The osa‐miR164 target OsCUC1 functions redundantly with OsCUC3 in controlling rice meristem/organ boundary specification

Summary The specification of the meristem/organ boundary is critical for plant development. Here, we investigate two previously uncharacterized NAC transcription factors: the first, OsCUC1, which is negatively regulated by osa‐miR164c, dimerizes with the second, OsCUC3, and functions partially redundantly in meristem/organ boundary specification in rice (Oryza sativa). We produced knockout lines for rice OsCUC1 (the homolog of Arabidopsis CUC1 and CUC2) and OsCUC3 (the homolog of Arabidopsis CUC3), as well as an overexpression line for osa‐miR164c, to study the molecular mechanism of boundary specification in rice. A single mutation in either OsCUC1 or OsCUC3 leads to defects in the establishment of the meristem/organ boundary, resulting in reduced stamen numbers and the fusion of leaves and filaments, and the defects are greatly enhanced in the double mutant. Transgenic plants overexpressing osa‐miR164c showed a phenotype similar to that of the OsCUC1 knockout line. In addition, knockout of OsCUC1 leads to multiple defects, including dwarf plant architecture, male sterility and twisted‐rolling leaves. Further study indicated that OsCUC1 physically interacts with leaf‐rolling related protein CURLED LEAF AND DWARF 1 (CLD1) and stabilizes it in the nucleus to control leaf morphology. This work demonstrated that the interplay of osa‐miR164c, OsCUC1 and OsCUC3 controls boundary specification in rice.

Although many NAC genes have been characterized in rice, whether they are involved in meristem or organ boundary specification remains unknown. To clarify this question, we knocked out the homologues of CUC genes in rice using CRISPR/Cas9. Our results indicated that OsCUC1, which is negatively regulated by osa-miR164c, together with OsCUC3, is involved in rice meristem/organ boundary specification in a partially redundant manner. In addition, OsCUC1 physically interacts with a previously known leaf rolling related protein CURLED LEAF AND DWARF 1 (CLD1) and stabilizes it in the nucleus to control leaf morphology.

Plant materials and growth conditions
Rice (Oryza sativa L.) plants were grown in a glasshouse at 30°C (for daytime)/25°C (for nighttime) under a 13.5 h : 8.5 h, light : dark photoperiod (>3000 lux) with 60% humidity. The genetic background of all transgenic plants used was Nipponbare. omtn4 ZH11 and omtn6 ZH11 were obtained from Biogle GeneTech (Hangzhou Biogle Co. Ltd, Hangzhou, Zhejiang, China) Phylogenetic tree and sequence alignment Rice OsCUC1, OsCUC3 and 70 homologues from 13 species were chosen for phylogenetic analysis. Protein sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov/) according to their accession number. The sequence alignment was performed with CLUSTALX and the phylogenetic tree was constructed using MEGA7 with the neighbor-joining method and bootstrap analysis (1000 replicates). The accession numbers are listed in Supporting Information Table S1.

Vector construction
To knock out OsCUC1 and OsCUC3, target sites were designed online (http://cbi.hzau.edu.cn/crispr/), and the optimal target sites with low off-target scores and high sgRNA scores were selected. We prepared the CRISPR/Cas9 binary constructs as described previously . To overexpress osa-miR164c, a polymerase chain reaction (PCR) fragment amplified from Nipponbare genomic DNA using primers OE-miR164cF and OE-miR164cR was cloned into the PstI/SpeI sites of binary vector pOX between the maize Ubi1 promoter and Nos terminators.
Generation of pOsCUC1::GUS was achieved by amplifying a 2 kb OsCUC1 promoter from Nipponbare genomic DNA using primers OsCUC1pgus-F and OsCUC1pgus-R, which was then inserted into pCAMBIA1300-GN at the HindIII/XbaI site. To generate pOsCUC1::OsCUC1-GUS, a PCR fragment harboring the 2 kb upstream promoter and gDNA (without a stop codon) of OsCUC1 was amplified from Nipponbare genomic DNA using primers pro-cds-gus-F and pro-cds-gus-R, and was then inserted into pCAMBIA1300-GN at the SalI/XbaI site. To generate pOsCUC1::mOsCUC1-GUS, two overlapped fragments harboring the 2 kb upstream promoter and gDNA (without a stop codon) of OsCUC1 were amplified using two primer sets: procds-gus-F/mOsCUC1-R (carrying the 7 bp mutations) and mOsCUC1-F (carrying the 7 bp mutations)/pro-cds-gus-R, respectively. After purification, the two fragments were mixed in equal molar ratios and used as the PCR templates to amplify a fragment containing the 2 kb upstream promoter and gDNA (without a stop codon) of OsCUC1, into which the osa-miR164cresistant mutation was introduced, using pro-cds-gus-F and procds-gus-R. After sequencing, the fragment was inserted into pCAMBIA1300-GN at the SalI/XbaI site.
Primer sequences for the constructions are listed in Table S2.

Pollen viability test
Mature pollen grains from the unopened flowers were collected and immediately put on a microscope slide. A drop of IKI (iodine potassium iodide) solution was deposited onto the pollen, and the slide was covered with a coverslip. Pollen viability counts were made 5 min after the IKI solution was added to the pollen. Pollen grains exhibiting dark staining (dark red or black color) were counted as viable.

Histochemical analysis and b-glucuronidase (GUS) assay
Samples of the transgenic plants were incubated with GUS staining solution (50 mM sodium phosphate at pH 7.2, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 2 mM of X-Gluc, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide) overnight at 37°C. After staining, the tissues were rinsed several times with ethanol, then mounted on slides and photographed. For histochemical analysis, the samples were fixed with formaldehyde alcohol acetic acid (FAA) fixation solution at 4°C overnight, followed by dehydration and embedding in paraffin (Paraplast Plus, Sigma). They were then cut into 7 lm sections with a microtome, and stained with safranine and fast green FCF. Sections were observed under bright field with a microscope (CX31; Olympus, Tokyo, Japan).

Yeast-two-hybrid assay
The Matchmaker yeast-two-hybrid system (Clontech, Kusatsu, Japan) was used to study the interaction of OsCUC1, OsCUC3, OMTN4 and OMTN6 with CLD1. The deduced amino acid sequences of OsCUC1, OsCUC3, OMTN4 and OMTN6 were separately cloned into pGADT7 (AD) vectors, while CLD1 was inserted into pGBKT7 (BD). Yeast strain Y2HGold was transformed with bait plasmid and strain Y187 was transformed with prey plasmid. Co-transformants were plated on synthetic defined (SD)/-Leu/-Trp/-His/-Ade medium plates and SD/-Leu/-Trp/ -His/-Ade/X-a-Gal medium plates for examination of growth. Primer sequences for the constructions are listed in Table S2.

Transactivation assay
The deduced amino acid sequences of OsCUC1 and OsCUC3 were separately cloned into pGBKT7 (BD), resulting in fusions with the GAL4 binding domain. The fusion plasmids pBD-OsCUC1 and pBD-OsCUC3 were transformed into yeast strain AH109 and plated on SD/-Trp/-His/-Ade and SD/-Trp/-His/ -Ade/X-a-Gal medium plates for examination of growth. Primer sequences for the constructions are listed in Table S2.

Scanning electron microscopy (SEM)
The samples were fixed in FAA (50% ethanol/acetic acid/ formaldehyde, 9 : 0.5 : 0.5), and then dehydrated in an ethanol series and dried by supercritical fluid drying with CO 2 . The dried samples were mounted on copper supports and sputter-coated with gold, and then observed under SEM (S-3400N; Hitachi, Tokyo, Japan).

Subcellular localization assay
The coding sequences of OsCUC1, OsCUC3 and CLD1 (without stop codons) from Nipponbare were fused with green fluorescent protein (GFP) to generate the fusion protein. The fusion protein driven by the cauliflower mosaic virus (CaMV) 35S promoter was transcribed in rice protoplasts. The subcellular localization was evaluated using a confocal laser-scanning microscope (TCS-SP8MP; Leica). OsRac3-mCherry and Ghd7-mCherry were used as the membrane localization marker (Chen et al., 2010) and nuclear localization marker (Xue et al., 2008), respectively. Primer sequences for the constructions are listed in Table S2.

RNA extraction and quantitative real time (RT) polymerase chain reaction assay
To evaluate gene expression, total RNA was extracted using the RNeasy Plant Mini Kit (cat. no. 74904; Qiagen, Germany) following the manufacturer's instructions. First-strand cDNA was synthesized from 1 lg of total RNA using the PrimeScript RT Reagent Kit (cat. no. RR047A; Takara, Kusatsu, Japan) according to the manufacturer's instructions. ChamQ SYBR qPCR Master Mix (cat. no. Q311-01; Vazyme, Nanjing, Jiangsu, China) and a LightCycler 480II (Roche) were used for qRT-PCR, according to the manufacturers' instructions. Rice Ubiquitin (Os03g0234200) was used as the internal reference, and the level of gene expression was normalized to the Ubiquitin level. To evaluate the expression level of osa-miR164, RNA extraction and stem-loop qRT-PCR were performed as described previously . Rice miRNA U6 was used as the internal reference and the osa-miR164 level was normalized to U6. Primer sequences for the qRT-PCR are listed in Table S2.

In situ hybridization
To prepare the probe, a 500-bp fragment of OsCUC1-specific cDNA and a 500-bp fragment of OsCUC3-specific cDNA were amplified. The probes were labeled using a DIG RNA Labeling Kit (Roche). The in situ hybridization experiments were carried out as described previously (Brewer et al., 2006). Primer sequences for the in situ hybridization are listed in Table S2.

Co-immunoprecipitation (Co-IP) assay
To confirm the protein-protein interactions of OsCUC1, OsCUC3 and CLD1, OsCUC1-GFP, OsCUC1-MYC, OsCUC3-GFP and CLD1-Flag were artificially synthesized (GenScript Biotech Corp., Nanjing, Jiangsu, China) and transcribed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Twelve hours after co-transformation of the plasmids, the rice protoplasts were harvested by centrifugation at 400 g for 5 min. The collected protoplasts were homogenized in CelLytic immunoprecipitation (IP) buffer (B7345; Sigma-Aldrich), and incubated for 30 min on ice and then centrifuged at 15 000 g for 10 min at 4°C to remove aggregates. Next, 40 ll of Protein A/G Agarose Beads (nProtein A Sepharose 4 Fast Flow; GE Healthcare, Uppsala, Sweden) were added into the protein extract, which was diluted by IP buffer. The mixture was incubated for 3 h at 4°C with gentle shaking (40-50 rpm). After centrifugation at 14 000 g for 5 min at 4°C, the supernatant was separated from the beads, and the antibodies were added to the supernatant. After overnight incubation at 4°C, 40 ll of protein A-Sepharose was added and incubated for a further 2-3 h at 4°C. The beads were collected by centrifugation at 100 g for 3 min at 4°C, and then washed five times with ice-cold washing buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, and 1% protease inhibitor). The proteins were eluted from the beads by boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 5 min and analysed by Western blotting. The original blot images are shown in Fig. S1. Primer sequences for the constructions are listed in Table S2.

In vitro pull-down assay
To confirm the protein-protein interaction of OsCUC1 and OsCUC3, the coding sequences of OsCUC1 and OsCUC3 were New Phytologist (2021) 229: 1566-1581 Ó 2020 The Authors New Phytologist Ó 2020 New Phytologist Foundation www.newphytologist.com

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New Phytologist amplified and inserted into pGEX-6P-1 (in frame fusion with a glutathione-S-transferase (GST) tag) and pRSFDuet-MBP (in frame fusion with a maltose binding protein (MBP) tag), respectively, and then GST-OsCUC1 and MBP-OsCUC3 were amplified and cloned into pT N T (Promega). The recombinant GST-OsCUC1 and MBP-OsCUC3 were synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega) following the manufacturer's instructions. The recombinant GST-OsCUC1 fusion protein was immobilized on GST-Binding Resin (MagneGST Glutathione Particles, Promega) and incubated with MBP-OsCUC3 for 4 h at 4°C. After incubation, the beads were washed five times in washing buffer (4.2 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 140 mM NaCl and 10 mM KCl), and subsequently eluted using elution buffer (50 mM Tris-HCl, 50 mM glutathione). The supernatant was subjected to immunoblotting analysis with anti-MBP (TransGen, Beijing, China) and anti-GST (TransGen) antibodies.
To confirm the protein-protein interaction of OsCUC1 and CLD1, the coding sequence of CLD1 was amplified and inserted into pRSFDuet-His (in frame fusion with a His tag), and then His-CLD1 was amplified and cloned into pT N T (Promega). The recombinant His-CLD1 was synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega) following the manufacturer's instructions. The recombinant GST-OsCUC1 fusion protein was immobilized on GST-Binding Resin (MagneGST TM Glutathione Particles; Promega) and incubated with His-CLD1 for 4 h at 4°C. After incubation, the beads were washed five times in washing buffer (4.2 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 140 mM NaCl and 10 mM KCl), and subsequently eluted using elution buffer (50 mM Tris-HCl, 50 mM glutathione). The supernatant was subjected to immunoblotting analysis with anti-His (TransGen) and anti-GST (TransGen) antibodies.
The original blot images are shown in Fig. S1. Primer sequences for the constructions are listed in Table S2.

Protein extraction
For an in planta CLD1 accumulation assay, total proteins were extracted from 0.2 g of 12-d-old fresh leaves of non-KO1 OsCUC1 and oscuc1-KO1. The leaves were collected and ground in liquid nitrogen, then homogenized in protein extraction buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 4 M urea and 1 mM phenylmethylsulfonyl fluoride). Extracts were incubated at 4°C for 1 h, then centrifuged for 30 min at maximum speed. Supernatants were collected for further analysis. To obtain the nuclear-enriched fraction, samples were collected in the same quantity (0.2 g of 12-d-old fresh leaves), and the nuclear proteins were extracted as described previously (Kaufmann et al., 2010).
For the CLD1-GFP accumulation assay, to obtain total proteins, rice protoplasts were collected and treated with lysis buffer (cat. no. B7345; Sigma-Aldrich). After incubation at 4°C for 1 h, the extracts were centrifuged for 30 min at maximum speed. Supernatants were collected for further analysis. The nuclear proteins of the rice protoplasts were extracted using a Minute Cytoplasmic and Nuclear Kit (Invent Biotechnologies, Eden Prairie, MN, USA) according to the manufacturer's instructions.

Protein degradation assay
The recombinant His-CLD1 was synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega) according to the manufacturer's instructions. The equivalent amount of His-CLD1 was added to 120 ll crude total proteins (20 µg µl -1 ) extracted from indicated plants (non-KO1 OsCUC1 or oscuc1-KO1) using reaction buffer (50 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% (v/v) Tween 20, 10% (v/v) glycerol and 20 mM b-mercaptoethanol). The mixture was incubated at 25°C and sampled at indicated time points (0, 3, 6 and 9 h). The abundance of remaining His-CLD1 in these samples was detected by immunoblotting using an anti-His (TransGen) antibody. The original blot images are shown in Fig. S1.

Statistical analysis
Statistical analysis was performed by Student's t-test. P-values of < 0.05 were considered to indicate statistical significance. P-values of < 0.01 were considered to indicate statistically high significance. Statistical calculations were performed using Microsoft Excel 2010.

Results
Knocking out OsCUC1 and OsCUC3 in rice leads to defects in meristem/organ boundary specification To identify the OsCUC genes, we searched for homologs of CUC1, CUC2 and CUC3 in rice. Sequence alignment indicated that LOC_Os06g23650 (Os06g0344900) is the closest homolog of both CUC1 and CUC2, hereafter referred to as OsCUC1; LOC_Os08g40030 (Os08g0511200) shares the greatest similarity with CUC3, and we therefore named it OsCUC3. Both genes were previously uncharacterized. OsCUC1 encodes a 373 amino acid (aa) protein with a typical NAC domain; while OsCUC3 encodes a 340-aa NAC protein (Fig. S2a). Both OsCUC1 and OsCUC3 belong to the NAM subgroup of the NAC gene family Nuruzzaman et al., 2010). A phylogenetic tree analysis showed that OsCUC1 is the homologue of Arabidopsis CUC1, and it is also very similar to Arabidopsis CUC2, petunia NAM, tomato GOBLET, strawberry FveCUC2a, FveCUC2b, and FveCUC2c. OsCUC3 is the homologue of CUC3 in Arabidopsis and FvH4_5g12090 in strawberry (Fig. S2c).
To understand the biological function of OsCUC1 and OsCUC3, we employed CRISPR/Cas9 to produce two individual knockout lines for each of these two genes (Figs 1a, 2a). For OsCUC1, a 1 bp deletion for oscuc1-KO1 and a 1 bp insertion for oscuc1-KO2 (at different sites) lead to a frame-shift with premature transcription termination (Fig. S3a), while both oscuc3-KO1 and oscuc3-KO2 harbor a 1 bp insertion at different sites of the coding region resulting in a premature stop codon (Fig. S3b). In comparison with the non-KO control line, which harbored the intact OsCUC1, both homozygous oscuc1-KO lines showed multiple defects in rice development, including the fusion of leaves and filaments, decreased stamen numbers, reduced plant height and twisted-rolling leaves (Fig. 1b-p; Table S3). During the vegetative growth stage, fusion could be observed in the leaf blade (Figs 1g,h, S4a,b) as well as in the whole leaf sheath (Figs 1i-l, S4c,d) of the oscuc1-KO plants. The tube-like leaf

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New Phytologist sheath physically prevented the outgrowth of the new leaf or the panicle. During the reproductive growth stage, we also observed defects in floral development in oscuc1-KO, in which the florets displayed fusions of two or more filaments (Fig. 1m-p). This fusion in the mutants indicated the function of OsCUC1 in organ boundary specification. At the same time, most of the florets showed reduced stamen numbers (Fig. 1m-p; Table S3). An SEM assay showed that not all of the stamen primordia correctly formed in the early floral developmental stage in the oscuc1-KO plants, indicating that OsCUC1 also functions in meristem boundary specification (Fig. 1q-v). We did not obtain any seeds from any of the individual T 0 homozygous oscuc1-KO plants, while the non-KO plants displayed normal fertility. This result may be attributable to the development of aberrant stamens, which may lead to pollen defects in the mutant plants. The pollen viability test indicated that almost no pollen was produced in the KO plants (Fig. S5). Furthermore, pollen from wild-type plants (Nipponbare) was used to pollinate the gynoecia of oscuc1-KO1. In contrast with the self-pollinated florets, the cross-pollinated florets maintained normal development in later stages and yielded fertile seeds ( Fig. S6a-e). We further investigated whether these seeds could develop normally. No defects were detected in any developmental stage in these F 1 progeny (oscuc1-KO1/+). In the F 2 population, the homozygous oscuc1-KO1 plants showed the same phenotype as the T 0 transgenic plants, while the non-KO plants and heterozygous mutants developed normally (Fig. S6f,g). Both of the oscuc3-KO lines exhibited defects in meristem/organ boundary specification in the vegetative growth stage as well as in the reproductive growth stage, including the fusion of leaves and filaments, and defects in stamen identification (Fig. 2b-m ;  Table S3), which were highly similar to those of the oscuc1-KO lines. However, knocking out OsCUC3 did not lead to dwarf plant stature or twisted-rolling leaves. In addition, in contrast to the infertility of the oscuc1-KO plants, fertile seeds could be obtained from both of the oscuc3-KO lines. Similar to the oscuc1-KO1/+ plants, the heterozygous OsCUC3 mutant plants showed no defects during any developmental stages (Fig. S6h,i).
The expression profiles of OsCUC1 and OsCUC3 NAC family proteins have been shown to function as transcription factors (Xie et al., 1999;Vroemen et al., 2003). Since OsCUC1 and OsCUC3 encode NAC domain proteins, we used GFP fluorescence to determine their subcellular localization by transiently expressing OsCUC1-GFP or OsCUC3-GFP in-frame fusion proteins in rice protoplasts. The results revealed that both OsCUC1 and OsCUC3 are localized to the nucleus (Fig. 3a). In addition, to determine whether these genes have transactivation activity, OsCUC1 and OsCUC3 were fused to the GAL4 DNA binding domain and transformed into the yeast strain AH109. The yeast strains containing pBD-OsCUC1 or pBD-OsCUC3 grew in Trp-, His-and Ade-deficient SD medium while the negative control did not grow in triple-deficient SD medium (Fig. 3b), which indicated that both genes have transactivation activity. Taken together, these data indicated that OsCUC1 and OsCUC3 function as transcription factors.
We used qRT-PCR to study the expression patterns of OsCUC1 and OsCUC3 throughout plant development. OsCUC1 was highly expressed in the leaf, bract, flag leaf, and reproductive organs, especially in the stamens (Fig. 3c). In comparison to that of OsCUC1, the expression level of OsCUC3 was lower in all tested organs. However, OsCUC3 transcripts accumulated more abundantly in the vegetative organs than in the reproductive organs (Fig. 3d). To obtain the details of the expression patterns of these two genes, we carried out an in-situ hybridization assay (Fig. 3e). In the early floral developmental stage (In 5), OsCUC1 and OsCUC3 exhibited highly overlapping expression patterns, with both mRNAs accumulated mainly in the boundaries (between the leaves and between the meristems) as well as within the meristems (Fig. 3e1,e6). In early In 6 stage, OsCUC1 mRNA was strongly and specifically accumulated at the region where the stamen primordia will arise (Fig. 3e2). Afterward, the expression of OsCUC1 became diffused and uniform in the flower organs (Fig. 3e3,e4). For OsCUC3, in early In 6 stage, its mRNA was detected in the same region where OsCUC1 mRNA accumulated (Fig. 3e7); in addition, OsCUC3 was also detected in the primordia of sterile lemmas and rudimentary glumes (Fig. 3e7). In the later stages (late In 6 and In 7), in contrast to OsCUC1, OsCUC3 was mainly detected in the boundaries between the flower organs (Fig. 3e8,e9).

OsCUC1 and OsCUC3 dimerize and function in a partially redundant manner in boundary specification and SAM activity maintenance
To understand the relationship between OsCUC1 and OsCUC3, we first studied the protein-protein interaction of these two proteins. A bimolecular fluorescence complementation (BiFC) assay showed that OsCUC1 and OsCUC3 could form homodimers or heterodimers (Fig. 4a,b). An in vivo co-immunoprecipitation (Co-IP) assay and an in vitro pull-down assay further confirmed the dimerization between OsCUC1 and OsCUC3 (Fig. 4c,d). In Arabidopsis, due to the miR164 function in negatively regulating CUC1 and CUC2, the interaction between the CUC proteins cannot be detected in protoplasts by BiFC. However, we detected that the miR164-resistant versions of CUC1 and CUC2 (mCUC1 and mCUC2) formed homodimers, while mCUC1-mCUC2, mCUC1-CUC3 and mCUC2-CUC3 formed heterodimers (Fig. S7). These results were largely consistent with previous findings (Rubio-Somoza et al., 2014;Gonc ßalves et al., 2015), indicating the conserved protein-protein interaction behavior of CUC proteins in both rice and Arabidopsis.
Furthermore, we developed double mutant plants for these two genes. In contrast with the single mutant plants, which showed no defects during the early seedling stage (Fig. S6f, h), the oscuc1/+ oscuc3/oscuc3 plants exhibited leaf sheath fusion and twisted-rolling leaves; these defects were greatly enhanced in the oscuc1/oscuc1 oscuc3/oscuc3 plants (Fig. 4e-g). Although loss of function of OsCUC1 and OsCUC3 did not lead to defects in SAM initiation (Fig. S8a-

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New Phytologist plants was arrested in the early seedling stage, and they eventually died soon after that (Fig. S8e-l). Together, these results indicate that OsCUC1 and OsCUC3 dimerize and function in a partially redundant manner in boundary specification and SAM activity maintenance.

osa-miR164c is involved in organ boundary specification and leaf development by negatively regulating OsCUC1
In Arabidopsis, it has been demonstrated that miR164 negatively regulates CUC1 and CUC2 (Baker et al., 2005;Nikovics et al., 2006;Sieber et al., 2007;Raman et al., 2008). To test whether OsCUC1 is regulated by microRNA, we searched for the corresponding microRNA using OsCUC1 as a putative target in the Plant Non-coding RNA Database, (PNRD http://structuralbiol ogy.cau.edu.cn/PNRD/index.php) (Yi et al., 2014). The results indicated that OsCUC1 may be regulated by all members of the rice microRNA osa-miR164 family (osa-miR164a to osa-miR164f). These findings implied that osa-miR164 may also be involved in organ boundary specification and leaf development by negatively regulating OsCUC1. The bioinformatics analysis by PNRD indicated that OsCUC1 is considered to be the target of osa-miR164c with a high expectation value; moreover, only four genes, including OsCUC1, are predicted to be the potential targets of osa-miR164c, while the other osa-miR164 targets more genes. Thus, osa-miR164c was selected for further study. The results from RT-PCR followed by Sanger sequencing indicated that the real existence of the osa-miR164c is supported by  New Phytologist transcript evidence (Fig. S9a). Moreover, the qRT-PCR results indicated that osa-miR164c was accumulated highly in leaf tissues, but almost no expression could be detected in the mature flower organs, in which OsCUC1 is relatively highly expressed (Fig. S9b). To verify the functions of osa-miR164c in regulating boundary specification and leaf morphology, we overexpressed osa-miR164c in the genetic background of Nipponbare. In comparison to the control plants carrying the empty vector, OsCUC1 was significantly downregulated in both the vegetative and reproductive organs of OE-osa-miR164c plants (Fig. 5a-c). Similar to oscuc1-KO, the OE-osa-miR164c plants also showed defects in organ boundary separation and leaf development (Fig. 5d-h). However, unlike in oscuc1-KO, the stamens developed normally in OE-osa-miR164c, indicating that a low expression level of OsCUC1 is sufficient for maintaining normal organ separation in the reproductive growth stage but not in the vegetative growth stage. Notably, two transgenic lines with higher expression of osa-miR164c (OE-osa-miR164c-4 and OE-osa-miR164c-15) showed more severe defects than the others, and no seeds could be obtained from these two lines.

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OsCUC1 contains an osa-miR164-target sequence in the second exon. To further test the effects of osa-miR164 on OsCUC1, we generated transgenic lines carrying an OsCUC1 in-frame fusion with the b-glucuronidase (GUS) gene driven by its endogenous promoter with or without synonymous mutations in the osa-miR164-targeted site (pOsCUC1::OsCUC1-GUS and pOsCUC1::mOsCUC1-GUS) (Fig. 5i). The pOsCUC1:: mOsCUC1-GUS lines showed a relatively high level of GUS staining in most of the organs (Fig. 5j1-j5), while only minor staining was observed in the pOsCUC1::OsCUC1-GUS lines (Fig. 5j6-j10). These results strongly suggest that osa-miR164c functions by dampening the transcription level of OsCUC1. In agreement with this idea, the transcriptional reporter of OsCUC1 (pOsCUC1::GUS) showed a strong expression pattern, as expected ( Fig. 5j11-j15).
osa-miR164 targeting OMTN4 and OMTN6 may not be involved in meristem/organ boundary specification or leaf development In addition to OsCUC1, five other NAC (OMTN) genes, OMTN1 (ONAC027), OMTN2 (ONAC004/OsNAC2), OMTN3 (ONAC060), OMTN4 (ONAC011) and OMTN6 (ONAC104), are considered to be putative targets of osa-miR164 (Fang et al., 2014). We have shown that osa-miR164c plays a critical role in boundary specification and leaf development by downregulating OsCUC1. Our qRT-PCR results indicated that only OsCUC1and not the other five putative osa-miR164 targetswas significantly downregulated in all the organs tested in OE-osa-miR164c plants (Figs 5b, S10). In a previous study, OMTN4-RNAi (RNA interference) and OMTN6-RNAi transgenic plants were reported to show severe abnormal phenotypes, such as twisted-rolling leaves and fused organs, which were similar to those of the oscuc1-KO plants (Fang et al., 2014). To verify whether OMTN4 and OMTN6 function in controlling organ specification and leaf development, we knocked out these two genes using CRISPR/Cas9 in the Nipponbare genetic background. We did not observe any fused or twisted-rolling leaves for the homozygous knockout (KO) mutants of these two genes during the vegetative growth stage, and in the reproductive stage, defects in the florets were barely detected. Moreover, no significant defects could be observed from two other mutant lines with the Zhonghua11 background (omtn4 ZH11 and omtn6 ZH11 ) either ( Fig. S11; Table S4). Therefore, the defects in the two RNAi lines may be caused by RNAi off-target effects. Thus, we postulated that the targets of osa-miR164, OMTN4 and OMTN6 may not respond to meristem/organ boundary specification or leaf development as OsCUC1 does.
OsCUC1 physically interacts with CLD1 and maintains CLD1 stability in the nucleus to control leaf morphology Knocking out OsCUC1 in rice gives rise to defects in leaf development, which resemble the phenotype of the curled leaf and dwarf 1 (cld1) mutants (Li et al., 2017). However, the expression level of CLD1 showed no significant change in the KO plants (Fig. S12). Thus, we posited that OsCUC1 might interact with CLD1 and function in maintaining the stability of the CLD1 protein.
In previous research, it has been demonstrated that CLD1, also known as Semi-Rolled Leaf 1 (SRL1), encodes a glycophosphatidylinositol (GPI)-anchored membrane protein, which localizes predominantly at the plasma membrane and modulates leaf development (Xiang et al., 2012;Li et al., 2017). To verify whether CLD1 can interact with OsCUC1, we first tested whether CLD1 is located in the nucleus, as OsCUC1 is. We transiently co-expressed CLD1-GFP in-frame fusion proteins with a nuclear localization marker or membrane localization marker in rice protoplasts. Green fluorescence protein fluorescence indicated that CLD1 was located in both the membrane and the nucleus (Fig. 6a). Moreover, CLD1-GFP was detected in the nuclei-enriched fraction by immunoblotting (Fig. 6b). This result further confirmed the nuclear localization of CLD1. We then studied the protein-protein interactions between CLD1 and the NAC proteins; BiFC, yeast-two-hybrid (Y2H), in vivo Co-IP and in vitro pull-down assays revealed the interactions between CLD1 and OsCUC1 ( Fig. 6c-f). By contrast, we did not detect any interactions between CLD1 and OsCUC3 or two other NAC proteins (the putative osa-miR164 targets OMTN4 and OMTN6) by either BiFC or Y2H assays (Fig. S13). In the in vitro assay conditions, recombinant His-CLD1 was stable in the crude protein extract from non-KO1 OsCUC1 . By contrast, His-CLD1 proteins were rapidly degraded after the addition of crude protein extract from oscuc1-KO1, while the degradation of His-CLD1 was greatly slowed down when recombinant GST-OsCUC1 was first mixed with His-CLD1 (Fig. 7a). Moreover, we carried out an assay for CLD1 stability in planta. Immunoblotting assays revealed that CLD1 was not detected in the nucleus-enriched fraction from oscuc1-KO plants, indicating that the nuclear accumulation of CLD1 is dependent on OsCUC1 functioning (Fig. 7b). These results further confirmed the role of OsCUC1 in the stabilization of CLD1. Overall, the protein-protein interaction between OsCUC1 and CLD1 may stabilize CLD1 in the nucleus; loss of function of OsCUC1 may lead to acceleration of CLD1 degradation in the nucleus, resulting in a phenotype with twisted-rolling leaves, which is similar to that of the cld1 mutant.

New Phytologist
Arabidopsis are involved in SAM formation. Mutations in NAM, MtNAM and CUP lead to fusion in cotyledons, and no apical meristem can be formed. In Medicago, the development of the strong mutant of MtNAM is arrested in the fused cotyledon stage (Cheng et al., 2012). By contrast, in Petunia, the nam mutant occasionally produces escape shoots, which develop normally in the vegetative stage, but show defects in flower development (Souer et al., 1996). In Antirrhinum, the cup mutant always produces escape shoots; however, these escape shoots display defects during all developmental stages (Weir et al., 2004). In Arabidopsis, CUC1 and CUC2 function redundantly in SAM formation, and seedlings of the cuc1 cuc2 double mutant, which completely lacks a SAM, exhibit a cup-shaped cotyledon (Aida et al., 1997). Further study indicated that cuc2 OsCUC1 interacts with CLD1 in rice. (a) CLD1 is localized to the cell membrane as well as the nucleus. Upper row: co-localization of CLD1-GFP and an mCherry-tag nuclear localization marker. Upper middle row: co-localization of CLD1-GFP and an mCherry-tag membrane localization marker. Lower middle row: co-localization of green fluorescent protein (GFP) and an mCherry-tag membrane localization marker as a control. Lower row: co-localization of GFP and an mCherry-tag nuclear localization marker as a control. MLM, membrane localization marker (OsRac3-mCherry); NLM, nuclear localization marker (Ghd7-mCherry). Bars, 5 lm. (b) Accumulation of CLD1-GFP in the nucleus. Rice (Nipponbare) protoplast transiently expressed GFP or CLD-GFP. CLD1-GFP proteins were detected using anti-GFP antibodies in total proteins of the rice protoplast or in the nuclei-enriched fraction only. Histone H3 served as a nuclear marker and UGPase (UDP-glucose pyrophosphorylase) as a cytoplasmic marker. (c) BiFC assay. Co-expression of GFP C plus GFP N -CLD1, GFP C -CLD1 plus GFP N , GFP C -OsCUC1 plus GFP N , and GFP C plus GFP N -OsCUC1 were used as negative controls. Bars, 5 lm. (d) Y2H assay. The combination of BK-53 plus AD-T was used as a positive control, while BK-CLD1 plus AD, BK-LAM plus AD-T, and BK plus AD were used as negative controls. (e) In vivo co-immunoprecipitation (CoIP) assay. After the co-transformation of OsCUC1-GFP and CLD1-Flag in rice protoplasts, total proteins of protoplasts were immunoprecipitated using an anti-GFP antibody and were detected with anti-GFP and anti-Flag antibodies. IB, immunoblot; IP, immunoprecipitation. (f) In vitro pull-down assay. Recombinant GST-OsCUC1 and His-CLD1 proteins were used for the pull-down assay. IB, immunoblot.
Ó 2020 The Authors New Phytologist Ó 2020 New Phytologist Foundation New Phytologist (2021) 229: 1566-1581 www.newphytologist.com cuc3 double mutant seedlings, but not cuc1 cuc3 seedlings, have no functional SAM. Thus, only cuc2 in combination with cuc1 and/or cuc3 leads to the absence of the SAM (Vroemen et al., 2003). In our study, we found that the oscuc1 oscuc3 double mutant produces aberrant organs at the seedling stage, and the development of the double mutant seedling is arrested. These results indicated that, in addition to OsCUC1 and OsCUC3, other factor(s) may be involved in the regulation of SAM initiation, and loss of function of these two genes alone is not sufficient to prevent SAM formation. However, OsCUC1 and OsCUC3 are critical for maintaining normal SAM activity. Plants without functional OsCUC1 and OsCUC3 products stop developing and ultimately die at the seedling stage.

Functional divergence between OsCUC1 and OsCUC3
Although OsCUC1 and OsCUC3 function together in boundary specification and SAM maintenance, their functions remain different in other developmental processes. Loss of function of OsCUC1 leads to a dwarf plant structure, pollen defection and twisted-rolling leaves, while oscuc3 mutants show no defects in these related developmental processes. Our study indicates that only OsCUC1but not OsCUC3 or the other two NAC proteins (OMTN4 and OMTN6)interacts with the leaf-rolling related protein CLD1. These results are consistent with the fact that oscuc1 mutants but not oscuc3 or omtn4/6 mutants show defects in leaf development. Thus, the different interaction behaviors of OsCUC1 and OsCUC3 with the other gene may be partly responsible for the functional divergence of these two proteins.
Despite divergence, the mechanisms of meristem/organ boundary specification between Arabidopsis and rice are conserved In Arabidopsis, due to the redundant function of CUC1 and CUC2, neither the cuc1 nor the cuc2 single mutation results in a severe phenotype, but the cuc1 cuc2 double mutation causes a complete lack of embryonic shoot meristem formation. The regenerated cuc1 cuc2 mutant exhibits abnormal flowers, in which all the sepals and most of the stamens are severely fused (Aida et al., 1997(Aida et al., , 1999Takada et al., 2001). In rice, the oscuc1 single mutation causes severe defects in meristem/organ boundary specification, resulting in a reduction in stamen numbers and fusion of leaves/filaments. Consistent with this, systematic sequence analysis revealed that OsCUC1 is the only homolog of both CUC1 and CUC2 (Kondou et al., 2009), suggesting that the function of OsCUC1 may be executed redundantly by CUC1 and CUC2 in Arabidopsis. Despite the divergence between OsCUC1 and CUC1/2, the mechanisms of meristem/organ boundary specification are conserved between the dicot Arabidopsis and the monocot O. sativa (Fig. 8): one or more microRNA (osa-miR164/miR164) target(s) (OsCUC1/CUC1&CUC2) function redundantly with another NAC gene family member (OsCUC3/ CUC3) in meristem/organ boundary specification. In addition, OsCUC1 is involved in leaf development by affecting the stability of CLD1 in the nucleus. In vitro protein degradation assay. Recombinant His-CLD1 protein was incubated with total crude extract from non-KO1 OsCUC1 (left) and oscuc1-KO1 (middle, without GST-OsCUC1; right, with GST-OsCUC1). HSP (Hsp90) was used as a loading control. (b) The accumulation of CLD1 in the nucleus depends on OsCUC1 functioning; accumulation of CLD1 in non-KO1 OsCUC1 and oscuc1-KO1 plants is shown. CLD1 was detected with an anti-CLD1 antibody. Histone H3 served as a nuclear marker, and UGPase (UDP-glucose pyrophosphorylase) served as a cytoplasmic marker. HSP (Hsp90) was used as a loading control. Three independent plants of each line were used for the assay.
New Phytologist (2021) 229: 1566-1581 Ó 2020 The Authors New Phytologist Ó 2020 New Phytologist Foundation www.newphytologist.com Research New Phytologist Fig. 8 Interplay of a microRNA and CUP-SHAPED COTYLEDON genes controls meristem/organ boundary specification in rice and Arabidopsis. The microRNA (osa-miR164/miR164) negatively regulates the CUP-SHAPED COTYLEDON genes (OsCUC1/CUC1&CUC2), which function redundantly with another NAC gene family member (OsCUC3/CUC3) in meristem/organ boundary specification in both rice and Arabidopsis. In addition, OsCUC1 controls plant height, male fertility and leaf morphology in rice. Red represents Arabidopsis, and green represents rice.

Supporting Information
Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

Table S1
The accession numbers of the proteins listed in the phylogenetic tree.

Table S2
The primer sequences used in this study.  Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.
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