated at least three times with three biological samples. The RT-PCR primers used in these assays are listed in Table S1.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12030675/s1, Table S1: Sequences of primers used in this study; Figure S1. Length of panicle and internode; Figure S2. Evolutionary tree of LG5 in different cereal crops (the tree was constructed by the neighbor-joining method). A total of 33 species were constructed by Mega 6 and divided into three groups from Oryza sativa (Os), Zea mays (Zm), Triticum aestivum (Traes), Sorghum bicolor (Sobic), Hordeum vulgare (Horvu), and Glycine max (Glysopl).  Data Availability Statement: All data and conclusions are included in this paper.