A Recessive Mutant of argonaute1b/gsnl4 Leads to Narrow Leaf, Small Grain Size and Low Seed Setting in Rice

Song Mengqiu, Ruan Shuang, Peng Youlin, Wang Zhongwei, Jahan Noushin, Zhang Yu, Cui Yongtao, Hu Haitao, Jiang Hongzhen, Ding Shilin, Shen Lan, Gao Zhenyu, Hu Xingming, Qian Qian, Guo Longbiao

Letter

A Recessive Mutant ofLeads to Narrow Leaf, Small Grain Size and Low Seed Setting in Rice

Song Mengqiu1, 2, #, Ruan Shuang1, 2, #, Peng Youlin3, #, Wang Zhongwei1, Jahan Noushin1, Zhang Yu1, Cui Yongtao1, Hu Haitao1, Jiang Hongzhen1, Ding Shilin1, Shen Lan1, Gao Zhenyu1, Hu Xingming2, Qian Qian1, Guo Longbiao1

(State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China; College of Agronomy, Anhui Agricultural University, Hefei 230036, China; Rice Research Institute, Southwest University of Science and Technology, Mianyang 621010, China; These authors contributed equally to this work)

Amutant characterized by small grain size, narrow leaf and low seed-setting rate was obtained by ethyl methane sulfonate (EMS) mutagenesis of arice variety Wuyunjing 21. Genetic analysis showed thatis a loss-of-function mutant. A single-base mutation inresulted in the substitution of Ser to Asn in the Piwi domain of OsAGO1b protein. CRISPR/Cas9-mediated editing ofyieldeda mutant phenotypically resembling. Furthermore, miRNA-Seq analysis showed that the transcript expression levels of miRNAs in the signal transduction pathways related to pollen development, leaf morphology and hormone activation were significantly different between themutant and the wild type (WT) plants. Several miRNAs were downregulated, and their target genes were upregulated inmutants. The auxin content in the root tips of themutant decreased, and the expression of most auxin-related genes was altered. In summary,not only regulates organ development by controlling cell division and expansion, but also plays an important role in regulating auxin transport in rice.

ARGONAUTE (AGO) proteins are widely distributed in eukaryotes and are the central components of the RNA-induced silencing complex (RISC) (Baulcombe, 2004). AGO proteins bind to small non-coding RNAs, such as siRNAs and miRNAs, and play an important role in the silencing mechanism of RNAs by affecting protein synthesis and RNA stability. AGO proteins contain four conserved domains: N-terminal domain, PAZ domain, middle domain (MID) and Piwi domain (Song et al, 2004). Previous studies showed that the Piwi domain is the catalytic core of AGO, which shares structural similarity with ribonuclease H (RNase H) in the form of a conserved catalytic center of amino acid quadruple Asp-Glu-Asp-His/Asp (DEDH/D) (Song et al, 2004). The function of the catalytic center is to cut the target sequence of small RNA. Rice contains 19 AGO proteins. Among them, the mutation ofcauses the leaves to curl upwards, extends the upright time of leaves, and promotes the formation of an upright leaf crown (Shi et al, 2007).directly regulates the expression ofthrough DNA methylation and regulates the development of anthers (Zheng et al, 2019).may be a key protein in the siRNA pathway, which positively regulates rice grain size and weight, and promotes rice stem development (Zhong et al, 2020). OsAGO1 has four homologs, named OsAGO1a/b/c/d, which may be functionally redundant. RNA interference on the conservative regions ofresults in dwarfism of plants, narrowed and curled leaves, as well as low seed-setting rate (Wu et al, 2009). Recently,was reported to be a key regulator of growth and development in rice but did not participate in the establishment of leaf polarity (Li et al, 2019). However, these studies were based on reverse genetics, and the effects of individualgene mutation on development of plant organs is still unclear through forward genetics.

In this study, a pleiotropic mutant() was isolated from arice Wuyunjing 21 after EMS-induced mutagenesis. Themutant showed narrow leaves, small grain size, low pollen fertility, low seed- setting rate and thin culm (Fig. 1-A to -F; Fig. S1). To determine the effect of thegene on the development of various tissues, histological analysis of multiple organs was performed. The transverse sections of the middle part of the second blades from top at the maturity stage showed that the total numbers of small veins and large veins were significantly reduced in theplants compared with WT (Fig. 1-G to -I; Fig. S2-A and -B). Transverse sections of internode III indicated that the distribution of vascular bundles in theplants was severely damaged, an irregular arrangement was apparent (Fig. 1-J and -K), and the number of vascular bundles and the length of epidermal cells were significantly reduced (Fig. 1-L; Fig. S2-C and -D). Additionally, the number of inner parenchyma cells as well as the average width and number of outer epidermal cells in themutant spikelet cells were lower than those in WT (Fig. 1-M to -O; Fig. S2-E and -F). In summary, these results revealed that thegene also plays an important role in regulating cell division and cell elongation.

To determine whether thegene is responsible for thepleiotropic phenotypes, theplant was crossed with TN1 (anrice). The F1progenies exhibited normal phenotype.In the F2population, the separation ratio was 3:1 (normal: pleiotropic = 2297:720; χ2= 2.07,> 0.05), indicating that thephenotype was controlled by a single recessive gene. Map-based cloning documented that themutant contained a single-nucleotide G to A mutation in thegene (), resulting in the substitution of Ser to Asn in the Piwi domain (Fig. 1-P). Intriguingly, we found the mutational site ofin theplants was the same as the mutational site ofin themutant(Cuperus et al, 2010; Poulsen et al, 2013). The amino acid sequence alignment confirmed that the residue S810N in the Piwi domain was very conserved between rice and(Fig. S3). Although the exact functional position ofis unknown, their common mutation sites provided genetic evidence to further dissect its biochemical function, and the mutant provided good materials for further research on the function ofin the future. To further verify the function of thegene, the non-mutatedgene of WT was knocked out in WT rice using a CRISPR/Cas9 system. Eight different types of homozygous mutant lines were obtained (Fig. 1-Q), and all of which showed narrower leaves and smaller grains (Fig. 1-R to -T). Taken together, these results confirmed thatis the candidate gene of. The expression of thegene was measured by qRT-PCR, and the expression of thegene in themutant, F1(obtained by crossing WT and) and three-knockout lines increased significantly in comparison with WT (Fig. 1-U). These results indicated that the loss-of- function ofin the mutant may lead to negative feedback regulation, thus promoting the transcription of thegene.

Fig. 1. Phenotype analysis, histological analysis, genetic analysis and transcription analysis of wild type (WT) andplants.

A, Gross morphology at the heading stage. Scale bar, 10 cm. B, Leaf morphology of the uppermost three leaves at the maturity stage. Scale bar, 5 cm. C andD, Phenotypes of seeds. Scale bars, 1 cm.E, Leaf width of the uppermost three leaves. F, Seed-setting rate. G, Transverse sections of the middle part of the second leaf blades from the top at the maturity stage. Scale bars, 500 μm. H, Regions between two large veins in G. lv, Large vein; sv, Small vein. Scale bars, 100 μm. I, Scanning electron microscopy analysis of mature flag leaves. Scale bars, 100 μm. J, Transverse sections of the middle part of internode III at the maturity stage. Scale bars, 500 μm. K, Regions between two vascular bundles in J. Scale bars, 100 μm. L, Longitudinal sections of the middle part of internode III at the maturity stage. Scale bars, 50 μm. M, Spikelet hulls. The dashed lines indicate the sites of the transverse sections. Scale bar, 2 mm. N, Cross-sections of spikelet hulls. Scale bars are 1 mm in the left and 200 μm in the right close-up views of the boxed regions. O, Scanning electron microscopy analysis of spikelet hulls of the outer glume. Scale bars, 200 μm. P,Fine mapping of the gene on chromosome (Chr) 4, and the structure and mutation site of thegene. Red arrow indicates the mutation site.Q, Mutations in the T1generation ofby CRISPR/Cas9. Red letters and dashed lines indicate inserted and deleted bases, respectively. R, Morphology of WT and three-knockout lines. Scale bar, 10 cm. S, Phenotypes of seed embryos of WT,and-knockout lines. Scale bar, 0.5 cm. T, Flag leaf morphology of WT and a-knockoutline. Scale bar, 5 cm. U, Expression ofgene inWT,, F1(obtained by crossing WT and) and three-knockout lines. V,Relative expression of eight miRNAs related to leaf development. W, Relative expression of OsHB family genes. X, Relative expression ofandinvolved in leafdevelopment. Data are Mean ± SD (= 3). * and **, Significant differences at< 0.05 and< 0.01 compared with WT by the Student’stest, respectively.

To understand the function of, we analyzed its expression pattern in different tissues of WT.expressed in every organ but predominantly in young panicles (Fig. S4-A). Additionally, rice leaf sheath protoplasts were transformed with the pCA1301-35S-s65T-vector to detect the subcellular localization ofGSNL4. The green fluorescent signal of GFP- GSNL4 protein was localized in the nucleus and cytoplasm, while the signal of an empty GFP vector was distributed in the entire cell (Fig. S4-B).

To study the regulation mechanism of, we performed miRNA-Seq to analyze the effect ofon miRNA expression. Transcriptome sequencing was performed in triplicates using young panicles of WT and. Compared with WT, there were approximately 29 upregulated and 23 downregulated miRNAs in theplants (Fig. S5-A and Table S1). The enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes identified that the target genes of miRNAs expressed differently between the WT andplants were related to the signal transduction pathways involved in pollen development, leaf morphology, chlorophyll and carotenoid synthesis as well as hormone activation (Fig. S5-B to -E). In addition, the accumulation of eight miRNAs in leaves at the tillering stage was assessed by qRT-PCR. The results showed that the levels of,,andinwere markedly lower than those in WT (Fig. 1-V). Sincegene negatively regulates the expression of HD-ZIP III family genes by combining withto regulate leaf differentiation (Liu et al, 2009). The expression of five members of this family,to, in leaves at the tillering stage was evaluated. The results showed that the expression of the five OsHB genes was significantly higher in theleaves than in WT (Fig. 1-W). We also measured the expression of 11 OsARF genes in theleaves at the tillering stage. Except for thegene, the expression of the other 10 OsARF genes was increased compared to WT (Fig. S6-A). Among them,andare related to, implying that the mutation ofmay affect the expression of, thus affecting the expression of OsARF genes. In addition,() is homologous toin.not only regulates leaf development and maintains apical meristem characteristics, but also plays an important role in vascular bundle development and leaf formation (Nishimura et al, 2002).can be regulated by the feedback from miR168, which binds to AGO10, to maintain the homeostasis of small RNA and AGO protein (Vaucheret et al, 2004). AtAGO7 binds miR390 to form RISC, and cleaves the transcript of() to produce ta-siRNA (trans-acting small interfering RNA), which targetsgenes to regulate leaf development (Hunter et al, 2003). Therefore, their expression was determinedin leaves at the tillering stage. In compared with WT, the expression ofinwas significantly increased, while the expression ofwas significantly decreased (Fig. 1-X). These findings indicated that the mutation ofmay, to a certain extent, disrupt the regulatory pathways of miRNAs, increase the expression of their target genes and induce pleiotropic effects ofmutation in rice.

Endogenous hormones play an important role in regulating leaf and root development and differentiation (Tsiantis et al, 1999). Therefore, the content of endogenous IAA (indole-3- acetic acid) in 1 cm of the root tip of WT andseedlings was measured. The IAA content inwas significantly lower than that in WT (Fig. S6-B). Thegene plays an important role in the development of auxin-dependent adventitious roots (Xu et al, 2005). The expression ofwas significantly downregulated, but the expression ofandwas significantly upregulated in theplants (Fig. S6-C). In addition, the YUCCA and YABBY family genes play important roles in the development of various plant organs (Sarojam et al, 2010). We documented that the expression of,,,andwas decreased (Fig. S6-D and -E). These findings indicated thatmay indirectly regulate the expression of auxin pathway genes, thereby affecting the transport or accumulation of IAA in rice, thus controlling cell expansion and division in various organs.

In this study, a pleiotropic mutant,, was isolated,which showed narrow leaves, small grains, low seed-setting rate and thin culm. Genetic analysis showed that a single-base mutation inresulted in the substitution of Ser to Asn in the Piwi domain of OsAGO1b protein. This study reported thatplays an essential function in regulating the development of multiple organs and plant growth in rice through forward genetics research.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant Nos. 31771887, 31671761 and 32001491).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

File S1. Methods.

Fig. S1. Comparison of agronomic traits of wild type (WT) andplants.

Fig. S2. Comparison of histology statistics between wild type (WT) andplants.

Fig. S3. Structural mapping of AGO mutations.

Fig. S4. Expression pattern and subcellular localization of GSNL4.

Fig. S5.RNA-Seq analysis of wild type (WT) andplants.

Fig. S6. Determination of indole acetic acid (IAA) content and relative expression of auxin-related genes.

Table S1. Differentially expressed miRNAs in wild type (WT) and.

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Guo Longbiao (guolongbiao@caas.cn); Qian Qian (qianqian188@hotmail.com); Hu Xingming (huxingmingx@126.com)

5 February 2021;

13 May 2021

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http://dx.doi.org/10.1016/j.rsci.2021.05.012