SMALL PLANT AND ORGAN 1 (SPO1) Encoding a Cellulose Synthase-like Protein D4 (OsCSLD4) Is an Important Regulator for Plant Architecture and Organ Size in Rice

Plant architecture and organ size are considered as important traits in crop breeding and germplasm improvement. Although several factors affecting plant architecture and organ size have been identified in rice, the genetic and regulatory mechanisms remain to be elucidated. Here, we identified and characterized the small plant and organ 1 (spo1) mutant in rice (Oryza sativa), which exhibits narrow and rolled leaf, reductions in plant height, root length, and grain width, and other morphological defects. Map-based cloning revealed that SPO1 is allelic with OsCSLD4, a gene encoding the cellulose synthase-like protein D4, and is highly expressed in the roots at the seedling and tillering stages. Microscopic observation revealed the spo1 mutant had reduced number and width in leaf veins, smaller size of leaf bulliform cells, reduced cell length and cell area in the culm, and decreased width of epidermal cells in the outer glume of the grain. These results indicate the role of SPO1 in modulating cell division and cell expansion, which modulates plant architecture and organ size. It is showed that the contents of endogenous hormones including auxin, abscisic acid, gibberellin, and zeatin tested in the spo1 mutant were significantly altered, compared to the wild type. Furthermore, the transcriptome analysis revealed that the differentially expressed genes (DEGs) are significantly enriched in the pathways associated with plant hormone signal transduction, cell cycle progression, and cell wall formation. These results indicated that the loss of SPO1/OsCSLD4 function disrupted cell wall cellulose synthase and hormones homeostasis and signaling, thus leading to smaller plant and organ size in spo1. Taken together, we suggest the functional role of SPO1/OsCSLD4 in the control of rice plant and organ size by modulating cell division and expansion, likely through the effects of multiple hormonal pathways on cell wall formation.


Introduction
Rice (Oryza sativa L.) is one of the most important food crops in the world.Increasing plant biomass, especially grain yield, has always been a primary goal of rice breeding and germplasm improvement [1].Plant architecture including leaf morphology, plant height, tiller patterning, and reproductive organ structure, etc., is closely associated with rice cultivation, harvest index, biomass, and grain yield, being of great agronomic importance [2,3].At the cellular level, these agronomic traits, such as leaf shape, plant height, and grain size, also known as organ size, are determined by cell number and cell size, resulting from the processes of cell proliferation and cell expansion [4,5].Therefore, elucidation of the genetic and molecular mechanisms controlling plant architecture and organ size may contribute to rice breeding and improvement.
The leaf is the main photosynthetic and gas-exchange organ in rice.Leaf shape affects energy capture and other important physiological activities that have a close connection with plant architecture and grain yield [6,7].So far, a large number of genes have been reported to regulate leaf shape, particularly leaf width and leaf rolling.A series of characterized genes in rice, such as NAL1 [8], NAL2/3 [9], NAL9 [10], NAL11 [11], NRL2 [12], DNL-4 [13], NAL21 [14], WL1 [15], and NAL22 [16], control leaf width mainly by regulating the number of leaf veins, the distance between veins, and the width of veins.Rice leaf rolling is often associated with the development of bulliform cells (BCs) in the adaxial side of the leaves.Changes in the size and/or number of BCs may cause varying degrees of leaf rolling, such as NAL7 [17], SLL1 [18], ACL1 and ACL2 [19], ROC5 [20], SRL1 [21], RL14 [22], OsZHD1 [23], OsHox32 [24], and REL2 [25].Moreover, some genes not only regulate leaf shape but also play a role in determining plant height and/or grain size, such as NRL2 [12], DNL-4 [13], NAL21 [14], REL2 [25], NAL1 [26], miR159 [27] AH2/SLL1 [28], and CLD/SRL1 [29].It is suggested that leaf shape, plant height, and grain size are affected by the number and/or size of cells.For example, the nal1 exhibits narrow leaves due to defects in anticlinal cell division and reduced abaxial epidermal cell size [26,30].The semi-dwarf mutant cpb1 is characterized by shorter stem internodes, mainly resulting from shorter cell length [31].Rice OsCBL5 promotes grain size by affecting the expansion of spikelet hull cells [32].Rice GS9 regulates grain shape by affecting the number of cells in the spikelet hull [33].Therefore, genes regulating cell division and/or expansion play important roles in determining plant architecture and organ size, which are determined by cell number and/or size.Furthermore, in plants, hormones can also play critical roles in plant architecture and organ size regulation.Many hormone-related genes have been characterized as important regulators of plant architecture and organ size in rice [34,35].Among the hormones, gibberellins (GAs) and auxin (IAA) are mainly responsible for cell division and expansion in controlling organ size [36,37].Blocked GA synthesis and signaling usually show reduced height and small grain [36,38].Auxin deficiency and/or insensitivity may alter plant height, leaf width, and grain size [39,40].Rice plants overexpressing OsGA2ox5, a gene encoding gibberellin (GA) 2-oxidases, exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems resulting in shorter and smaller cells of leaf sheaths and later development of reproductive organs [41].GW6 (GRAIN WIDTH 6) encodes a GA-regulated GAST family protein and positively regulates grain width by promoting cell expansion in the spikelet hull and by modulating GA response and biosynthesis in rice [42].The OsARF11 gene contributes to the reduced growth of roots and leaf blades with a reduced response to exogenous auxin, which is consistent with its role in mediating a response to auxin to stimulate cell division and/or cell expansion [43].Activation of BIG GRAIN1 (BG1), a rice gene regulating auxin transport, results in large grains due to increased cell proliferation and cell expansion in spikelet hulls [44].Furthermore, some hormones may play overlapping roles and have crosstalk in regulating plant architecture and organ size [45][46][47][48].It is suggested that OsSHI1 can act as a transcriptional regulatory hub that orchestrates the integration and self-feedback regulation of multiple hormone signaling pathways including auxin, BRs, and ABA to coordinate plant architecture and other aspects [49].OsIAA1-overexpressing transgenic plants have decreased plant height and loose plant architecture, followed by reduced auxin sensitivity but increased sensitivity to BR [50].These studies suggest that the sophisticated regulatory mechanism coordinates plant architecture and organ size by integrating various plant hormone signaling pathways to regulate cell division and/or cell expansion in rice.
Plant cell walls mainly comprise cellulose, hemicellulose, pectin, and structural proteins [51].Cellulose is produced in the plasma membrane by cellulose synthase complexes, and cellulose synthase-like (CSL) superfamily genes play a very important role in this process [52].Besides the functions in regulation of cell wall and cellulose biosynthesis, CSL members are also involved in other aspects of plant growth and development, such as the roles of maize CSLD1 [53] and Arabidopsis SOS6/AtCSLD5 [54] in cell division, expansion, and abiotic stress response, etc.As one of the five CSLD genes in rice, the OsCSLD4 gene has been extensively studied in recent years [55][56][57][58][59][60][61][62][63][64].Although the different mutant alleles display more or less difference in plant phenotype, a common characteristic in OsCSLD4 gene mutation is the defects in plant and leaf morphology.For instance, the dnl1 mutant displayed dwarf and narrowed phenotype [60], the nrl1 mutant showed fewer veins and smaller adaxial bulliform cells in leaf blade and decreased in plant height [56], and the nrl1 mutant showed a decrease in the number of vascular bundles of leaf [57].It was revealed that the loss of OsCSLD4 gene function altered the structure of arabinoxylan and the content of cellulose and homogalacturonan in nd1 mutant [55] and altered the cellulose content and the level of xylose in cd1 mutant [58].Yoshikawa et al. (2013) [59] suggested that the product of OsCSLD4 plays a pivotal role in the M phase to regulate cell proliferation.Shi et al. (2016) [61] indicated the role of OsCSLD4 in gibberellin signaling by characterization of the dnl3 mutant.Furthermore, it demonstrated the function of OSCSLD4 in the regulation of abiotic stresses including salt, salt-alkali, and drought stress [62][63][64].These results indicate that OsCSLD4 is an important regulator in plant growth and development, abiotic stress response, and other aspects.
Although genes regulating plant architecture and organ size have been extensively identified in rice, the underlying regulatory mechanisms are still under investigation.In this study, we identified and characterized small plant and organ 1 (spo1), a new mutant allele of OsCSLD4, which showed narrow and rolled leaves, decreased plant height, root length, and grain width, and other morphological defects.Our investigation revealed that the lossof-function of SPO1/OsCSLD4 led to a reduced number and width in leaf veins, smaller size of leaf bulliform cells, reduced cell length and cell area in the culm, and decreased width of epidermal cells in the grain, and was accompanied with significant changes in multiple hormone contents and altered expressions of genes associated with plant hormone signal transduction, cell cycle progression, and cell wall formation.Considering that SPO1/OsCSLD4 encoding cellulose synthase-like D4 protein is required for cell wall cellulose formation, these results suggested that SPO1/OsCSLD4 plays an important role in regulating plant and organ size through cell division and expansion, possibly mediated by the effects of multiple hormone pathways on cell wall formation.

Phenotypic Characterization of the spo1 Mutant
At the seedling stage, the spo1 mutant exhibited abnormal phenotypes including reduced plant height and narrowed and curled leaves (Figure 1a).At the mature stage, the spo1 mutant showed completely different plant architecture, including plant height, leaf length and width, tiller number, and grain size, as compared with the WT (Figure 1b-e).The plant height of spo1 was 77.18% of that of WT (Figure 1b,g), and the reduced plant height of spo1 was mainly due to shortened I, II, and IV internodes of the culm (Figure 1d,h).Compared to WT, the spo1 mutant has more tillers per plant (Figure 1b and Table 1).The width of leaf blades in the spo1 mutant was significantly reduced (Figure 1c,i) and the length of leaf blades was also significantly reduced in the second and third leaf in spo1 mutant (Figure 1c and Table 1).Furthermore, the spo1 mutant exhibited significantly increases in leaf-rolling index (LRI, Figure 1c,j) and leaf angle (Table 1).Associated with the characterization above, it was showed that the spo1 mutant also has significant reductions including grain width (Figure 1e,k), 1000-grain weight, panicle length, and fertility rate (Table 1) as compared with WT.Moreover, we noticed that the spo1 mutant exhibited abnormalities in roots (Figure 1f and Table 1) and reduced content of chlorophyll a (Chl a) in leaves (Figure S1).These observations indicated that the spo1 mutant has small organs and multiple morphological defects.Table 1.Comparison differences of some agronomic traits between WT and spo1 mutant.

Map-Based Cloning Revealed That SPO1 Gene Encodes Cellulose Synthase-like D4
To isolate the SPO1 gene by map-based cloning, we developed a genetic population by crossing the spo1 mutant with the indica cultivar Kasalath.All the F 1 plants displayed wild-type phenotype.In the F 2 mapping population, 221 normal individuals and 79 spo1 individuals were obtained, fitting to the 3:1 Mendel's separation ratio, indicating that spo1 was controlled by a single recessive gene.Genetic mapping of the SPO1 gene using F 2 mutant individuals revealed that SPO1 was linked with molecular markers R12M10 and RM101 on rice chromosome 12 (Figure 2a).Using 32 F 2 mutant individuals, the SPO1 gene was initially mapped to a region on the long arm of chromosome 12 between molecular markers RM277 and RM17 (Figure 2a).The SPO1 gene was further fine-mapped to a 90-kb physical region between molecular markers RM28433 and RM28449 by genotyping of 160 F 2 mutant individuals (Figure 2b).According to gene annotation in the IRGSP 1.0 database (https://rapdb.dna.affrc.go.jp/, accessed on 1 May 2020), there are 15 candidate genes in the 90-kb mapping region (Figure 2c).The candidate OsCSLD4/Os12g0555600, reported previously, encodes cellulose synthase-like D4 and is essential for normal cellwall biosynthesis, plant growth, and abiotic resistance [55][56][57][58][59][60][61][62][63].Gene sequencing analysis revealed that spo1 contains C to T substitution in the second exon of OsCSLD4, resulting in an amino acid substitution from Ala to Val (Figure 2d).RT-PCR and qRT-PCR analyses further showed that the mRNA expression of SPO1/OsCSLD4 was greatly reduced in the spo1 mutant (Figure 2e,f).Predictions of protein structure revealed that the spo1 mutation (C to T substitution) caused a change of OsCSLD4 three-dimensional structure in the direction of rotation (Figure S2a).Then, phylogenetic analysis and multiple sequence alignments were performed among the SPO1/OsCSLD4 and its homologous proteins.The phylogenetic analysis showed that the SPO1/OsCSLD4 protein had the closest relationship with BdCSLD4, ZmCSLD1, and AtCSLD5 (Figure S2b).The result from multiple sequence alignment analysis showed that the amino acid Ala at the SPO1 mutation site is highly conserved in higher plants (Figure S2c), implying that the amino acid substitution from Ala to Val had a profound effect on the SPO1/OsCSLD4 protein function.
To further confirm SPO1 function, complementation experiment was performed by genetic transformation of the wild-type SPO1/OsCSLD4 gene into the spo1 mutant.Compared with that of the spo1 mutant, the phenotype of complementary transgenic lines (Com-SPO1) is similar to that of wild-type plants including increased plant height and reduced leaf rolling (Figure 2g), and increased leaf width (Figure 2g,h).These results indicated that the spo1 mutant phenotype is caused by the loss-of-function mutation of SPO1/OsCSLD4.We then overexpressed SPO1 in Nipponbare.However, there was no significant phenotypic difference between the overexpression lines and wild-type Nipponbare (Figure S3a-i).

The Tissue-Specific and Stressed-Induced Expression Patterns of SPO1
We examined the detailed tissue-specific expression patterns by qRT-PCR in different tissues of Nipponbare.It was revealed that the expression of SPO1 was highly tissuespecific, with the highest expressions in roots at seedling stage and tillering stage of rice plants (Figure 3a).However, the expression level of SPO1 was relatively low in the leaves at all stages tested (Figure 3a).On the whole, SPO1 showed higher expression levels in vigorous growth organs.
Analysis of cis-elements in the promoter region of SPO1 was performed on PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 1 May 2020).This revealed that the SPO1 promoter contains several elements, such as auxin-responsive element TGA-element, ABA-response element (ABRE), gibberellin-response element TATCbox, and drought-response element MBS, etc., (Table S1), suggesting that SPO1 might be regulated by hormones and respond to abiotic stress.We then performed the stimulusinduced expression analysis of SPO1.On the whole, the SPO1 showed up-or downregulation in response to different treatments or stresses.We analyzed the expression of SPO1 by exogenous applications of hormone IAA, GA, ABA, and MeJA.It was showed that SPO1 was strongly induced by GA at 1 h but was decreased to a low level at 6 h (Figure 3b).However, the expression of SPO1 increased again at 12 and 24 h after GA treatment (Figure 3b).Exogenous application of hormone ABA also induced the expression of SPO1, but application of hormone IAA significantly decreased the expression of SPO1 (Figure 3b).The expression of SPO1 was up-regulated from 1 to 9 h by MeJA treatment (Figure 3b).We further analyzed the expression of SPO1 under different stress conditions, including NaCl, drought, oxidative, heat, and cold stress.The SPO1 expressions were progressively induced at 3, 6, 9, and 12 h after treatment with NaCl (Figure 3c).Under drought stress mimicked by PEG-6000 treatment, the expression of SPO1 was increased at 3, 6, and 9 h (Figure 3c).Under oxidative stress via MV treatment, SPO1 expression was rapidly increased at 0.5 h, but decreased to the pre-treatment level at 1 h (Figure 3c).The expression of SPO1 was increased after 6 h by high-temperature stress, but the expression of SPO1 was not significantly affected under low temperature conditions (Figure 3c).ment (Figure 3b).We further analyzed the expression of SPO1 under different ditions, including NaCl, drought, oxidative, heat, and cold stress.The SPO1 e were progressively induced at 3, 6, 9, and 12 h after treatment with NaCl (Figu der drought stress mimicked by PEG-6000 treatment, the expression of SPO creased at 3, 6, and 9 h (Figure 3c).Under oxidative stress via MV treatment, SP sion was rapidly increased at 0.5 h, but decreased to the pre-treatment level at 3c).The expression of SPO1 was increased after 6 h by high-temperature stre expression of SPO1 was not significantly affected under low temperature cond ure 3c).

SPO1 Regulates Leaf Shape, Plant Height, and Grain Size through Affecting Cell Division and/or Cell Expansion
Because spo1 showed abnormalities in leaf shape, plant height, and grain size, we therefore conducted cytological characterization on the wild type and the mutant.According to cross-sections on the leaf blade, it was showed that the number of leaf veins, especially small veins, and the mean width of large and small veins were significantly reduced in spo1 mutant (Figure 4a,c,d,e).The spo1 mutant also showed a reduced size of bulliform cells (BCs), but no difference in the number of BCs (Figure 4b,f,g).According to the observation on longitudinal sections of culms, it was demonstrated that the cell length and areas were significantly reduced in spo1 mutant (Figure 4h-j).It is suggested that spikelet hulls can limit grain growth and affect grain size [65].Because the spo1 mutant showed a reduction in grain width, we further analyzed the cell width and length of spikelet hulls.According to observation by SEM, it is demonstrated that the width of the longitudinal cells on the outer epidermis of spikelet hulls was significantly reduced in the spo1 mutant (Figure 4k,l).However, there is no difference in the length of the longitudinal cells on the outer epidermis of spikelet hulls between the WT and spo1 mutant (Figure 4k,m).Taken together, these data indicated that SPO1 regulates leaf shape, plant height, and grain size, mainly by affecting cell division and/or cell expansion.
eas were significantly reduced in spo1 mutant (Figure 4h-j).It is suggested th hulls can limit grain growth and affect grain size [65].Because the spo1 mutan reduction in grain width, we further analyzed the cell width and length of sp According to observation by SEM, it is demonstrated that the width of the lo cells on the outer epidermis of spikelet hulls was significantly reduced in the s (Figure 4k,l).However, there is no difference in the length of the longitudinal outer epidermis of spikelet hulls between the WT and spo1 mutant (Figure 4k together, these data indicated that SPO1 regulates leaf shape, plant height, and mainly by affecting cell division and/or cell expansion.

Hormone Contents Were Significantly Altered in the spo1 Mutant
To investigate if hormone-related processes were affected in the spo1 mutant, we further analyzed hormone contents in the leaf of spo1 and WT.Various hormones such as IAA, ABA, GA 3 , GA 4 , ZR, and dhZR content are significantly affected.The IAA content is about 2 times lower in spo1 than in wild-type plants.The ABA content is also decreased in spo1 compared to WT (Table 2).A 29.84% and 31.6% increase in the content of GA 3 and GA 4 , respectively, is measured in the spo1 mutant compared to the wild type (Table 2).Similarly, ZR and dhZR were also increased in the spo1 mutant (Table 2).These results indicated that the loss-of-function of SPO1 has a significant impact on hormone contents.The values were shown as mean ± SD (n ≥ 3).Double asterisk (**) and indicates that the difference between the WT and spo1 is statistically significant at p < 0.01.

Loss-of-Function of SPO1 Affects the Expression of Leaf-Shape-Related Genes
To further investigate how SPO1 functions in regulation of leaf development, the expressions of leaf-shape-related genes, such as NAL1, NRL2, NAL7, NAL9, etc., were examined by qRT-PCR.The results showed that the loss of SPO1 gene function resulted in a strong increase in NAL7/COW1 (a flavin-containing monooxygenase gene, related to narrow and curled leaf phenotype) [17], ACL1, and ACL2 (relate to abaxial leaf curling phenotype) [19] expressions in the spo1 mutant (Figure 5).In contrast, the expressions of NRL2 (a novel protein with a conserved function, related to regulating leaf width) [12], ROC5 (homeodomain leucine zipper class IV gene, related to rolled leaf phenotype) [20], and RL14 (a 2OG-Fe (II) oxygenase family protein gene, related to narrow and rolled leaf phenotype) [22], were significantly down-regulated in the spo1 mutant (Figure 5).These results showed that the loss-of-function of SPO1 affects the expression of these genes in regulating leaf morphological development of rice.

Transcriptomic Analysis Revealed SPO1 Plays an Important Role in Hormone, Cell Cycle, and Cell Wall Formation Pathways
To further understand the molecular mechanism of SPO1 in the regulation of organ

Transcriptomic Analysis Revealed SPO1 Plays an Important Role in Hormone, Cell Cycle, and Cell Wall Formation Pathways
To further understand the molecular mechanism of SPO1 in the regulation of organ size, especially leaf shape, RNA-seq analysis was performed with the leaf blade of the spo1 mutant and wild-type plants at the tillering stage.A total of 2379 differentially expressed genes (DEGs) including 848 down-regulated genes and 1531 up-regulated genes were identified by RNA-seq (Figure S4, Table S3).Gene ontology (GO) enrichment analysis revealed that the 2379 DEGs were significantly enriched in 10 terms at the biological process category, 8 terms at the cellular component category, and 12 terms at the molecular function category (Figure 6a).In the biological process category, the most enriched GO term is metabolic process (Figure 6a).In the cellular component category, the top two most enriched GO terms are the membrane part and membrane (Figure 6a).In the molecular function category, the top three most enriched GO terms include catalytic activity, binding, and transporter activity (Figure 6a).According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the DEGs, the top five most enriched pathways are biosynthesis of amino acids, carbon metabolism, plant hormone signal transduction, starch and sucrose metabolism, and cysteine and methionine metabolism (Figure 6b).
(OsRR6/LOC_Os04g57720), and cytokinin receptor (OHK4/LOC_Os03g50860) were icantly up-regulated in the spo1 mutant compared to the wild type (Table 3).These r indicated that the loss of SPO1 gene function affected auxins, GAs, and CKs pathw In addition, many of the genes associated with cell cycle and cell wall formation significantly differently expressed in the spo1 mutant.For example, two (LOC_Os07g30240，LOC_Os12g04980) involved in meiosis were up-regulated in spo one gene (LOC_Os05g41880) related to meiotic reciprocal recombination was down lated in the spo1 mutant (Table 3).Four genes (LOC_Os04g53680, LOC_Os01g LOC_Os05g33040, LOC_Os09g38768) encoding cell cycle proteins were differentia pressed in the spo1 mutant (Table 3).Moreover, a large number of genes related t wall-related pathways were affected in the spo1 mutant (Table 3).For example, six involved in cell wall formation had significantly altered expressions in the spo1 mut NAC transcription factor OsSWN3 (LOC_Os08g01330) and OsCAD3 (LOC_Os10g2 which participate in cell wall lignin biosynthesis, were significantly up-regulated spo1 mutant.A R2R3-type MYB transcription factor (LOC_Os04g50770) and a NAC scription factor OsSWN2 (LOC_Os08g02300), both of which regulate the cell wall cel biosynthesis pathway, were down-regulated in the spo1 mutant.The NAC transcr factor OsSWN1 (LOC_Os06g04090) and the 2OG-Fe (II) oxygenase family protein (LOC_Os10g40960), which regulate secondary wall biosynthesis, were down-regula the spo1 mutant.These results indicated the loss of SPO1 gene function affected th cesses associated with cell cycle and cell wall formation pathways.According to KEGG enrichment analysis, plant hormone signal transduction is one of the most enriched pathways.We found a great number of genes related to auxins, GAs, and CKs pathways were affected in the spo1 mutant (Table 3).It is demonstrated that auxin response factor (OsARF11/LOC_Os04g56850), auxin-responsive SAUR gene (LOC_Os02g07110), auxin receptor (OsAFB6/LOC_Os03g08850), auxin efflux transport carrier (OsPIN5a/LOC_Os01g69070), and indole-3-acetic acid-amido synthetase (Os-MGH3/LOC_Os07g40290) were significantly up-regulated in the spo1 mutant, but the AUX1/LAX (OsAUX4/LOC_Os10g05690, OsAUX3/LOC_Os05g37470) and two auxinresponsive Aux/IAA genes (LOC_Os02g56120/LOC_Os01g53880) and IAA synthesis gene OsYUCCA6 (LOC_Os07g25540) were significantly down-regulated in the spo1 mutant (Table 3).Genes involved in GA-related pathways, such as three genes encoding gibberellin oxidase (OsGA2ox5/LOC_Os07g01340, OsGA20ox2/LOC_Os01g08220, CYP714B2/ LOC_Os03g21400), one gene encoding gibberellin 3β-hydroxylase (OsGA3ox2/ LOC_Os01g08220), and two genes encoding gibberellin receptors GID1L2 (LOC_Os07g44900, LOC_Os09g28230) were significantly up-regulated in the spo1 mutant (Table 3).Two genes encoding UDP-glucosyltransferase (LOC_Os05g08480, LOC_Os07g30610), two genes belonging to the cytokinin oxidase/dehydrogenase family (OsCKX3/LOC_Os10g34230, Os-CKX4/LOC_Os01g71310), type-A response regulator gene (OsRR6/LOC_Os04g57720), and cytokinin receptor (OHK4/LOC_Os03g50860) were significantly up-regulated in the spo1 mutant compared to the wild type (Table 3).These results indicated that the loss of SPO1 gene function affected auxins, GAs, and CKs pathways.In addition, many of the genes associated with cell cycle and cell wall formation were significantly differently expressed in the spo1 mutant.For example, two genes (LOC_Os07g30240, LOC_Os12g04980) involved in meiosis were up-regulated in spo1 and one gene (LOC_Os05g41880) related to meiotic reciprocal recombination was downregulated in the spo1 mutant (Table 3).Four genes (LOC_Os04g53680, LOC_Os01g13260, LOC_Os05g33040, LOC_Os09g38768) encoding cell cycle proteins were differentially expressed in the spo1 mutant (Table 3).Moreover, a large number of genes related to cellwall-related pathways were affected in the spo1 mutant (Table 3).For example, six genes involved in cell wall formation had significantly altered expressions in the spo1 mutant.A NAC transcription factor OsSWN3 (LOC_Os08g01330) and OsCAD3 (LOC_Os10g29470), which participate in cell wall lignin biosynthesis, were significantly up-regulated in the spo1 mutant.A R2R3-type MYB transcription factor (LOC_Os04g50770) and a NAC transcription factor OsSWN2 (LOC_Os08g02300), both of which regulate the cell wall cellulose biosynthesis pathway, were down-regulated in the spo1 mutant.The NAC transcription factor OsSWN1 (LOC_Os06g04090) and the 2OG-Fe (II) oxygenase family protein RL14 (LOC_Os10g40960), which regulate secondary wall biosynthesis, were down-regulated in the spo1 mutant.These results indicated the loss of SPO1 gene function affected the processes associated with cell cycle and cell wall formation pathways.

Discussion
In this study, we characterized a spo1 mutant that exhibited small plant and organ size, including reduced plant height and root length, narrow and rolled leaf, and slender grain, etc.Through map-based cloning and genetic transformation, spo1 was identified as a novel allele of OsCSLD4, which belongs to the cellulose synthase-like superfamily and functions in cell wall formation, plant growth, and abiotic resistance based on previous functional studies [55][56][57][58][59][60][61][62][63].Our investigation indicated that SPO1 may control plant and organ size by modulating cell division and expansion possibly mediated by the effects of multiple hormone pathways on cell wall formation.
The spo1 mutant formed narrow and rolled leaves (Figure 1a-c) and showed vascular bundles and bulliform cells (BCs) defects in leaf blade (Figure 4a-g), indicating the SPO1 may act as an important factor influencing rice leaf morphology.Previous studies have shown that changes in leaf vein patterns can lead to leaf narrowing, for example, NAL1 [8], NAL2 [9,66], NAL3 [9,66], NAL9 [10], and NAL11 [11] affect leaf width by controlling the number of veins.Mutation of NRL22 reduces vein width in both large and small veins compared to the wild type, thereby producing narrow leaf phenotypes [16].In this study, the spo1 leaf showed a reduced number and width of veins, suggesting that SPO1 is involved in the formation of vascular bundles in the leaf.Furthermore, among the genes tested to regulate leaf morphology, NAL7 is strongly up-regulated in the spo1 mutant (Figure 5).Loss of function of NAL7, which encodes a member of the rice YUCCA gene family that regulates auxin biosynthesis, results in narrow and curled leaves and altered IAA content [17].In addition, the expression levels of RL14, ROC5, ACL1, and ACL2 appear to be down-or up-regulated in the spo1 mutant (Figure 5).These four genes cause leaf curling by directly or indirectly regulating the size or number of BCs.The NRL2, which interacts with RL14 and has a higher cellulose content and lower lignin content than the WT, is also down-regulated in the spo1 mutant.These results suggest that there are certain functional connections between SPO1 and these tested genes in regulating leaf morphology.Therefore, the function of the SPO1 gene is required for the normal formation of veins and BCs in the leaf blade.
Plant hormones such as auxin, gibberellin (GA), cytokinin (CK), and abscisic acid (ABA) have various functions in plant growth and development [67].Changes in hormone levels or homeostasis in plant may affect plant architecture and organ size.Several genes that regulate plant architecture and organ size in relation to hormone pathways have been identified and characterized in rice.For instance, the narrow leaf mutant nal7 [17], osarf24 [68], and tryptophan-deficient dwarf mutant tdd1 [39], which are all related to the auxin pathway.The dwarf mutants gid1 [69], gid2 [70], slr1 [71], d18 [72], and sd1 [73], and the small seeds mutants oscbl5 [32] and sgsd3 [74], are related to the gibberellin pathway.
In the present study, many traits related to organ size, including narrow leaf, dwarf plant, and small grain characteristics, may be associated with auxin and GAs.The content of IAA is decreased, but the contents of GA 3 and GA 4 are increased in the spo1 mutant as compared to the wild type (Table 2).Rice YUCCA (YUC) flavin-containing monooxygenaseencoding gene OsYUC11 has been shown to be a key factor for auxin biosynthesis.The osyuc11 showed reduced seed size and increased chalkiness, accompanied by reduced IAA level [75].The gid1 is a GA-insensitive mutant, which had a severe dwarf phenotype with wide, dark-green leaf blades and increased GA levels compared with the wild type [69].These results are consistent with the phenotype in the spo1 mutant.Given this knowledge, the spo1 mutant may be an IAA-deficient and GA-insensitive mutant.Our RNA-seq data revealed that the expression of auxin-responsive genes (OsIAA6, OsIAA9), IAA synthetic pathway gene (OsYUCCA6), and auxin influx transporter genes (OsAUX3, OsAUX4) were lower in the spo1 mutant compared to the wild type.Efflux carriers of polar auxin transporter (OsPIN5a), auxin-signaling F-Box gene (OsAFB6), and indole-3-acetate beta-glucosyl transferase were up-regulated in the spo1 mutant compared to the wild type.However, auxin response factor, OsARF11, auxin-responsive SAUR gene, OsSAUR6 and tryptophan amino transferase, and FIB were up-regulated in the spo1 mutant compared to the wild type.In addition, the expressions of genes related to the GA pathway (OsGA2ox5, D18, SD1, CYP714B2, and two GA receptor-like genes (GID1L2)) are higher in the spo1 mutant compared to the wild type.D18 and SD1 participate in the synthesis of GA and exhibit plant dwarfism [72,73].Overexpression of CYP714B2 can lead to semi-dwarfism in Arabidopsis plants and increase the content of 13-OH type GAs [76].Overexpressing OsGA2ox5, a gene involving in the GA catabolic pathway, exhibited dominant dwarf phenotype in rice [41].Therefore, the function of SPO1 may be related to the aforementioned genes and loss of SPO1 function may disrupt auxin and GA homeostasis and signaling, thus resulting in smaller organ size in the spo1 mutant.The increasing transcription levels of OsARF11, OsSAUR6, FIB, and OsGA2ox5 in the spo1 mutant may indicate that their regulations are required for plants to maintain the endogenous IAA and GAs levels stably.Moreover, the organ size is sustained by the coordination of the two basic cellular processes of cell division and cell expansion, which determine cell numbers and cell sizes, respectively [4,5].The leaf morphology is also influenced by cell proliferation and cell expansion [14,77].In fact, previous studies have revealed that sle1, the allelic mutant of spo1, had reduced cell proliferation beginning at the P3 primordial stage, resulting in narrow leaf blades [59].Consistent with this finding, according to our RNA-seq data, the transcripts levels of meiotic recombination genes (OsMSH4, OsDMC1A, OsMSH5) and cyclin genes (CycP1;1, CycA1;1, OsCYCP1;1) that control cell cycle processes were changed to varying degrees in the spo1 mutant.The results suggest that SPO1 affects the cell proliferation process by altering the expression of these cell cycle genes, and ultimately affecting rice architecture and organ size including leaf development.In addition to changes in leaf morphology, the spo1 mutant also forms a dwarf and slender grain phenotype, which contributes to small organs (Figure 1a,b,d,e).The longitudinal sections of the shortened internodes in the spo1 mutant exhibited a reduction in the cell length and area (Figure 2h-j).In addition, the width of the longitudinal cells on the outer epidermis of the spikelet hulls was significantly reduced in the spo1 mutant, as determined by SEM observation (Figure 2k-m).These findings indicated that SPO1 regulates plant height and grain size by the progress of cell expansion.Auxin and GAs are mainly responsible for cell division and expansion [36,37].NAL1 regulates cell division and affects polar auxin transport and vascular patterns, and mutation of NAL1 resulting in a reduction in leaf blade width [8,26].Activation of rice BIG GRAIN1 (BG1), which is involved in auxin transport, results in large grains due to increased cell proliferation and cell expansion in spikelet hulls [44].BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice [78].Consistent with these, it is very likely that altered expressions of genes relating to auxins and GAs pathway in the spo1 mutant were owing to disrupted auxin and GA homeostasis and signaling, which are involved in the cellular processes of cell division and cell expansion.In addition to auxin and GAs, CKs also mediate organs size in plant growth and development including cell division and cell expansion [79].In the spo1 mutant, the expression of OsCKX4 and OsCKX3 was increased.Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis [80].OsRR6 acts as a negative regulator of CK signaling, the overexpression of which leads to dwarf phenotypes [81].Therefore, it was possible that abnormal CK signaling in the spo1 mutant might also be attributed to altered morphological characters, such as plant height and grain size.
In fact, the role of hormones in plant growth cannot be easily summarized by a linear signaling pathway.Some hormones such as ABA, and BR are also involved in regulating organ size and plant architecture through crosstalk and integration with multiple hormone pathways [49,82].For instance, OsIAA1 may play important roles in the crosstalk between auxin and the BR signaling pathways during plant morphogenesis.OsIAA1 overexpression in transgenic plants showed decreased plant height and loose plant architecture due to decreased auxin sensitivity and increased BR sensitivity [50].In the present study, SPO1 may be induced to varying degrees by different hormones, and hormone levels such as IAA, GAs, CKs, and BR were also changed in the spo1 mutant.In addition, genes involved in different hormone pathways have been identified by RNA-seq.According to these results, we suggest that SPO1 may regulate hormone homeostasis by integrating multiple hormone pathways, thereby regulating cell division and expansion, and ultimately regulating plant architecture and organ size.
Previous studies have reported that OsCSLD4 plays an important role in cell wall formation by altering xylan and cellulose content, and indeed the cellulose content was reduced in OsCSLD4 mutant [55].In this study, a large number of DEGs involved in cell wall formation were identified in spo1 mutant (Table S3).For instance, OsSWN3 (LOC_Os08g01330) and OsCAD3 (LOC_Os10g29470) which participate in cell wall lignin biosynthesis, a R2R3-type MYB transcription factor (LOC_Os04g50770) and a NAC transcription factor OsSWN2 (LOC_Os08g02300) which regulate the cell wall cellulose biosynthesis pathway, and a NAC transcription factor OsSWN1 (LOC_Os06g04090) and 2OG-Fe (II) oxygenase family member RL14 (LOC_Os10g40960) which regulate secondary wall biosynthesis, were differentially expressed in spo1 mutant.This finding suggests that SPO1 is involved in regulating the synthesis of cell walls.Studies have shown that a decrease in cellulose content can lead to weakened mechanical support and increased leaf angle [83].In breeding, rational dense planting is advocated to increase yield, while increased leaf angle requires more space for growth, which affects yield and leads to decreased fertility rate and biomass.The spo1 mutant has significantly reduced leaf width, which may affect its photosynthetic capacity and overall growth.This may also be the reason for the decreased fertility rate.Root length was significantly shorter in the mutant, which may lead to inadequate nutrient uptake and thus affect yield.SPO1 has a very obvious "one-causemultiple-effect" role, suggesting that this gene plays an important role at all stages of rice development.The data reported here that SPO1 is allelic with SPO1/OsCSLD4.As shown in Figure S5, the spo1 mutant has one single nucleotide substitution (C2006T) that is same as dnl1 and nd1, but each of their mutation positions is different.Additionally, the mutated amino acids are different between the spo1 and dnl1 mutant.Therefore, we demonstrated that spo1 is a novel allele of SPO1/OsCSLD4.The mutated amino acid Ala in spo1 is highly conserved in CSLD members of plants (Figure S2c); the amino acid Ala was mutated to Val by the single base mutation in the SPO1/OsCSLD4 gene, but the polarity of the amino acids did not change.However, it led to severe mutations in multiple phenotypes, indicating the importance of this site.In addition, SPO1/OsCSLD4 has noteworthy biological functions in the regulation of plant architecture and organ size in rice.

Plant Materials and Growth Conditions
The spo1 mutant was isolated from the rice cultivar Nipponbare (Oryza sativa L. japonica) by mutagenesis with ethyl methanesulfonate.Overexpressing and complementary transgenic plants were obtained by infecting the rice calli of the Nipponbare variety and the spo1 mutant, respectively, with Agrobacterium tumefaciens carrying related vectors.All plant materials were grown in pots or in paddy fields under natural conditions and in a greenhouse with a 14 h light (30 • C)/10 h dark (26 • C) cycle, and with 70-80% relative humidity.

The Mapping of the SPO1 Gene
For genetic mapping, the spo1 mutant was crossed with the rice cultivar Kasalath (Oryza sativa L. indica).We identified 318 mutant individuals from the F 2 population.Preliminary genetic mapping was performed by genotyping of 32 F 2 mutant plants with SSR markers (Table S2).For the fine-mapping of spo1, 20 new SSR markers was designed and 160 mutant individuals of the F 2 population was used.To identify the mutation site, a candidate gene was amplified using genomic DNA extracted from the spo1 mutant and Nipponbare and sequenced.The primer sequences used in the fine-mapping and candidate gene analyses are listed in Table S2.

Vector Construction and Plant Transformation
For the functional complementation test, the full-length CDS of SPO1 was cloned into the binary vector pUN-1301.The recombinant plasmids were introduced into the spo1 mutant using the Agrobacterium tumefaciens-mediated transformation method, as described previously [25].To generate the overexpression transgenic lines of SPO1, the full-length CDS of SPO1 was cloned into the binary vector p35S-1301-GFP.The recombinant plasmids were introduced into wild-type Nipponbare calli by the Agrobacterium-mediated transformation method [25].The positive transgenic plants above were identified by the evaluation of hygromycin resistance and GUS array.The primer sequences used are listed in Table S2.

Microscopy Analysis
The fresh culm segments and the second top leaves of WT and spo1 at tilling stage were collected and immediately fixed in formaldehyde alcohol acetic acid (FAA) solution at 4 • C overnight.Microscopic analysis was performed using both hand-cut sections and paraffin sectioning methods [12,29].The sections were examined and photographed using an Olympus BX51 microscope (Tokyo, Japan).At least three independent experiments were carried out for each analysis.The number and area of the bulliform cells were measured by using the ImageJ software (https://imagej.net/downloads,accessed on 1 May 2020).

RNA Extraction and Gene Expression Analysis
To study the tissue expression patterns of SPO1, total RNA was extracted from various tissues of cultivar Nipponbare at different developmental stages using RNAiso TM Plus Trizol (TaKaRa, Dalian, China).For expression analyses of SPO1 gene under various hormone treatment and stress conditions, total RNA was extracted from 2-week-old Nipponbare seedlings by using the above method.For the analysis of transcripts of SPO1 and some leaf-shape-related genes, total RNA was isolated from leaf blades of WT, spo1 mutant, and transgenic plants in the tillering stage using the same method.First-strand cDNAs were synthesized from 2.0 µg total RNA using Integrated First-strand cDNA Synthesis Kit (One-Step gDNA Remover) (DiNing, Beijing, China) following the manufacturer's instructions.RT-PCR analysis was conducted with the gene-specific primers.The rice OsActin1 gene was used as an internal control in RT-PCR analysis.The qRT-PCR was performed on a Bio-Rad CFX96 real-time system (Bio-Rad Laboratories Inc., Hercules, CA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Beijing, China) following the manufacturer's instructions.The relative expression level of the target gene was obtained by normalization to internal reference gene OsActin1.Each set of experiments had three biological replicates, and the quantitative variation was evaluated by the 2 −∆∆Ct relative quantification method [84].The primer sequences were listed in Table S2.

Measurement of Endogenous Hormone Content
Endogenous hormone content of samples was measured according to the enzymelinked immunosorbent assay (ELISA) method described by Li et al. (2021) [85] with some modifications.Approximately 500 mg leaves of WT and spo1 plants at tilling stage were ground to a fine powder with liquid nitrogen for testing.At least three independent replicates were tested.

RNA-seq Analysis
The leaves of WT and spo1 plants at tillering stage were collected, with three biological replicates of each to test.The experimental procedures for RNA-seq and data analysis were performed according to the description given by Li et al. (2021) [85].

Measurements
For testing leaf rolling index (LRI), Lw (expand the leaf blade and determine the greatest width) and Ln (the natural distance of leaf blade margins at the same position) were measured.The LRI was calculated as LRI (%) = (Lw − Ln)/Lw × 100 [29].The chlorophyll content was measured as described by Chen et al. (2013) [86], and the method slightly modified.An equal weight of the second top leaf tissues was ground in 95% ethanol under dark conditions and filtered.Then, the concentrations of chlorophyll were measured at 665 nm and 649 nm.The contents of chlorophyll a and chlorophyll b were calculated according to the following formula: Ca = 13.95A665 − 6.88A 649 , Cb = 24.96A649 − 7.32A 665 .

Phylogenetic Analysis and Protein Structure Prediction
The homologous proteins of SPO1 were identified by the Blastp search program of the National Center for Biotechnology Information (NCBI https://www.ncbi.nlm.nih.gov/ and Phytozome 13 https://phytozome-next.jgi.doe.gov/,accessed on 1 May 2020).All of the sequences in the CSLD subfamily from Oryza sativa, Arabidopsis thaliana, Brachypodium distachyon, and Zea mays were used to construct a phylogenetic tree using MEGA 7.0 software by the neighbor-joining (NJ) method with 1000 bootstrap replications.Multiple sequence alignments were conducted using DNAMAN version 6.0 software.The threedimensional structure of proteins was constructed with Phyre2 software (http://www.sbg.bio.ic.ac.uk/~phyre2/html/, accessed on 1 October 2023) using default parameters.

Figure 1 .
Figure 1.Characterization of WT (Nipponbare) and spo1 mutant morphology.(a) Two-week-old WT and spo1 seedlings.(b) Gross morphology of WT and spo1 plants at the mature stage.(c) Morphology of the top three leaf blades between WT and spo1 mutant and the transverse sections of WT and spo1 mature leaves.(d) Features of internodes (I, II, III, IV, and V) among WT and spo1 mutant at the mature stage.(e) Panicles, paddy rice grains, and brown rice grains of WT and spo1 plants.(f) Roots of WT and spo1 plants.(g-l) Quantification data of plant height (g), internode length (h), leaf width (i), leaf rolling index (LRI) (j), grain width (k) and length (l) of WT and spo1 mutant at mature stage.At least 12 samples of WT and spo1 mutant were measured for each.Data are means ± SD, asterisks indicate significant differences according to Student's t-test (* p < 0.05; ** p < 0.01).Scale bars: (a) 2.5 cm; (b) 10 cm; (c) 0.35 cm and 6 cm; (d) 5 cm; (e) 4 cm, 0.35 cm, and 0.4 cm; (f) 2 cm.

Figure 1 .
Figure 1.Characterization of WT (Nipponbare) and spo1 mutant morphology.(a) Two-week-old WT and spo1 seedlings.(b) Gross morphology of WT and spo1 plants at the mature stage.(c) Morphology of the top three leaf blades between WT and spo1 mutant and the transverse sections of WT and spo1 mature leaves.(d) Features of internodes (I, II, III, IV, and V) among WT and spo1 mutant at the mature stage.(e) Panicles, paddy rice grains, and brown rice grains of WT and spo1 plants.(f) Roots of WT and spo1 plants.(g-l) Quantification data of plant height (g), internode length (h), leaf width (i), leaf rolling index (LRI) (j), grain width (k) and length (l) of WT and spo1 mutant at mature stage.At least 12 samples of WT and spo1 mutant were measured for each.Data are means ± SD, asterisks indicate significant differences according to Student's t-test (* p < 0.05; ** p < 0.01, ns means no significance).Scale bars: (a) 2.5 cm; (b) 10 cm; (c) 0.35 cm and 6 cm; (d) 5 cm; (e) 4 cm, 0.35 cm, and 0.4 cm; (f) 2 cm.

Figure 2 .
Figure 2. Map-based cloning of SPO1.(a) Linkage map of the gene SPO1 on the long arm of ch mosome 12.(b) Fine-mapping of the SPO1 locus.The genetic linkage map is derived from 3 mutant individuals and 160 F2 mutant individuals for fine-mapping.Marker names are above vertical lines and the number of recombinants is displayed under the vertical lines.(c) Accordin IRGSP1.0 database annotation, the 90-kb region contains 15 annotated genes.(d) Gene structur SPO1/OsCSLD4/Os12g0555600 and the corresponding positions of intron and exons.The w boxes indicate the 5′ and 3′ untranslated regions, the black boxes indicate the exons, and the b line between the two black boxes indicate the intron.The start codon (ATG) and the stop co (TAG) are indicated.The spo1 mutant has a base substitution (C to T) in the second exon at posi 2006 of the coding regions.(e) RT-PCR analysis of SPO1 expression in spo1.(f) qRT-PCR analys SPO1 expression in spo1.Data are means ± SD, asterisks indicate significant differences accord to Student's t-test (** p < 0.01).(g) Genetic complementation of spo1.Three representative lines (c 3, com-5, and com-11) of complementation with young plants are shown.(h) Statistical analysi the leaf blades width of WT, spo1, and complementary lines (com-3, com-5, and com-11) at seed stage.Data presented are means ± SD: different letters indicate significant differences betw

Figure 2 .
Figure 2. Map-based cloning of SPO1.(a) Linkage map of the gene SPO1 on the long arm of chromosome 12.(b) Fine-mapping of the SPO1 locus.The genetic linkage map is derived from 32 F 2 mutant individuals and 160 F 2 mutant individuals for fine-mapping.Marker names are above the vertical lines and the number of recombinants is displayed under the vertical lines.(c) According to IRGSP1.0 database annotation, the 90-kb region contains 15 annotated genes.(d) Gene structure of SPO1/OsCSLD4/Os12g0555600 and the corresponding positions of intron and exons.The white boxes indicate the 5 and 3 untranslated regions, the black boxes indicate the exons, and the black line between the two black boxes indicate the intron.The start codon (ATG) and the stop codon (TAG) are indicated.The spo1 mutant has a base substitution (C to T) in the second exon at position 2006 of the coding regions.(e) RT-PCR analysis of SPO1 expression in spo1.(f) qRT-PCR analysis of SPO1 expression in spo1.Data are means ± SD, asterisks indicate significant differences according to

Figure 3 .
Figure 3. qRT-PCR analysis of the tissue-specific and stressed-induced expression patte (a) The tissue-specific expression pattern of SPO1.The expression of SPO1 in leaves at see was set to 1.(b) Expression of SPO1 in leaves of two-week-old seedlings treated with 1 50 μM GA, 100 μM ABA, and 100 μM MeJA at different time points.(c) Expression of SP of two-week-old seedlings treated with 200 mM NaCl, 20%PEG6000, 15 μM MV, heat cold (4 °C) at different time points.The OsActin1 gene was used as a control.The data ar the mean ± SD with three biological replicates.

Figure 3 .
Figure 3. qRT-PCR analysis of the tissue-specific and stressed-induced expression pattern of SPO1.(a) The tissue-specific expression pattern of SPO1.The expression of SPO1 in leaves at seedling stage was set to 1.(b) Expression of SPO1 in leaves of two-week-old seedlings treated with 100 µM IAA, 50 µM GA, 100 µM ABA, and 100 µM MeJA at different time points.(c) Expression of SPO1 in leaves of two-week-old seedlings treated with 200 mM NaCl, 20%PEG6000, 15 µM MV, heat (42 • C), and cold (4 • C) at different time points.The OsActin1 gene was used as a control.The data are shown by the mean ± SD with three biological replicates.

Figure 4 .
Figure 4. Histological analysis of the leaf blades, stems, and grains between WT and plants.(a) Cross sections of leaf blades of WT and spo1 mutant.(b) Cross section of m vein, and small vein at the middle of the second leaf from the top of WT and spo1 muta stage.(c-g) Statistics analysis of a number of large veins (c) and small veins (d) per l veins (e), number of BCs (f), area of BCs (g).(h) Longitudinal sections of stems betw spo1 mutant.(i) The length of stem cells.(j) The area of stem cells.(k) Grain morpholo ning electron microscopy of the outer surfaces of WT and spo1 glumes.(l,m) Cell width ( (m) of outer glume of the grain.At least 30 samples of WT and spo1 mutant were measu Data are means ± SD, asterisks indicate significant differences according to Student's 0.05; ** p < 0.01, ns means no significance).Abbreviations: Ab, abaxial surface; Ad, ada

Figure 4 .
Figure 4. Histological analysis of the leaf blades, stems, and grains between WT and spo1 mutant plants.(a) Cross sections of leaf blades of WT and spo1 mutant.(b) Cross section of midrib, large vein, and small vein at the middle of the second leaf from the top of WT and spo1 mutant at tillering stage.(c-g) Statistics analysis of a number of large veins (c) and small veins (d) per leaf, width of veins (e), number of BCs (f), area of BCs (g).(h) Longitudinal sections of stems between WT and spo1 mutant.(i) The length of stem cells.(j) The area of stem cells.(k) Grain morphology and scanning electron microscopy of the outer surfaces of WT and spo1 glumes.(l,m) Cell width (l) and length (m) of outer glume of the grain.At least 30 samples of WT and spo1 mutant were measured for each.Data are means ± SD, asterisks indicate significant differences according to Student's t-test (* p < 0.05; ** p < 0.01, ns means no significance).Abbreviations: Ab, abaxial surface; Ad, adaxial surface; BC, bulliform cell; EC, epidermal cell; LV, large vein; SV, small vein.Scale bars: (a) 500 µm; (b) 100 µm; (h) 100 µm; (k) 0.2 cm and 50 µm.

21 Figure 5 .
Figure 5. qRT-PCR analysis of the expression of genes related to leaf shape regulation in the spo1 mutant.Total RNA was extracted from leaves of WT and spo1 mutant at tillering stage.OsActin1 gene was used as a control.Data are the mean ± SD with three biological replicates, asterisks indicate significant differences according to Student's t-test (* p < 0.05; ** p < 0.01).

Figure 5 .
Figure 5. qRT-PCR analysis of the expression of genes related to leaf shape regulation in the spo1 mutant.Total RNA was extracted from leaves of WT and spo1 mutant at tillering stage.OsActin1 gene was used as a control.Data are the mean ± SD with three biological replicates, asterisks indicate significant differences according to Student's t-test (* p < 0.05; ** p < 0.01).

Figure 6 .
Figure 6.Transcriptomic analysis of spo1 mutant.(a) Gene Ontology (GO) enrichment anal DEGs in spo1.(b) KEGG pathway enrichment of DEGs in spo1.Leaves of spo1 mutant and wil plants in the tillering stage are used as test samples.

Figure 6 .
Figure 6.Transcriptomic analysis of spo1 mutant.(a) Gene Ontology (GO) enrichment analysis of DEGs in spo1.(b) KEGG pathway enrichment of DEGs in spo1.Leaves of spo1 mutant and wild-type plants in the tillering stage are used as test samples.

Table 1 .
Comparison differences of some agronomic traits between WT and spo1 mutant.

Table 2 .
Hormone content of the wild type (WT) and spo1 mutant.

Table 3 .
Differentially expressed genes associated with multiple hormone pathways, cell cyc cell wall formation.

Table 3 .
Differentially expressed genes associated with multiple hormone pathways, cell cycle, and cell wall formation.