Mol. Cells 2019; 42(12): 858-868
Published online November 27, 2019
https://doi.org/10.14348/molcells.2019.0141
© The Korean Society for Molecular and Cellular Biology
Correspondence to : genean@khu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Shoot branching is an essential agronomic trait that impacts on plant architecture and yield. Shoot branching is determined by two independent steps: axillary meristem formation and axillary bud outgrowth. Although several genes and regulatory mechanism have been studied with respect to shoot branching, the roles of chromatin-remodeling factors in the developmental process have not been reported in rice. We previously identified a chromatin-remodeling factor OsVIL2 that controls the trimethylation of histone H3 lysine 27 (H3K27me3) at target genes. In this study, we report that loss-of-function mutants in
Keywords bud outgrowth, chromatin modification, rice
Shoot branching is one of the critical agronomic traits, contributing to biomass and grain yield in crops (Tian and Jiao, 2015). Rice tillers are specialized shoot branches that are formed on the unelongated basal internode and grow independently from the main culm (Li et al., 2003). Rice tillering affects panicle development such as the number of primary and secondary branches, so that branching determines grain number per panicle (Wang and Li, 2011).
Tillers arise from the axillary bud apical meristem (AM), which develops in the axils of leaves during the vegetative stage in rice (Wang and Li, 2011). The development of tillers undergoes two processes: formation of the axillary bud and outgrowth of the axillary (tiller) bud (Wang and Li, 2011). At Stage 1, the AM consists of cytoplasm-dense cells which form as a bulge in the axil of the leaf primordium. The bulge develops into a cone-like structure at Stage 2. The dome and prophylls become gradually more evident at Stage 3, and the axillary bud is formed at Stage 4 (Tanaka et al., 2015).
In rice,
Not all of the axillary buds develop immediately into tillers and some of them stay dormant until maturity. Final tiller number depends mainly on the capability of axillary bud outgrowth (Wai and An, 2017; Wang and Li, 2011), which is affected by various environmental factors and hormones (Deshpande et al., 2015).
Auxins and cytokinins influence the outgrowth of tiller buds (Wai and An, 2017). Auxins are synthesized at the shoot apex and transported by the polar auxin transport system. In rice, a polar auxin transporter, OsPIN1, is essential for auxin-dependent tiller bud emergence. Reduction of
Strigolactone (SL), a carotenoid phytohormone, suppresses axillary bud outgrowth (Wang et al., 2013). Rice mutants defective in the biosynthesis or signaling of SLs display increased branching phenotypes (Wai and An, 2017; Zhang et al., 2010; Zhao et al., 2014; Zou et al., 2006).
Several transcription factors control outgrowth of axillary buds. Rice
Although many of the chromatin-remodeling factors are found to control plant development such as flowering time and panicle architecture in rice, their roles in shoot branching have not been studied. Polycomb repressive complex 2 (PRC2), which represses target gene expression by regulating the trimethylation of histone 3 lysine 27 (H3K27me3) of the chromatin of the target gene, plays essential roles as an epigenetic repressor (Mozgova and Henning, 2015). The rice VERNALIZATION INSENSITIVE 3-like (VIL3) protein OsVIL2 associates with the PRC2 complex to suppress target gene expression by mediating H3K27me3 (Yang et al., 2013; 2019). The
We previously reported the T-DNA null mutants,
The cDNAs were synthesized from 2 μg of total RNA isolated from basal parts of the shoots using RNAiso Plus (Takara, Japan). Moloney murine leukemia virus reverse transcriptase (Promega, USA), RNasin Ribonuclease Inhibitor (Promega), 10 ng of oligo (dTs) and 2.5 mM deoxiribonucleotide triphosphates were included in the reaction mixture. Quantitative RT-PCR (RT-qPCR) was performed with a Rotor-Gene 6000 (Corbett Research, Australia) using SYBR Green I Prime Q-Mastermix (GeNet Bio, Korea) as described previously (Cho et al., 2016; 2018b). The internal control was
Basal parts of the seedlings were fixed in a formaldehyde-acetic acid-alcohol solution at 4°C. Samples were dehydrated with an ethanol series, treated with a tert-butyl alcohol and infiltrated with paraffin. After embedding, samples were cut into 10-μm thick slices with a microtome (Leica Microsystems, Germany). Slices were stained with toluidine blue and observed under a BX61 microscope (Olympus, Japan) as previously reported (Yoon et al., 2017). For GUS staining, samples were incubated in a GUS staining solution containing and 0.1% X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, cyclohexylammonium salt), 5% methanol, 2% dimethyl sulfoxide (DMSO), 100 mM sodium phosphate (pH 7.0), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.5% Triton X-100, and 10 mM EDTA (pH 8.0) (Yoon et al., 2017). Chlorophylls were removed from the stained samples by immersion in 70% ethanol for 2 h and in 95% ethanol overnight at 65°C.
To prepare the RNA probe, a gene-specific fragment was amplified by PCR and cloned in pBluescript II. Digoxigenin (DIG)-labeled sense and antisense RNA probes were prepared as previously reported (Lee and An, 2012; Lee et al., 2007; Yoon et al., 2014). RNA
Basal parts of at least 10 plants at 28 days after germination (DAG) were harvested for the assay. ChIP was performed as previously reported (Haring et al., 2007). For cross-linking, 1-g samples were incubated with 3% formaldehyde solution. After nuclei were isolated, chromatin was sheared to approximately 500- to 1,000-bp length by sonication. Anti-trimethyl-histone H3 (Lys27) monoclonal antibody was used for immunoprecipitation (07-449; Millipore, USA) (Yang et al., 2019). For normalization, we used the fold enrichment method in which the values obtained from the antibody reaction were divided by values from no-antibody controls (Haring et al., 2007). The primers for this analysis are listed in Supplementary Table 2.
Plasmid vector for
The data were analyzed by one-way ANOVA, with pairwise multiple comparison tests carried out by the Tukey honestly significant difference test to compare samples, using the R program (Cohen and Cohen, 2008).
To determine whether tiller development is dependent on flowering time in rice, we studied tiller phenotypes of various flowering mutants that we had generated in
Neither
Axillary tillers are produced by two steps. The first involves formation of axillary buds and the second involves outgrowth of the axillary buds (Wai et al., 2017). To investigate whether the phenotype of fewer tillers in
Using transgenic plants expressing the
To investigate the genes that were affected by
We investigated the expression patterns of genes involved in tiller development in the basal parts of culm tissues at 28 and 30 DAG, at which time point axillary tillers had developed in WT but not in the
We then studied the genes controlling axillary bud outgrowth. Tiller bud outgrowth is controlled by hormones such as auxins, cytokinins, and SLs. Expression levels of auxin-related genes
We also analyzed expression levels of transcription factors that control axillary bud outgrowth. Expression of a MADS-box gene
To study the expression patterns of
Previous studies suggest that OsVIL2 interacts with PRC2 to repress target chromatins by regulating H3K27me3. The analysis of expression patterns of tiller outgrowth genes revealed that
To confirm that OsVIL2 directly binds to
To obtain a genetic relationship between
At heading stage in the paddy field,
In this study, we provided evidence that OsVIL2 promotes tiller outgrowth by suppressing expression of
We observed that the expression level of
The PRC2 complex plays important roles in the development of plants and animals by suppressing target gene expression through histone modification (Jeong et al., 2015). OsVIL2 is highly homologous to
We showed that new tillers were developed during the vegetative phase and that tiller development stopped at the floral transition stage. In most late-flowering mutants that have a longer vegetative period than the WT, tiller numbers were higher than in the WT, due probably to increased vegetative growth. It is also possible that the floral transition signal directly inhibits a major regulatory gene that controls tiller bud growth. Alternatively, floral signals could affect biosynthesis of or signaling by the hormones that control tiller development. Interestingly, the
This work was supported in part by a grant from the Next Generation BioGreen 21 Program (Plant Molecular Breeding Center; No. PJ013210), Rural Development Administration, Republic of Korea to GA and by the Republic of Korea Basic Research Promotion Fund to JY (grant No. NRF-2018R1A6A3A11047894).
Mol. Cells 2019; 42(12): 858-868
Published online December 31, 2019 https://doi.org/10.14348/molcells.2019.0141
Copyright © The Korean Society for Molecular and Cellular Biology.
Jinmi Yoon1, Lae-Hyeon Cho1,2, Sichul Lee3, Richa Pasriga1, Win Tun1, Jungil Yang1, Hyeryung Yoon1, Hee Joong Jeong1, Jong-Seong Jeon1, and Gynheung An1,*
1Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 17104, Korea, 2Department of Plant Bioscience, Pusan National University, Miryang 50463, Korea, 3Center for Plant Aging Research, Institute for Basic Science, Daegu 42988, Korea
Correspondence to:genean@khu.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Shoot branching is an essential agronomic trait that impacts on plant architecture and yield. Shoot branching is determined by two independent steps: axillary meristem formation and axillary bud outgrowth. Although several genes and regulatory mechanism have been studied with respect to shoot branching, the roles of chromatin-remodeling factors in the developmental process have not been reported in rice. We previously identified a chromatin-remodeling factor OsVIL2 that controls the trimethylation of histone H3 lysine 27 (H3K27me3) at target genes. In this study, we report that loss-of-function mutants in
Keywords: bud outgrowth, chromatin modification, rice
Shoot branching is one of the critical agronomic traits, contributing to biomass and grain yield in crops (Tian and Jiao, 2015). Rice tillers are specialized shoot branches that are formed on the unelongated basal internode and grow independently from the main culm (Li et al., 2003). Rice tillering affects panicle development such as the number of primary and secondary branches, so that branching determines grain number per panicle (Wang and Li, 2011).
Tillers arise from the axillary bud apical meristem (AM), which develops in the axils of leaves during the vegetative stage in rice (Wang and Li, 2011). The development of tillers undergoes two processes: formation of the axillary bud and outgrowth of the axillary (tiller) bud (Wang and Li, 2011). At Stage 1, the AM consists of cytoplasm-dense cells which form as a bulge in the axil of the leaf primordium. The bulge develops into a cone-like structure at Stage 2. The dome and prophylls become gradually more evident at Stage 3, and the axillary bud is formed at Stage 4 (Tanaka et al., 2015).
In rice,
Not all of the axillary buds develop immediately into tillers and some of them stay dormant until maturity. Final tiller number depends mainly on the capability of axillary bud outgrowth (Wai and An, 2017; Wang and Li, 2011), which is affected by various environmental factors and hormones (Deshpande et al., 2015).
Auxins and cytokinins influence the outgrowth of tiller buds (Wai and An, 2017). Auxins are synthesized at the shoot apex and transported by the polar auxin transport system. In rice, a polar auxin transporter, OsPIN1, is essential for auxin-dependent tiller bud emergence. Reduction of
Strigolactone (SL), a carotenoid phytohormone, suppresses axillary bud outgrowth (Wang et al., 2013). Rice mutants defective in the biosynthesis or signaling of SLs display increased branching phenotypes (Wai and An, 2017; Zhang et al., 2010; Zhao et al., 2014; Zou et al., 2006).
Several transcription factors control outgrowth of axillary buds. Rice
Although many of the chromatin-remodeling factors are found to control plant development such as flowering time and panicle architecture in rice, their roles in shoot branching have not been studied. Polycomb repressive complex 2 (PRC2), which represses target gene expression by regulating the trimethylation of histone 3 lysine 27 (H3K27me3) of the chromatin of the target gene, plays essential roles as an epigenetic repressor (Mozgova and Henning, 2015). The rice VERNALIZATION INSENSITIVE 3-like (VIL3) protein OsVIL2 associates with the PRC2 complex to suppress target gene expression by mediating H3K27me3 (Yang et al., 2013; 2019). The
We previously reported the T-DNA null mutants,
The cDNAs were synthesized from 2 μg of total RNA isolated from basal parts of the shoots using RNAiso Plus (Takara, Japan). Moloney murine leukemia virus reverse transcriptase (Promega, USA), RNasin Ribonuclease Inhibitor (Promega), 10 ng of oligo (dTs) and 2.5 mM deoxiribonucleotide triphosphates were included in the reaction mixture. Quantitative RT-PCR (RT-qPCR) was performed with a Rotor-Gene 6000 (Corbett Research, Australia) using SYBR Green I Prime Q-Mastermix (GeNet Bio, Korea) as described previously (Cho et al., 2016; 2018b). The internal control was
Basal parts of the seedlings were fixed in a formaldehyde-acetic acid-alcohol solution at 4°C. Samples were dehydrated with an ethanol series, treated with a tert-butyl alcohol and infiltrated with paraffin. After embedding, samples were cut into 10-μm thick slices with a microtome (Leica Microsystems, Germany). Slices were stained with toluidine blue and observed under a BX61 microscope (Olympus, Japan) as previously reported (Yoon et al., 2017). For GUS staining, samples were incubated in a GUS staining solution containing and 0.1% X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, cyclohexylammonium salt), 5% methanol, 2% dimethyl sulfoxide (DMSO), 100 mM sodium phosphate (pH 7.0), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.5% Triton X-100, and 10 mM EDTA (pH 8.0) (Yoon et al., 2017). Chlorophylls were removed from the stained samples by immersion in 70% ethanol for 2 h and in 95% ethanol overnight at 65°C.
To prepare the RNA probe, a gene-specific fragment was amplified by PCR and cloned in pBluescript II. Digoxigenin (DIG)-labeled sense and antisense RNA probes were prepared as previously reported (Lee and An, 2012; Lee et al., 2007; Yoon et al., 2014). RNA
Basal parts of at least 10 plants at 28 days after germination (DAG) were harvested for the assay. ChIP was performed as previously reported (Haring et al., 2007). For cross-linking, 1-g samples were incubated with 3% formaldehyde solution. After nuclei were isolated, chromatin was sheared to approximately 500- to 1,000-bp length by sonication. Anti-trimethyl-histone H3 (Lys27) monoclonal antibody was used for immunoprecipitation (07-449; Millipore, USA) (Yang et al., 2019). For normalization, we used the fold enrichment method in which the values obtained from the antibody reaction were divided by values from no-antibody controls (Haring et al., 2007). The primers for this analysis are listed in Supplementary Table 2.
Plasmid vector for
The data were analyzed by one-way ANOVA, with pairwise multiple comparison tests carried out by the Tukey honestly significant difference test to compare samples, using the R program (Cohen and Cohen, 2008).
To determine whether tiller development is dependent on flowering time in rice, we studied tiller phenotypes of various flowering mutants that we had generated in
Neither
Axillary tillers are produced by two steps. The first involves formation of axillary buds and the second involves outgrowth of the axillary buds (Wai et al., 2017). To investigate whether the phenotype of fewer tillers in
Using transgenic plants expressing the
To investigate the genes that were affected by
We investigated the expression patterns of genes involved in tiller development in the basal parts of culm tissues at 28 and 30 DAG, at which time point axillary tillers had developed in WT but not in the
We then studied the genes controlling axillary bud outgrowth. Tiller bud outgrowth is controlled by hormones such as auxins, cytokinins, and SLs. Expression levels of auxin-related genes
We also analyzed expression levels of transcription factors that control axillary bud outgrowth. Expression of a MADS-box gene
To study the expression patterns of
Previous studies suggest that OsVIL2 interacts with PRC2 to repress target chromatins by regulating H3K27me3. The analysis of expression patterns of tiller outgrowth genes revealed that
To confirm that OsVIL2 directly binds to
To obtain a genetic relationship between
At heading stage in the paddy field,
In this study, we provided evidence that OsVIL2 promotes tiller outgrowth by suppressing expression of
We observed that the expression level of
The PRC2 complex plays important roles in the development of plants and animals by suppressing target gene expression through histone modification (Jeong et al., 2015). OsVIL2 is highly homologous to
We showed that new tillers were developed during the vegetative phase and that tiller development stopped at the floral transition stage. In most late-flowering mutants that have a longer vegetative period than the WT, tiller numbers were higher than in the WT, due probably to increased vegetative growth. It is also possible that the floral transition signal directly inhibits a major regulatory gene that controls tiller bud growth. Alternatively, floral signals could affect biosynthesis of or signaling by the hormones that control tiller development. Interestingly, the
This work was supported in part by a grant from the Next Generation BioGreen 21 Program (Plant Molecular Breeding Center; No. PJ013210), Rural Development Administration, Republic of Korea to GA and by the Republic of Korea Basic Research Promotion Fund to JY (grant No. NRF-2018R1A6A3A11047894).
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