DH2 regulates the development of lateral organs in rice

doi:10.21203/rs.3.rs-5016600/v1

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DH2 regulates the development of lateral organs in rice

https://doi.org/10.21203/rs.3.rs-5016600/v1

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In most of crops, the development of lateral organs such as leaves and floral organs play important roles in architecture of plant and grain, and then determine the yield. Establishment of polarityin these lateral organs is one of the most critical events for their morphogenesis. However, the molecular mechanisms about this in rice is still not clear enough. Here, we isolated two allelic mutants named degenerated hull 2-1, -2 (dh2-1, -2) in rice, exhibiting abaxially rolled leaves and rod-shaped lemmas. DH2 encoded the relatively conservative ARGONAUT 7 (AGO7) protein in plants, and expressed in the lateral organs including leaf and floral organs. When the knock-out of DH2 gene resulted in the same defects with the two allelic mutants, the over-expression lines of DH2showed adaxially rolled leaves. Next, it was proved that DH2 was involved in the synthesis of tasiR-ARFs, the expression level of which was decreased sharply in lateral organs of dh2 mutants. And then it was found that the expression of OsARF2, OsARF3, OsARF14, and OsARF15, the potential targets oftasiR-ARFs, was increased in lateral organs of dh2 mutants. However, it was not as expected that results of in situ hybridization showed the four ARF genes were not expressed in WT lemma, whereas they were all ectopically expressed in rod-shaped lemma in dh2 mutants. Meanwhile tasiR-ARFs was expressed in the whole lemma but not abaxial side.That means there was not a opposite expression of tasiR-ARFs and ARFs in adaxial–abaxial of lemma. Therefore, according our data, we think that the pathway of OsAGO7—tasiR-ARFs in rice was more likely involved in the whole development of lemma but not only abaxial side by restricting ectopical expression of OsARFs in the whole lemma, which was different with that in lateral organs of Arabidopsis.

AGO7

polarity establishment

ta-siRNAs

lateral organs

Seed plants have two different organ growth patterns: a root system and a shoot system (Bowman et al. 2002, Golz et al. 2002). The shoot system includes stems and lateral organs (leaves and flowers), and lateral organs are formed by the lateral flanks of shoot apical meristem (SAM), then exhibiting lateral growth and significant asymmetric development along three (proximal-distal, adaxial-abaxial, and medial-lateral) axes (Moon et al. 2011). Rice leaves are the main organ of photosynthesis, then improving leaf morphology is one of effective ways to increase yield (Xu et al. 2018). The flower organs in rice eventually develop into grains, in which lemma and palea limits grain shape and size, and then affects grain yield to a great degree (Xiang et al. 2015; Yu et al. 2018). Therefore, the development of leaves and floral organs has an important role in the vegetative and reproductive growth stages of rice.

In the initial stage of lateral organs formation, after founder cells that are designated to develop into lateral organs are recruited from the SAM, the axes of the three polarity of adaxial-abaxial, medial-lateral and proximal-distal, are established orderly (Bowman et al. 2002; Braybrook et al. 2010; Moon et al. 2011). The molecular mechanism underlying the establishment of lateral organs adaxial-abaxial polarity in Arabidopsis and rice has been extensively studied.

In Arabidopsis, three HD-ZIP Ⅲ genes (PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTE (REV)), and a LOB-domain (LBD) transcription factor AS2, all predominantly express on the adaxial side and regulate adaxial cell development in leaves. And the expression of HD-ZIPIII genes was restricted by miR165/166, which accumulate on the abaxial side (Tang et al. 2003; Juarez et al. 2004). Then dominant mutations of HD-ZIPIII genes cause a lateral organ adaxialization (McConnell et al. 2001; Prigge et al. 2005; Otsuga et al. 2008). In rice, adaxially rolled leaves were commonly observed in the plants expressing OSHB1m, OSHB3m, and OSHB5m (microRNA165/166-resistant HD-ZIPIII genes versions) genes, imply miR165/166-HD-ZIP Ⅲ genes pathway is key to adaxial cell fate and conserved in plants (Itoh et al. 2008; Li et al. 2016; Zhang et al. 2021). However, although OsAS2 overexpression resulted in the defects of leaf structure, it is likely that the adaxial–abaxial patterning is not affected obviously. In addition, the OsAS2 transcripts were identified throughout leaf primordia. Thus, either OsAS2 in rice or AS2 in Arabidopsis may have a divergent role in adaxial polarity establishment of leaf (Ma et al. 2009).

It has been reported that KANADIs (KAN), YABBYs (YAB), and ARFs family genes are involved in the establishment of abaxial polarity of lateral organs. In Arabidopsis, KAN1, KAN2, YAB1, YAB2, YAB3, and ARF2, ARF3/ETT and ARF4, are all expressed in the abaxial side, the mutations of which lead to adaxialization or reduced abaxial identity in lateral organs (Yu et al. 2017; Eshed et al. 2001; Kerstetter et al. 2001; Emery et al. 2003; Sawa et al. 1999; Siegfried et al. 1999; Stahle et al. 2009; Sarojam et al. 2010; Pekker et al. 2005; Guan et al. 2017). In rice, KAN-like gene SHALLOT-LIKE1 (SLL1), accumulates in the abaxial epidermis of leaf and regulates leaf abaxial cell development (Zhang et al. 2009). OsYAB1 is expressed in the abaxial sclerenchyma in the leaves, and the palea and lemma, and the mutation of OsYAB1 causes the abaxial-rolling leaves. However, OsYAB1 was recognized as a regulator of sclerenchymatous cells types but not an abaxial polarity regulator (Toriba et al. 2007; Dai et al. 2007). A recently study found that SLL1 could directly regulate the proper expression of OsARF2/OsARF3a/OsETT and then contribute to lemma polarity development in rice (Si et al. 2022).

It has been broadly reported that TAS3 trans-acting small interfering RNA (ta-siRNA, generally termed tasiR-ARFs ) participate in specifying abaxial–adaxial fates, which express in the adaxial domain and represses the expression of ARFs there (Garcia et al. 2006; Takahashi et al. 2013; Adenot et al. 2006). During the generation of tasiR-ARFs, TAS3 transcripts are firstly cleaved by miR390 and ARGONAUTE7 (AGO7) into fragments, which then are converted to double-stranded RNA by RNADEPENDENT RNA POLYMERASE 6 (RDR6) with SUPPRESSOR OF GENE SILENCING 3 (SGS3), and then are further processed into 21-nucleotide tasiRNAs by DICER-LIKE 4 (DCL4) (Liu et al. 2020; Song et al. 2019; Song et al. 2012; Toriba et al. 2010; Yoshikawa et al. 2005; Liu et al. 2007; Si et al. 2022). Finally, mature tasiR-ARFs are loaded into AGO1 protein to target ARFs (Allen et al. 2005; Xie et al. 2004; Yoshikawa et al. 2005; Si et al. 2022). Therefore, these genes related to the tasiR-ARFs generation are also involved in the establishment of abaxial-adaxial polarity. In rice, the mutations of AGO7, OsSGS3a/PDL1, SHOOTLESS2(SHO2)/OsRDR6 and SHO1/OsDCL4 exhibits obvious defects in polarity development of lateral organs, specially in lemma (Zhang et al. 2023; Itoh et al. 2000; Nagasaki et al. 2007; Liu et al. 2007; Toriba et al. 2010; Song et al. 2012). And these mutants all displays reduced accumulation of tasiR-ARFs, and over-/ectopic- expression of ARF genes. However, due to too many genes in this pathway, the molecular mechanism of AGO7—tasiR-ARFs—ARFs in regulating the polarity development of lateral organs in rice still remain some unsolved questions.

In this study, we characterized two polarity defect of lateral organs mutant named degenerated hull 2 − 1, -2 (dh2-1, -2), which exhibits abaxial leaves and rod-like lemmas caused by defects in polarity development. DH2 encodes the AGO7 protein and is highly expressed in the lateral organs including leaf and floral organs. Our data indicates that DH2 is involved in tasiR-ARFs synthesis, thereby affecting the organ but not polarity distribution of ARFs transcript in lateral organs, like lemma in rice. Therefore, our findings provides some new views about the mechanism level that how the DH2-mediated tasiR-ARFs pathways regulating lateral organ development in rice.

2.1. Plant materials

The dh2-1, dh2-2 mutant was respectively isolated from an ethylmethane sulfonate-treated population of Xian cultivar ‘Jinhui 10’, ‘Xi Da 1B’, which was used as the wild type (WT). dh2-1 mutant was used for rice genetic transformation, among which transgenic plants exhibit overexpression of DH2. The plant materials used in other experiments were grown in experimental fields of Southwest University in Chongqing, China.

2.2. Morphological and Histological analysis

Leaf at the seedling stage, panicles at the heading stage, and spikelets at the flowering stage of dh2-1, -2 and WT (J10, 1B) were used to investigate the phenotypic characteristics. Take fresh material from the field, stick it on the conductive adhesive and observe. Nikon SMZ1500 stereomicroscope (Nikon, Tokyo, Japan) and a Hitachi SU3500 scanning electron microscope (Hitachi, Tokyo, Japan) were used (low vacuum, -20°C, 5 kV). Leaf of WT (J10) and OE-DH2 lines at the seedling stage were observed by Nikon SMZ1500 stereomicroscope (Nikon, Tokyo, Japan).

For paraffin sectioning, tissues of the dh2-1, -2, WT (J10, 1B) and OE-DH2 lines were collected in FAA solution (total 50ml volume: 5ml 37% formaldehyde, 2.5ml 0.9 M glacial acetic acid, 25ml ethanol and 17.5ml ddH₂O) for suitable time (24-48 h) after vacuum infiltration in room temperature. The fixed rice tissues were dehydrated in an ethanol gradient series (50% ethanol, 70% ethanol, 85% ethanol, 95% ethanol and three times 100% ethanol), for about 30-60 minutes for every time. After alcohol treatment, the materials were treated with a 3:1, 1:1, 1:3 ratio of alcohol and xylene, as well as three times of 100% xylene, for 30-60 minutes each time. In order to embed the materials in paraffin, the materials were mixed with xylene and melted paraffin (1:1) (Sigma, St Louis, MO, USA) in 42℃ oven for overnight, as well as four rounds of paraffin in 65 ℃ oven for 6-12 hours each time. Samples were sectioned into 10μm thickness and stained with 1% safranin and 1% Fast Green (Amresco), and observed using an Eclipse E600 light microscope (Nikon).

2.3. Phenotypic Statistics

The leaf rolling index (LRI) and leaf erect index (LEI) of the top three leaves (flag leaf, second upper leaf, and third upper leaf) were determined at the grain-filling stage as described previously (Shi et al. 2007; Zhang et al. 2009). All the phenotypic data shown in this study were measured using 1B, dh2-1, -2, J10 and two or three independent transgenic lines (DH2-OE), and ten individual plants per line were used for the measurement.

2.4. Anatomical Observation of Leaves

Take leaves of 30-day-old seedlings leaves of 1B, dh2-1, -2, J10 and two or three independent transgenic lines (DH2-OE) from the field, embed them with frozen embedding agent, and make slices of about 10µm thickness on a Leica frozen slicer; the leaf slices were directly imaged using visible or ultraviolet light excitation, under the Eclipse E600 light microscope (Nikon).

2.4. Genetic and complementary analysis of DH2

Gene messages information was downloaded from the Gramene (http://www.gramene.org/). BLAST tool on the National Center for Biotechnology Information website was used to conduct homology analysis. To construct the complementary vector of DH2, a fragment containing a 7623-bp wild-type genomic fragment of DH2 was amplified using the primers DH2-com-F1, DH2-com-F2, DH2-com-R1, DH2-com-R2 (7623-bp wild-type genomic fragment was divided into two segments and amplify them separately). DNA extracted from J10 was used to prepare PCR amplification template using Prime STAR® Max DNA Polymerase (TaKaRa, Dalian, China). The two PCR amplification fragments were purified by universal DNA Purification Kit (TianGen, Beijing, China), and recombined into pCAMBIA1300 using the Minerva Super Fusion Cloning Kit (UElandy, Suzhou, China). After conducting sequence analysis for verification purposes, the vector was subjected to dh2-1 using the Agrobacterium tumefaciens-mediated technique. The primer sequences used are listed in Table1. All the experiments above were performed according to the manufacturer's protocol.

2.5. RNA isolation and RT-qPCR analysis

Total RNA was extracted from individual organs using the KK Fast Plant Total RNA Kit (Zoman, Beijing, China), and was used to synthesize the first-strand cDNA by using the UEIris RT mix with DNase (All-in-One) (UElandy, Suzhou, China). RT-qPCR analysis was performed using the SYBR® Premix ExTaq™ II 4 Kit (TaKaRa, Dalian, China) in a CFX ConnectTM Real-Time System (BIO-RAD, USA). To obtain the average expression levels of each gene, at least three replicates were examined. ACTIN (LOC_Os01g12900) and U6 were used as endogenous controls.

2.6. In situ hybridization

In situ hybridization was performed according to previous methods (Zhang et al. 2017). DIG RNA Labeling Kit (Roche, Basel, Switzerland) was used to label the gene-specific OsARF2, OsARF3, OsARF4, OsARF14, OsARF15 probe. A TAS complementary LNA-modified DNA was used as the TAS detection probe. Shanghai Jie-Li Biotechnology Company synthesized a TAS complementary LNA-modified DNA probe. The primers sequences used are listed in Table S1.

2.7. Protein sequence and phylogenetic analysis

GenBank (http://www.ncbi.nlm.nih.gov/genbank/) was used to download the 12 AGO7 proteins sequences form different plant species by blasting DH2 candidate gene Os03g33650 as a query. MEGA 10.0 software was used to construct the phylogenetic tree by the neighbor-joining method with 1000 replicates (Tamura et al. 2011). MEME (http://alternate.meme-suite.org/) was used to predict the protein motifs. Accession numbers and plant species was listed in Additional file 3.

Two allelic recessive mutants of DH2, designated dh2-1, and dh2-2, were identified in this study. Given that dh2-1 showed typical defects, the remainder of this study focuses on the dh2-1 mutants.

3.1. dh2 mutantsexhibited defective lemmas

In rice, the wild-type (WT) spikelet was composed of a pair of rudimentary glumes, a pair of sterile lemmas and a fertile terminal floret, which contained four whorls of floral organs from outside to inside. Lemma and palea were shell-shaped and have 5 and 3 vascular bundles, respectively. The lemma had two inward hook-like edges, when the palea had two inward hook-like structures between the body of palea and margin region of palea. Then they hooked together perfectly to provided a protective environment for the internal floral organs (Fig. 1-A1, A2, A3).

In all the dh2-1 spikelets, it was observed that the lemma and palea could not hooked together mainly because of defective lemma. Based on the lemma phenotype, then dh2-1 spikelets were classified into two types. In type Ⅰ spikelets, although it still showed shell-shaped, the lemma becomed significantly narrower than that of the WT. (Fig. 1-B1, B2, B3). In type Ⅱ spikelets, the whole lemma almost degraded into a rod-shaped awn structure, losing its shell-shaped structural characteristics and exposing the inner floral organs (Fig. 1-C1, C2, C3).

For further clarifying the defects in dh2-1 lemma, the histology was analyzed between WT and dh2-1 mutants. Firstly, the lemma of WT had 5 vascular bundles and wider than the palea. However, the dh2-1 lemma became narrower, because the numbers of cells and vascular bundles were significantly reduced (Fig. 1-A8, A9). Secondly, the WT lemma had different epidermis cell types between the abaxial and the adaxial sides. The abaxial epidermis covered a silicified cell layer bearing trichomes and protrusions, when the adaxial epidermis consist of a layer of vacuolated cells (Fig. 1-A7, A8, A9). In lemma of type I spikelets, the abaxial and the adaxial sides were similar to the wild type. However, in lemma of type II spikelets, the ectopic silicified cells were observed in the adaxial epidermis, even some lemmas completely lost epidermis and showed rod-shaped with a silicified abaxial epidermis (Fig. 1-B7-B9, C7-C9). In general, the dh2-1 mutant showed defectivemedial-lateral and adaxial-abaxial polarities of lemma to different degrees, which suggests that DH2 plays a vital role in the establish of lemma polarity.

3.2 The defects of lemma in dh2-1 occured in the early stage of spikelet development

The development of WT and dh2-1 mutants during the early spikelet development stage was observed by SEM. In the Sp4 stage, the lemma primordia had already formed and the palea primordia appears. In the stage of Sp5-6, while the lodicules and stamens primordia of the WT spikelet began to formed, the lemma and palea primordia continue to differentiate, and the bottom edges of the palea and lemma began to hook together. Then, the lemma and palea primordia continued to differentiate and finally closed completely at the Sp8 (Fig. 2-A, B, C, D). It should be pointed out that the lemma and palea showed shell-shaped from the beginning of their primordia formation.

At the stage of Sp4 when lemma just formed, compared with WT, there were no obvious abnormalities in the dh2-1 mutants (Fig. 2-E, I). However, from the stage of Sp5, the dh2-1 spikelet displayed obvious defects. In type I spikelet, the lemma began to become narrower in the medial-lateral direction than that of WT at the stage of Sp5, although it still showed the shell-shaped (Fig. 2-F). Next, from Sp6 to Sp8, it was observed that the lemma primordia could not differentiate enough in medial-lateral direction and showed narrower than that of WT, resulting the inner organ primordia partially exposed (Fig. 2-G). In type II spikelet, the lemma almost fail to differentiate in medial-lateral direction at the Sp5 and showed rod-like in its upper part (Fig. 2-F). Then, the lemma primordia of the dh2-1 mutants further developed into a rod-like structure, and the stamens and pistil primordia were completely exposed from Sp6 to Sp8 (Fig. 2-K, L). Therefore, the entire data above indicated that the establish of medial-lateral and adaxial-abaxial polarities of lemma were seriously affected at the early stage of spikelet development, which was consistent with the observations at the mature stage.

3.3 dh2-1 mutants exhibited defective leaves

Under normal physiological conditions, the WT leaf blade was flat (Fig. 3- A, B, C), whereas the dh2-1 mutants showed narrow and abaxially-rolled leaves throughout the whole growth period (Fig. 3-A, I, J). At the mature period, the LRIs of the top three leaves was investigated between the WT and dh2-1 mutants. The results showed that the abaxial LRIs of the top three leaves of dh2-1 mutants were significantly increased compared with that of the WT (Fig. 3-P). Meanwhile, it was observed that the width of all top three leaves of dh2-1 mutants were significantly decreased compared with that of the WT (Fig. 3-Q, R). In addition, it was also found that the length of flag leaves in dh2-1 mutants was significantly decreased while that of other leaves showed no obvious difference compared with that of the WT (Fig. 3-Q, R).

For further clarifying the defects of dh2-1 leaves at the histological level, the SEM and freezing microtome section were conducted. Firstly, by using SEM, there were no obvious differences in both adaxial and abaxial surface between the WT and dh2-1 mutants leaves (Fig. 3-D, E, K, L). Next, by analyzing the cross sections, it was found that both the number and area of bulliform cells on the adaxial surface in dh2-1 mutants were increased significantly, between large and small veins, and among small veins, compared with those of the WT (Fig. 3-F, G, H, M, N, O, T, U). As a result, the leaves in dh2-1 were rolled abaxially. We also observed that the numbers of both large and small veins in dh2-1 were significantly decreased compared with that of the WT, which accounting for the narrow leaves. In general, in dh2-1 leaves, the adaxial cell feature was disordered and cell differentiation along medial-lateral direction was repressed, which means DH2 also played crucially important role in the establish of leaf polarity in rice.

Besides of exhibiting abaxially rolled leaves and rod-like lemmas, dh2-1 showed a series of growth and development defects. Compared with WT, the plant height of dh2-1 was lightly reduced (Fig. S3-A), the panicle length was significantly reduced (Fig. S3-B, G), the number of primary and secondary branches, the number of spikelets per panicle , the number of grains per panicle, and the seed setting rate were both decreased (Fig. S3-H-L). Meanwhile, we noted that mature grains of dh2-1 were also affected. Both the length and width of grain and brown grain of dh2-1 were significantly reduced, which resulted the decrease of the 1000-grain weight of dh2-1. (Fig. S3-C, D, E, F, M, N, O, P, Q, R). Taken together, the abnormal development of lateral organs has a significant effect on yield in rice.

3.4 Similarity of Defects in the dh2-2 Mutants to Those of the dh2-1 Mutant

The dh2-2 mutants showed very similar defects in morphology, histology, and agronomy, compared with those of the dh2-1 mutant (Fig. S1, Fig. S2, Fig. S4, Fig. S5). However, some differences were observed. dh2-2 spikelets were also classified into two types. In type Ⅰ spikelets, the degree of narrowing of the lemma in dh2-2 was stronger than that of dh2-1, and dh2-2 produced the elongated rod-like awn at the top of lemma (Fig. S1-B1, Fig. 1-B1). In the same planting environment, the changes in dh2-2 mutants are more severe compared to that of dh2-1 mutants. The spikelet of dh2-2 mutants usually presented a completely rod-shaped lemma, while the lemma of dh2-1 mutants was only slightly narrowed (Fig. S1-C1, Fig. 1-C1). The LRIs of the top three leaves of dh2-2 mutants leaves were higher than that of dh2-1 mutants leaves (Fig. S4-P, Fig. 3-P1). At the same time, the mature brown grains of type I dh2-1 spikelets had a complete grain morphology, while the morphology of the mature brown grains of dh2-2 mutants had changed. The shape of the brown grains of dh2-2 mutants were similar to that of water droplets (Fig. S5-D, E; Fig. S3-D, F). Taken together, these observations demonstrate that the dh2-2 mutant showed more severe defects than the dh2-1 mutant.

3.5. DH2 encode a Rice Argonaute7 (AGO7) protein

Previous studies showed that DH2 was located between the INDEL markers of IND7 and IND12 on chromosome 3, and there are total of 13 annotation genes related to flower development within this interval (Guo et al. 2013). In the present study, we performed gene cloning and sequence alignment analysis among those 13 annotation genes between WT and dh2 mutants. Sequencing analysis showed that a C to A transversion in the 1661th base of second exon of LOC_Os03g33650 occurred in dh2-1, which causes the replacement of amino acids Pro to Gln. Meanwhile, in dh2-2, there was a C-T transition at the 2861th base of the third exon of LOC_Os03g33650, leading to Ser-Phe substitution of the 221th amino acid (Fig. 4-A).

To further determine whether the phenotype of dh2 were due to the mutation of LOC_Os03g33650, we constructed the complementary vector containing 7623bp genomic sequence of wild-type LOC_Os03g33650 and introduced it into dh2-1 mutants. It was found that in positive transgenic lines, the mutation phenotype of dh2-1 was restored completely. Therefore, the results verified that the DH2 gene was LOC_Os03g33650 (Fig. 4-B).

We also utilize CRISP-CAS9 technology to perform targeted mutagenesis in coding frame of the LOC_Os03g33650 in the ZH11 (a geng-type) background. Compared with the ZH11, the leaves of DH2-cas9-1 lines were slightly abaxial curled (Fig. S4-A-E2). Meanwhile, the width and length of DH2-cas9-1 leaves were significantly decreased (Fig. S4-O, P, Q). Furthermore, the number and area of bulliform cells on the adaxial surface of DH2-cas9-1 were also increased (Fig. S4-F1, G1, F2, G2). Compared with the spikelet of ZH11 (Fig. S4-H1, I1, J1), it was obvious that the development of lemma in DH2-cas9-1 was affected, which resulted in lemma and palea primordia could not closed completely (Fig. S4-H2, I2, J2). It was worth noting that we found the plant height, and the length of each internode of DH2-cas9-1 were significantly decreased. Meanwhile, the panicle length, the number of primary and secondary branches, the number of spikelets per panicle, the number of grains per panicle, and the setting rate of DH2-cas9-1 were both decreased. Both the length and width of grain and brown grain of DH2-cas9-1 were significantly reduced, which resulted the decrease of the 1000-grain weight of DH2-cas9-1 (Fig. S7-A-P). Those phenotypes of DH2-cas9-1 were similar to the phenotypes in type I of dh2-1, and dh2-2, further verifying that LOC_Os03g33650 was the DH2 gene (Fig. S4-K1, L1, M1, K2, L2, M2).

DH2 encodes an Argonaute-like protein, which has four conserved domains, Piwi_ago-like domain, PAZ_argonaute_like domain, ArgoL1 domain, and ArgoN domain (Fig.4-C). Piwi_ago-like domain is the C-terminal portion of Argonaute7, which provides the 5' anchoring of the guide RNA and the catalytic site for slicing. PAZ_argonaute_like domain functions as a nucleic acid binding domain, with a strong preference for single-stranded nucleic acids (RNA or DNA) or RNA duplexes with single-stranded 3' overhangs. Phylogenetic tree analysis showed that DH2 and its homologous genes were widely present, and highly conserved in monocotyledonous and dicotyledonous plants. Those homologous genes could be classified into four Types, and DH2 protein belongs to the Type Ⅰ. Therefore, it indicated that DH2 encodes an OsAGO7 protein and may be act as the similar role with AtAGO7 (Fig. 4-D).

3.6. Overexpression of DH2 Causes Adaxially Rolled Leaves

As analyzing the phenotype of complementary transgenic plants, it was interesting that some of these lines showed adaxially curled leaves, which was just the opposite of abaxially curled leaves in loss-of-function mutants of DH2, in addition to being able to restore the phenotype of the dh2-1 mutant (Fig. 5-A, B, C, D). We further detected the expression level of DH2 in these complementary transgenic lines, OE1 to OE6 and WT. The result displayed that in OE1, OE3, OE5, and OE6 lines, the expression of DH2 increased by at least 100 times, while OE2 and OE4 increased about 50 times (Fig. 5-J). Then, it was suggested that adaxially curled leaves in these transgenic plants might be caused by overexpression of DH2.

For further clarifying the function of DH2, we analyzed the phenotypes of overexpression lines OE-2 and OE-6 in detail. During the mature period, the LRIs of the top three leaves of OE lines were significantly increased. In OE-2 , OE-4 and OE-6 lines, the average values of the LRIs of the top three leaves are 50%, 70% and 75%, respectively (Fig. 5-K), but the LEIs of the top three leaves of OE lines have no obvious changes (Fig. 5-L). In addition, the width and number of large or small vein of leaves in OE lines were extremely significantly decreased while the length of leaves were slightly decreased or unchanged, compared with those in the WT (Fig. 5-M, N, O). Next, by using SEM, it was found that the adaxial surface of OE-2, and OE-6 was similar to that of WT. However, in the abaxial surface of OE-2 and OE-6, prickly hair cells massively and regularly produced on the surface of dumbbell cells, while it was rare in the abaxial surface of WT but generally occurred in the adaxial surface of WT(Fig. 5-E1, E2, E3, F1, F2, F3). Finally, by the freezing microtome sections of the leaves in WT and OE-2, OE-6, it was observed that the number and area of bulliform cells on the adaxial surface between large and small vein, and among small veins, decreased significantly compared with that in WT (Fig. 5-H1, H2, H3, I1, I2, I3, P, Q).

Therefore, the results mentioned above showed that overexpression of DH2 could cause defects on development of abaxial-adaxial and medial-lateral axiles, further verifying that DH2 played crucially important role in the establish of leaf polarity in rice.

3.7. Overexpression of DH2 affects the panicle and grain development

Moderate leaf rolling, which is helpful to establish ideal plant architecture, plays a key role contributing to the high yields in rice. For evaluating the leaf rolling of overexpression of DH2 in breeding, we investigated the agronomy traits of the WT and overexpression lines.

It was found that in OE lines the panicle morphology and grains development were changed. Compared with the WT, The panicle length of OE-2, OE-4, OE-6 significantly decreased (Fig. 6-A, F). The number of primary branches, secondary branches, spikelets per panicle, grain per panicle of OE-2, OE-4 and OE-6 increased significantly. The setting rates of OE-2, OE-4, OE-6 were gently decreased (Fig. 6-G, H, I, J, K). The length and width of grain and brown grain of OE-2, OE-4, OE-6 were delightedly decreased, while the weight of 1000 grains and brown grains were decreased in the OE lines (Fig. 6- B, C, D, E, L, M, N, O, P, Q). The results showed that overexpression of DH2 may affect the development of rice panicles and grain.

3.8. DH2 affects the expressions of tasiR-ARF and OsARFs on the lemma

In order to further understand the functions of DH2 gene, we examined the spatial expression patterns of DH2 by RT-qPCR. The results indicated that DH2 were expressed mainly in leave blades and young panicle, while it showed very low expression level in root, shoot and leave sheath. Further, in spikelets, it was found that DH2 expressed mainly in lemma but not palea, lodicule, pistil (Fig. S8- A, B). Then, the expression pattern was constant with the role of DH2 in regulating the development of leave blades and lemma.

In Arabidopsis, AtAGO7, the DH2 homologous, had been verified to function on the production and silencing complex formation of tasiR-ARFs, then mediate the post-transcriptional gene silencing of AtARF3/4 by specially degrading the mRNA of ARF3/4 on adaxial side of leaves to function on the adaxial identity establishment (Allen et al. 2005; Axtell et al. 2006; Montgomery et al. 2008; Peragine et al. 2004; Iwakawa et al. 2021; Sakurai et al. 2021).

In this study, the expression of tasiR-ARFs was detected firstly by qPCR. The results showed that it was decreased significantly in the leaf, lemma and panicle of dh2-1 compared with that in WT (Fig. 8-A). Then, we detected the expression of OsARF2, OsARF3, OsARF14, and OsARF15, the homologous of the AtARF3/4, between dh2-1 mutants and the WT. The results showed that their expressions were all increased significantly in the leaf and lemma in dh2-1 compared with those in WT (Fig. 8-B, C, D), whereas their expression in the leaf of OE-2, OE-4 were decreased significantly compared with those in the WT (Fig. 8-D). Meanwhile, expression levels of these genes in young panicles were detected. The results showed that their expression were all increased significantly in panicles with different length, compared with those in the WT (Fig. 8-E, F, G, H). Therefore, these data indicated that DH2/OsAGO7 also mediated the formation of tasiR-ARFs, and then repress the expression of ARFs in rice, which may be quite conserved in monocots and dicots.

For further clarify the expression pattern of tasiR-ARFs and its targets OsARFs, we conducted in situ hybridization. The results showed that tasiR-ARFs is widely expressed in the floral organ primordia of the WT (Fig. 8-I1). However, there was no obvious signal of tasiR-ARFs in the rod-like lemma primordia in dh2-1 mutants, while it was still expressed in the other floral organ primordia in dh2-1 mutants (Fig. 8-I2). In the WT spikelets, OsARF2, OsARF3, OsARF14, and OsARF15 shared a similar expression pattern, mainly expressing on the abaxial surface of the margin region of palea, and weak signals of these genes could be detected in the stamens and pistils, whereas they were not almost expressed in the lemma primordia (Fig. 8-K1, L1, M1, N1). In dh2-1 spikelets, the signal of OsARF2, OsARF3, OsARF14, or OsARF15 was slightly enhanced in the abaxial surface of the margin region of palea, pistil and stamen. Most importantly, strong ectopic signals of these genes were detected in the rod-shaped lemma primordia (Fig. 8-K2, L2, M2, N2). In addition, we also detect the expression of OsARF4. In the WT flowers, OsARF4 specifically expresses in the lemma and palea primordia. However, it could not be detected in the rod-like lemma primordia in the dh2-1 spikelets (Fig. 8-J1, J2). Together, these results indicated DH2 was mainly responsible for the repressing the expression of OsARF2, OsARF3, OsARF14 and OsARF15 in lemma in a way of mediating the production of ta-siRNA.

4.1 The DH2 mediated tasiR-ARFs synthesis and ARFs repressing to affect polarity of lateral organs in rice.

tasiR-ARFs are unique to plants, and plays an important role in their growth and development, which includes the regulation of the adaxial–abaxial polarity of lateral organs, as well as SAM formation (Nagasaki et al. 2007; Toriba et al. 2010; Arikit et al. 2013; Garcia et al. 2006; Fahlgren et al. 2006; Adenot et al. 2006). AGO7 specifically loads miR390 to target TAS3 transcripts, leading to their cleavage, which primarily determines the synthesis of mature tasiR-ARFs although next several proteins like SGS3, RDR6 and DCL4 are also important (Allen et al. 2005; Axtell et al. 2006; Montgomery et al. 2008; Peragine et al. 2004; Iwakawa et al. 2021; Sakurai et al. 2021). In Arabidopsis and rice, the mutations of AGO7, RDR6, and DCL4 lead to decreased accumulation of tasiR-ARFs and increased expression of target genes ARFs, and then affects adaxial–abaxial polarity of leaf and/or floral organs (Xu et al. 2006; Li et al. 2005; Fahlgren et al. 2006; Yoon et al. 2010; Marin et al. 2010; Nagasaki et al. 2007; Toriba et al. 2010; Song et al. 2012).

In this study, DH2 encodes the OsAGO7 in rice. In dh2 mutants, the rod-shaped lemma with abaxial epidermics was observed, meaning the adaxial side of lemma was abaxialized. It was also found the leaf showed outward rolling in dh2 mutants, due to the development defects of bulliform cell at adaxial side. Moreover, the expression of tasiR-ARFs was reduced in the lemma, leaf and spikelet primordia, the expression of OsARF2, OsARF3, OsARF14, and OsARF15 was significantly increased. Therefore, it was seem that AGO7—tasiR-ARFs—ARFs was conserved in establishment of polarity of lateral organs between rice and Arabidopsis.

However, we found the transcripts of four OsARF genes showed organ speciality but not organ polarity in lemma in the present study. More narrowly, they were all expressed in palea but not lemma in the WT, whereas they were all ectopically expressed in rod-shaped lemma. Although the four genes show organ-polarity expression pattern (expressing at the abaxial side of margin region of palea ) in the WT, they showed the same expression pattern in the dh2 palea. As a result, we also observed no obvious defects in the dh2 palea. Meawhile, we also found that tasiR-ARFs was expressed in the whole lemma and palea but not abaxial side. That means there was not a opposite expression of tasiR-ARFs and ARFs in adaxial–abaxial of lemma. In the previous studies about the pathway of AGO7—tasiR-ARFs—ARFs, researches commonly used qRT-PCR and/or norther-blot to detected the expression of tasiR-ARFs and ARFs in lateral organs, so it was difficult to clarify how the expression patterns of them was between the WT and mutants (Zhang et al. 2023; Itoh et al. 2000; Nagasaki et al. 2007; Liu et al. 2007; Song et al. 2012). In sho2/osrdr6 mutant, by in situ hybridization, it was found that OsARF3 was expressed in the whole rod-shaped lemma, while in the wild-type lemma, OsARF3 was observed in some parts of the abaxial sides of lemma (Toriba et al. 2010). So, the expression pattern of OsARF3 in the WT described in this study was different with our data. Then we read the supplemental Fig. 2 carefully in this paper, and found it is doubtful about whether OsARF3 is expressed in the WT lemma. Therefore, according to these studies, we think that the pathway of OsAGO7—tasiR-ARFs in rice was more likely involved in the whole development of lemma but not only abaxial side by restricting ectopical expression of OsARFs in the whole lemma, which was different with that in lateral organs of Arabidopsis. In dh2 mutants, while tasiR-ARFs was decreased, OsARF2, OsARF3, OsARF15 was ectopically expressed in the whole lemma while OsARF14 was ectopically expressed in the abaxial side of lemma, which finally resulted in the rod-shaped abaxialized lemma.

4.2 DH2 affects the rice yield

Moderate leaf rolling, which is helpful to establish ideal plant architecture, plays a key role contributing to the high yields in rice. As the organ of photosynthesis, the leaf morphology is directly related to factors such as photosynthesis area and effective photosynthetic efficiency (Xu et al. 2018; Du et al. 2018). And the development of hull (lemma and palea) affects grain shape, because it limits the length and width of grain (Xiang et al. 2015; Yu et al. 2018). Therefore, reasonable leaf and hull morphology are important factors for high yield in rice.

In this study, most of agronomy traits in dh2 mutants and DH2-cas9 lines, like height, number of spikelets per panicle, setting rate and grain weight were all decreased compared with WT, indicating that the dh2 mutation negatively affected yield, due to changes of morphology of leaf and lemma. Interesting, we identified some over-expression of DH2 lines in the complementary test. These lines showed adaxially rolling leaves throughout the whole growth period, which was opposed to abaxially rolling leaves in the dh2 mutants. By statistical analysis, it was found that number of branches, number of spikelets per panicle, and grains weight was increased significantly in over-expression of DH2 lines compared with that dh2 mutants, while the other main agronomy traits showed no obvious differences. In a previous study, a R05 mutant showed the upregulated expression of OsAGO7 gene, caused by the T-DNA inserting into the promoter region of the gene. The R05 mutant showed the upward rolling leaf and the increasing LRI, which enhanced erect-leaf habit and elevated the value of LEI (Shi et al. 2007). Now it is a common sense that the appropriate rolled leaf is regarded as a critical element for the ideal rice phenotype (Yuan et al. 1997; Chen et al. 2001; Zhang et al. 2009). Moderate leaf rolling helps to maintain the erectness of leaves, which can increase the light transmission rate and light saturation point without affecting the light compensation point, resulting in a well-proportioned leaf area for photosynthesis (Duncan. 1971; Zhang et al. 2009). Moderate leaf rolling improves photosynthetic efficiency, accelerates dry-matter accumulation, increases yield, reduces the solar radiation on leaves, and decreases leaf transpiration under drought stress (Lang et al. 2004; Zhang et al. 2009). Therefore, it is of great practical value that enhance the DH2 expression in future molecular design breeding.

We identified two alleles, named degenerated hull 2 − 1, -2 (dh2-1, -2), which encode a relatively conservative Argonaut7 (AGO7) protein in plants, which is involved in the synthesis of tasiR-ARFs. The production of tasiR-ARF depends on the function of AGO7, and tasiR-ARFs form a silencing complex with AGO7 to inhibit downstream ARFs, thereby regulating the lateral organ polarity development. In rice, the decreasing of tasiR-ARFs in dh2-1,-2, the increasing of OsARF2, OsARF3, OsARF14, and OsARF15, and the disrupting of their polarity distribution of lateral organ indicates that DH2-mediated tasiR-ARFs synthesis regulates lateral organ polarity development in rice.

Accession numbers

Sequence data from this article can be accessed in the GenBank database under the following accession numbers: DH2 (OsAGO7), ACTIN (LOC_Os03g50885), OsARF2, OsARF3, OsARF14, and OsARF15 are AP014968, AB071291, AK072330, NM_001062573, and AK062170, respectively. Locus identifications in the Rice Genome Annotation Project Database are as follows: DH2/OsAGO7 (LOC_Os03g33650), OsARF2 (LOC_Os01g48060), OsARF3 (LOC_Os01g54990), OsARF14 (LOC_Os05g43920), and OsARF15 (LOC_Os05g48870).

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Availiability of supporting data

All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no conflicts of interest associated with this work.

CRediT authorship contribution statement

Jun Tang: Phenotypic experiment, Material preparation, Data collection and analysis, Writing - original draft, Writing - review & editing. Tianye Li: Material preparation, Data collection and analysis, Writing - original draft, Writing - review & editing. Yuanzhuo Gao: Phenotypic experiment, Data collection and analysis. Xinghang Li: Material preparation. Ziheng Huang: Data collection. Hui Zhuang: Material preparation. Hongfa Luo: Methodology, Supervision. Yunfeng Li: Methodology, Project administration, Supervision, Funding acquisition, Writing - review & editing.

Funding

This work was supported by the National Natural Science Foundation of China (32172044 and 31971919), Sichuan Science and Technology Program (2024NSFSC0321), Natural Science Foundation of Chongqing, China (cstc2020jcyj-jqX0020), Chongqing Modern Agricultural Industry Technology System (CQMAITS202301), and Fundamental Research Funds for the Central Universities (SWU-XDJH202315).

Adenot X, Elmayan T, Lauressergues D, Boutet S, Bouché N, Gasciolli V, Vaucheret H. 2006. DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr Biol, 16,927-32. Agricultural Res, 22, 88–91.
Allen E, Xie Z, Gustafson AM, Carrington JC. 2005. MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121, 207-221.
Arikit S, Zhai J, Meyers BC. 2013. Biogenesis and function of rice small RNAs from non-coding RNA precursors. Curr Opin Plant Biol, 16, 170-9.
Axtell MJ, Jan C, Rajagopalan R, Bartel DP. 2006. A two-hit trigger for siRNA biogenesis in plants. Cell, 127, 565-577.
Bowman, Y, Eshed, S.F, Baum. 2002. Establishment of polarity in angiosperm lateral organs. Trends Genet, 18, 134-141.
Braybrook SA, Kuhlemeier C. 2010. How a plant builds leaves. The Plant Cell, 22, 1006-18.
Chen Z X, Pan X B, Hu J. 2001. Relationship between rolling leaf and ideal plant type of rice (Oryza sativa L.) (in Chinese). Jiangsu Agricultural Res, 22, 88–91.
Dai M, Zhao Y, Ma Q, Hu Y, Hedden P, Zhang Q, Zhou D X. 2007. The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism. Plant Physiol, 144, 121-33.
Du F, Guan C, Jiao Y. 2018. Molecular mechanisms of leaf morphogenesis. Molecular Plant, 11, 1117-1134.
Duncan W G. 1971. Leaf angle, leaf area, and canopy photosynthesis.Crop Sci, 11, 482–485.
Emery John F, Floyd Sandra K, Alvarez John, Eshed Yuval, Hawker Nathaniel P, Izhaki Anat, Baum Stuart F, Bowman John L. 2003. Radial Patterning of Arabidopsis Shoots by Class III HD-ZIP and KANADI Genes. Current Biology, 13, 1768–1774.
Eshed Yuval, Baum Stuart F, Perea John V, Bowman John L. 2001. Establishment of polarity in lateral organs of plants. Current Biology, 11, 1251–1260.
Fahlgren N, Montgomery TA, Howell MD, Allen E, Dvorak SK, Alexander AL, Carrington JC. 2006. Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr Biol, 16, 939-44.
Garcia D, Collier SA, Byrne ME, Martienssen RA. 2006. Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway. Curr Biol, 16,933-8.
Golz John F, Hudson Andrew. 2002. Signalling in plant lateral organ development. The Plant Cell, 14, 277-288.
Guan C M, Wu B B, Yu T, Wang Q Q, Krogan Naden T, Liu X G, Jiao Y L. 2017. Spatial Auxin Signaling Controls Leaf Flattening in Arabidopsis. Current Biology, 27, 2940–2950.
Guo S, Li Y F, Ren D Y, Zhang T Q, He G H. 2013. Phenotypic analysis and gene mapping of degenerated hull 2 (dh2) mutant in rice (Oryza sativa L.). Acta Agronomica Sinica, 39, 10391044 (in Chinese)
Itoh J I, Kitano H, Matsuoka M, Nagato Y. 2000. Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice. The Plant Cell, 12, 2161–2174.
Itoh J I, Hibara K, Sato Y, Nagato Y. 2008. Developmental role and auxin responsiveness of Class III homeodomain leucine zipper gene family members in rice. Plant Physiol, 147, 1960-75.
Iwakawa Hiro-Oki, Lam Andy Y W, Mine Akira, Fujita Tomoya, Kiyokawa Kaori, Yoshikawa Manabu, Takeda Atsushi, Iwasaki Shintaro, Tomari Yukihide. 2021. Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants. Cell Reports, 35, 109300.
Juarez Michelle T, Kui Jonathan S, Thomas Julie, Heller Bradley A, Timmermans Marja C. P. 2004. microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature, 428, 84–88.
Kerstetter Randall A, Bollman Krista, Taylor R. Alexandra, Bomblies Kirsten, Poethig R. Scott. 2001. KANADI regulates organ polarity in Arabidopsis. Nature, 411, 706-9.
Lang Y Z, Zhang Z J, Gu X Y, Yang J C, Zhu Q S. 2004. A physiological and ecological effect of crimpy leaf character in rice (Oryza sativa L.) II. Photosynthetic character, dry mass production and yield forming. Acta Agron. Sin, 30, 883–887.
Li H, Xu L, Wang H, Yuan Z, Cao X, Yang Z, Zhang D, Xu Y, Huang H. 2005. The Putative RNA-dependent RNA polymerase RDR6 acts synergistically with ASYMMETRIC LEAVES1 and 2 to repress BREVIPEDICELLUS and MicroRNA165/166 in Arabidopsis leaf development. The Plant Cell, 17, 2157-71.
Li Y Y, Shen A, Xiong W, Sun Q L, Luo Q, Song T, Li Z L, Luan W J. 2016. Overexpression of OsHox32 Results in Pleiotropic Effects on Plant Type Architecture and Leaf Development in Rice. Rice, 9, 46.
Liu B, Chen Z, Song X, Liu C, Cui X, Zhao X, Fang J, Xu W, Zhang H, Wang X, Chu C, Deng X, Xue Y, Cao X. 2007. Oryza sativa dicer-like4 reveals a key role for small interfering RNA silencing in plant development. The Plant Cell, 19, 2705-18.
Liu Y L, Teng C, Xia R, Meyers Blake C. 2020. PhasiRNAs in Plants, Their Biogenesis, Genic Sources, and Roles in Stress Responses, Development, and Reproduction. The Plant Cell, 32, 3059–3080.
Ma Y, Wang F, Guo J, Zhang X S. 2009. Rice OsAS2 Gene, a Member of LOB Domain Family, Functions in the Regulation of Shoot Differentiation and Leaf Development. J. Plant Biol, 52, 374–381.
Marin E, Jouannet V, Herz A, Lokerse AS, Weijers D, Vaucheret H, Nussaume L, Crespi MD, Maizel A. 2010. miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. The Plant Cell, 22, 1104-17.
McConnell JR, Emery J, Eshed Y, Bao N, Bowman J, Barton MK. 2001. Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature, 411, 709-13.
Montgomery Taiowa A, Howell Miya D, Cuperus Josh T, Li D W, Hansen Jesse E, Alexander Amanda L, Chapman Elisabeth J, Fahlgren Noah, Allen Edwards, Carrington,James C. 2008. Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell, 133, 128–141.
Moon J, Hake S .2011. How a leaf gets its shape. Curr Opin Plant Biol, 14, 24-30.
Nagasaki H, Itoh J, Hayashi K, Hibara K, Satoh-Nagasawa N, Nosaka M, Mukouhata M, Ashikari M, Kitano H, Matsuoka M, Nagato Y, Sato Y. 2007. The small interfering RNA production pathway is required for shoot meristem initiation in rice. Proc Natl Acad Sci U S A, 104, 14867-71.
Otsuga D, DeGuzman B, Prigge MJ, Drews GN, Clark SE. 2008. REVOLUTA regulates meristem initiation at lateral positions. Plant J, 25, 223–236.
Pekker Irena, Alvarez John Paul, Eshed Yuval. 2005. Auxin response factors mediate Arabidopsis organ asymmetry via modulation of KANADI activity. The Plant Cell, 17, 2899-910.
Peragine Angela, Yoshikawa Manabu, Wu G, Albrecht Heidi L, Poethig R Scott. 2004. SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev, 18, 2368-79.
Prigge MJ, Otsuga D, Alonso JM, Ecker JR, Drews GN, Clark SE. 2005. Class III homeodomain-leucine zipper gene family members have overlapping, antagonistic, and distinct roles in Arabidopsis development. The Plant Cell, 17, 61-76.
Sakurai Yuriki, Baeg Kyungmin, Lam Andy Y W, Shoji Keisuke, Tomari Yukihide, Iwakawa Hiro-Oki. 2021. Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants. Proc Natl Acad Sci U S A, 118, e2102889118.
Sarojam Rajani, Sappl Pia G, Goldshmidt Alexander, Efroni Idan, Floyd Sandra K, Eshed Yuval, Bowman John L. 2010. Differentiating Arabidopsis shoots from leaves by combined YABBY activities. The Plant Cell, 21, 3105–3118.
Sawa Shinichiro, Watanabe Keiro, Goto Koji, Eiko Kanaya, Morita Eugene Hayato, Okada Kiyotaka. 1999. FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMG-related domains. Genes Dev, 13, 1079-1088.
Shi Z, Wang J, Wan X, Shen G, Wang X, Zhang J. 2007. Over-expression of rice OsAGO7 gene induces upward curling of the leaf blade that enhanced erect leaf habit. Planta, 226, 99-108.
Si F Y, Yang C, Yan B, Yan W, Tang S J , Yan Y, Cao X F, Song X W. 2022. Control of OsARF3a by OsKANADI1 contributes to lemma development in rice. The Plant Journal, 110, 1717–1730.
Siegfried Kellee R, Eshed Yuval, Baum Stuart F, Otsuga Denichiro, Drews Gary N,Bowman John L. 1999. Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development, 126, 4117-4128.
Song X W, Li Y, Cao X F, Qi Y J. 2019. MicroRNAs and Their Regulatory Roles in Plant–Environment Interactions. Annu. Rev. Plant Biol, 70, 489–525.
Song X, Li P, Zhai J, Zhou M, Ma L, Liu B, Jeong DH, Nakano M, Cao S, Liu C, Chu C, Wang XJ, Green PJ, Meyers BC, Cao X. 2012. Roles of DCL4 and DCL3b in rice phased small RNA biogenesis. Plant J, 69, 462-74.
Song X, Wang D, Ma L, Chen Z, Li P, Cui X, Liu C, Cao S, Chu C, Tao Y, Cao X. 2012. Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development. Plant J, 71, 378-89.
Stahle Melissa I, Kuehlich Janine, Staron Lindsay, Arnim Albrecht G von, Golz John F. 2009. YABBYs and the Transcriptional Corepressors LEUNIG and LEUNIG_HOMOLOG Maintain Leaf Polarity and Meristem Activity in Arabidopsis. The Plant Cell, 21, 3105–3118.
Takahashi H, Iwakawa H, Ishibashi N, Kojima S, Matsumura Y, Prananingrum P, Iwasaki M, Takahashi A, Ikezaki M, Luo L, Kobayashi T, Machida Y, Machida C. 2013. Meta-analyses of microarrays of Arabidopsis asymmetric leaves1 (as1), as2 and their modifying mutants reveal a critical role for the ETT pathway in stabilization of adaxial-abaxial patterning and cell division during leaf development. Plant Cell Physiol, 54, 418-31.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5, Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731-2739.
Tang G L, Reinhart Brenda J, Bartel David P , Zamore Phillip D. 2003. A biochemical framework for RNA silencing in plants. Genes Development, 17, 49-63.
Toriba T, Harada K, Takamura A, Nakamura H, Ichikawa H, Suzaki T, Hirano HY. 2007. Molecular characterization the YABBY gene family in Oryza sativa and expression analysis of OsYABBY1. Mol Genet Genomics, 277, 457-68.
Toriba T, Suzaki T, Yamaguchi T, Ohmori Y, Tsukaya H, Hirano HY. 2010. Distinct regulation of adaxial–abaxial polarity in anther patterning in rice. The Plant Cell, 22, 1452-1462.
Xiang, C Y, Liang, X X, Chu, R Z. et al. 2015. Fine mapping of a palea defective 1 (pd1), a locus associated with palea and stamen development in rice. Plant Cell Rep, 34, 2151–2159.
Xie Z X, Johansen Lisa K, Gustafson Adam M, Kasschau Kristin D, Lellis Andrew D, Zilberman Daniel, Jacobsen Steven E, Carrington James C. 2004. Genetic and Functional Diversification of Small RNA Pathways in Plants. PLoS BIOLOGY, 2, 5-0642.
Xu L, Yang L, Pi L, Liu Q, Ling Q, Wang H, Poethig RS, Huang H. 2006. Genetic interaction between the AS1-AS2 and RDR6-SGS3-AGO7 pathways for leaf morphogenesis. Plant Cell Physiol, 47, 853-63.
Xu P, Ali A, Han B, Wu X. 2018. Current Advances in Molecular Basis and Mechanisms Regulating Leaf Morphology in Rice. Front Plant Sci, 9,1528.
Yoon EK, Yang JH, Lim J, Kim SH, Kim SK, Lee WS. 2010. Auxin regulation of the microRNA390-dependent transacting small interfering RNA pathway in Arabidopsis lateral root development. Nucleic Acids Res, 38,1382-91.
Yoshikawa Manabu, Peragine Angela, Park Mee Yeon, Poethig R. Scott. 2005. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev, 19, 2164-2175.
Yu T, Guan C M ,Wang J, Sajjad Muhammad, Ma L J, Jiao Y L. 2017. Dynamic patterns of gene expression during leaf initiation. Journal of Genetics and Genomics, 44 ,599-601.
Yu, J P, Ma, J L, Zhang, Z Y. et al. 2018. Alternative splicing of OsLG3b controls grain length and yield in japonica rice. Plant Biotechnol. J, 16, 1667–1678.
Yuan L P. 1997. Super-high yield hybrid rice breeding. Hybrid Rice,12, 1–6.
Zhang G H, Xu Q, Zhu X D, Qian Q, Xue H W. 2009. SHALLOT-LIKE1 Is a KANADI Transcription Factor That Modulates Rice Leaf Rolling by Regulating Leaf Abaxial Cell Development. The Plant Cell, 21, 719–735.
Zhang G, Xu Q, Zhu X, Qian Q, Xue H. 2009. SHALLOT-LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development. The Plant Cell, 21,719-35.
Zhang T, Li Y F, Ma L, Sang X C, Ling Y H, Wang Y, Yu P, Zhuang H, Huang J, Wang N, Zhao F M, Zhang C W, Yang Z L, Fang L, He G H. 2017. LATERAL FLORET 1 induced the three-floret spikelet in rice. Proc Natl Acad Sci U S A, 114, 9984-9989.
Zhang T, You J, Zhang Y, Yao W, Chen W, Duan Q, Xiao W, Ye L, Zhou Y, Sang X, Ling Y, He G H, Li Y F. 2021. LF1 regulates the lateral organs polarity development in rice. New Phytol, 231,1265-1277.
Zhang Y, You J, Tang J, Xiao W W, Wei M, Wu R H, Liu J Y, Zong H Y, Zhang S Y, Qiu J, Chen H, Ling Y H, Zhao F M, Li Y F, He G H, Zhang T. 2023. PDL1-dependent trans-acting siRNAs regulate lateral organ polarity development in rice. JIA, 10.1016.

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DH2 regulates the development of lateral organs in rice

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1. Introduction

2. Materials and methods

3. Results

4. DISCUSSION

4.2 DH2 affects the rice yield

5. Conclusion

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