Genome-wide analysis of microRNA156 and its targets, the genes encoding SQUAMOSA promoter-binding protein-like (SPL) transcription factors, in the grass family Poaceae

Genome-wide analysis of microRNA156 and its targets, the genes encoding SQUAMOSA promoter-binding protein-like (SPL) transcription factors, in the grass family Poaceae 禾本科microRNA156及其靶基因SPL转录因子的全基因组分析 1 first-author 2 1 corresp Jianhong XU, jhxu@zju.edu.cn Jianhong XU, jhxu@zju.edu.cn jhxu@zju.edu.cn https://orcid.org/0000-0002-5620-0410 Institute of Crop Science, Zhejiang Key Laboratory of Crop Germplasm, Zhejiang University, Hangzhou310058, China Institute of Crop Science, Zhejiang Key Laboratory of Crop Germplasm, Zhejiang University, Hangzhou310058, China Henan Agricultural Radio and Television School, Zhengzhou450008, China Henan Agricultural Radio and Television School, Zhengzhou450008, China MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play an important role in post-transcriptional gene regulation in plants and animals by targeting messenger RNAs (mRNAs) for cleavage or repressing translation of specific mRNAs. The first miRNA identified in plants, miRNA156 (miR156), targets the SQUAMOSA promoter-binding protein-like (SPL) transcription factors, which play critical roles in plant phase transition, flower and plant architecture, and fruit development. We identified multiple copies of MIR156 and SPL in the rice, Brachypodium , sorghum, maize, and foxtail millet genomes. Sequence and chromosomal synteny analysis showed that both MIR156 s and SPL s are conserved across species in the grass family. Analysis of expression data of the SPL s in eleven juvenile and adult rice tissues revealed that four non-miR156-targeted genes were highly expressed and three miR156-targeted genes were only slightly expressed in all tissues/developmental stages. The remaining SPL s were highly expressed in the juvenile stage, but their expression was lower in the adult stage. It has been proposed that under strong selective pressure, non-miR156-targeted mRNA may be able to re-structure to form a miRNA-responsive element. In our analysis, some non-miR156-targeted SPL s ( SPL5 / 8 / 10 ) had gene structure and gene expression patterns similar to those of miR156-targeted genes, suggesting that they could diversify into miR156-targeted genes. DNA methylation profiles of SPL s and MIR156 s in different rice tissues showed diverse methylation patterns, and hypomethylation of non-CG sites was observed in rice endosperm. Our findings suggested that MIR156 s and SPL s had different origination and evolutionary mechanisms: the SPL s appear to have resulted from vertical evolution, whereas MIR156 s appear to have resulted from strong evolutionary selection on mature sequences. 目的 2 为了探究microRNA156（miR156）及其靶基因 SPL 两者在禾本科基因组中拷贝数以及它们在植物基因组中的进化选择关系。 创新点 2 在5个已完成全基因组测序的禾本科作物中鉴定miR156及其靶基因 SPL 的拷贝数，利用比较基因组学和分子进化方法并结合DNA甲基化组数据阐明miR156及其靶基因 SPL 的起源和进化机制。 方法 2 基于全基因测序序列鉴定 SPL 基因和 MIR156 基因及其成熟序列在基因组中的拷贝数，利用MEGA X软件构建起进化树；下载水稻不同组织中 SPL 和 MIR156 基因的转录组和DNA甲基化组数据，并根据软件与程序检测其表达量与DNA甲基化水平。 结论 2 MIR156 和 SPL 具有不同的起源和进化机制， SPL 似乎是由垂直进化产生的，而 MIR156 似乎是对成熟序列的强烈进化选择产生的。 MicroRNA156 (miR156) SQUAMOSA promoter-binding protein-like ( SPL ) gene DNA methylation Gene expression Grass genome MicroRNA156 SPL 基因 DNA甲基化 基因表达 禾本科基因组 1 Introduction 1 MicroRNAs (miRNAs) are short non-coding RNAs that range in length from 20 to 24 nucleotides (nt) and are derived from single-stranded precursors with stable hairpin (HP) structures ( Bartel, 2004 Bartel, 2004 ; Voinnet, 2009 Voinnet, 2009 ). In plants, miRNAs are highly complementary to their targets, and their binding can cause cleavage of the target ( Reinhart et al., 2002 Reinhart et al., 2002 ). miRNAs have been identified in nearly all eukaryotes and act in concert with transcription factors to achieve a dynamic equilibrium of gene expression and to allow adaptation to environmental stressors ( Zhang et al., 2006 Zhang et al., 2006 ; Martin et al., 2010 Martin et al., 2010 ; Sollome et al., 2016 Sollome et al., 2016 ). miRNA156 (miR156) was originally identified in Arabidopsis thaliana because of its striking similarity to the corresponding animal miRNA ( Reinhart et al., 2002 Reinhart et al., 2002 ). miR156 is relatively conserved in higher plants ( Willmann and Poethig, 2007 Willmann and Poethig, 2007 ) and regulates the expression of more than half of the SQUAMOSA promoter-binding protein-like ( SPL ) genes in rice ( Oryza sativa ) and Arabidopsis ( Xie et al., 2006 Xie et al., 2006 ; Guo et al., 2008 Guo et al., 2008 ; Miao et al., 2019 Miao et al., 2019 ). SPLs are plant-specific transcription factors that are characterized by a highly conserved SQUAMOSA promoter-binding protein (SBP) domain which binds to a specific cis -element glycidyltrimethylammonium chloride (GTAC)-binding domain ( Birkenbihl et al., 2005 Birkenbihl et al., 2005 ; Kropat et al., 2005 Kropat et al., 2005 ). They have essential roles in plant phase transition ( Fornara and Coupland, 2009 Fornara and Coupland, 2009 ; Huijser and Schmid, 2011 Huijser and Schmid, 2011 ), flower and fruit development ( Manning et al., 2006 Manning et al., 2006 ; Lal et al., 2011 Lal et al., 2011 ; Yang et al., 2019 Yang et al., 2019 ), plant architecture ( Jiao et al., 2010 Jiao et al., 2010 ; Miura et al., 2010 Miura et al., 2010 ), trichome distribution ( Yu et al., 2010 Yu et al., 2010 ), anthocyanin biosynthesis ( Gou et al., 2011 Gou et al., 2011 ), gibberellin signaling ( Zhang et al., 2007 Zhang et al., 2007 ), shade avoidance ( Xie et al., 2017 Xie et al., 2017 ), antiviral and disease defense ( Wang et al., 2018 Wang et al., 2018 ; Liu et al., 2019 Liu et al., 2019 ; Yao et al., 2019 Yao et al., 2019 ), and the copper response ( Kropat et al., 2005 Kropat et al., 2005 ; Gielen et al., 2016 Gielen et al., 2016 ; Yan et al., 2017 Yan et al., 2017 ; Ramamurthy et al., 2018 Ramamurthy et al., 2018 ). The 17 and 19 SPL s identified in Arabidopsis and rice, respectively, fall into several subgroups according to the amino acid sequences of their SBP domains and the exon-intron structure of their encoding genes ( Guo et al., 2005 Guo et al., 2005 ; Xie et al., 2006 Xie et al., 2006 ). Although SPL genes have been systematically studied in Arabidopsis and rice, there have been few studies in other grass species. The grass family Poaceae is a large and ubiquitous family of monocots, which includes species that are considered to be the most important crops for human nutrition and economies. With the advent of large-scale sequencing technologies, whole-genome sequences are now available for rice (International Rice Genome Sequencing Project and Sasaki, 2005), Brachypodium distachyon ( The International Brachypodium Initiative, 2010 The International Brachypodium Initiative, 2010 ), foxtail millet ( Setaria italica ) ( Bennetzen et al., 2012 Bennetzen et al., 2012 ; Zhang et al., 2012 Zhang et al., 2012 ), sorghum ( Sorghum bicolor ) ( Paterson et al., 2009 Paterson et al., 2009 ), and maize ( Zea mays ) ( Schnable et al., 2009 Schnable et al., 2009 ), which we used to analyze the copy number, genomic location, and synteny of MIR156 s and SPL s in the grass family. One possible alternative regulatory mechanism may be epigenomic modifications, including DNA methylation and histone modifications ( Feng et al., 2010 Feng et al., 2010 ; Xu et al., 2020 Xu et al., 2020 ). In plants, DNA methylation tends to occur at cytosine bases in the context of CG, CHG, and CHH (H refers to A, T, or C) ( Law and Jacobsen, 2010 Law and Jacobsen, 2010 ; Teixeira and Colot, 2010 Teixeira and Colot, 2010 ). In this study, we analyzed available methylation data for SPL s and MIR156 s in four rice tissues. Our systematic analysis of MIR156 s and SPL s in grasses provides insight into the conservation and evolution of these genes in plants and the complementary roles of miRNAs and DNA methylation in regulating gene expression. 2 Materials and methods 1 2.1 Sequence retrieval 2 The following databases were used to retrieve sequences for Arabidopsis (TAIR 9; http://www.arabidopsis.org http://www.arabidopsis.org ), rice ( http://rice.plantbiology.msu.edu http://rice.plantbiology.msu.edu (release 7) and http://www.phytozome.net/rice http://www.phytozome.net/rice ), Brachypodium ( http://www.brachypodium.org http://www.brachypodium.org and http://www.phytozome.net/brachypodium http://www.phytozome.net/brachypodium (JGI v1.2)), foxtail millet ( http://foxtailmillet.genomics.org.cn/page/species/index.jsp http://foxtailmillet.genomics.org.cn/page/species/index.jsp and http://www.phytozome.net/foxtailmillet http://www.phytozome.net/foxtailmillet (JGI v2.1)), sorghum ( http://www.phytozome.net/sorghum http://www.phytozome.net/sorghum (JGI v1.0)), and maize ( http://www.maizegdb.org http://www.maizegdb.org , http://www.maizesequence.org http://www.maizesequence.org (AGP v2), and http://www.phytozome.net/maize http://www.phytozome.net/maize ). 2.2 Identification of miR156 members in grass genomes 2 Precursor and mature miR156 sequences from grasses were retrieved from the miRBase database ( http://www.mirbase.org http://www.mirbase.org (release 22.1)) ( Kozomara and Griffiths-Jones, 2011 Kozomara and Griffiths-Jones, 2011 ). The reverse-complement of RNA sequences was determined by a custom Perl script when necessary. To identify all putative miR156s in grass genomes, BlastN with an E -value cutoff of 1×10 -3 was conducted. The upstream 20 nt and downstream 200 nt of the putative miR156 genes were also extracted for secondary structure analysis. The HP structure was computationally predicted with the Mfold package ( http://www.bioinfo.rpi.edu/applications/mfold http://www.bioinfo.rpi.edu/applications/mfold ) with manual modifications ( Zuker, 2003 Zuker, 2003 ). Potential miR156s that originated from intergenic and intronic loci were retained for further analysis. For the prediction of new miR156s, filtering criteria were similar to those of Meyers et al. (2008) Meyers et al. (2008) , as follows: (1) mature sequences of miR156 reside in the stem arm of the stem-loop structure; (2) no more than five unpaired nucleotides between the miRNA and miRNA * ; (3) no more than three consecutive unpaired nucleotides; (4) folding free energy should be lower than -25 kcal/mol; and (5) homologs of miR156s should be contained in related monocot plants. 2.3 Identification of SPL s in grass genomes 2 Nineteen O. sativa SPL ( OsSPL ) sequences were used as references to search for related orthologous SPL copies in Brachypodium , foxtail millet, sorghum, and maize ( Xie et al., 2006 Xie et al., 2006 ). BlastX with an E -value cutoff of 1×10 -3 was performed against respective grass genomes to locate the SPL s, and was combined with an HMMER ( http://hmmer.janelia.org http://hmmer.janelia.org ) search (Pfam: PF03110) to find all possible SBPs. Search results were manually modified to reduce the number of false positives. 2.4 miR156 target prediction 2 The potential targets of miR156 were predicted by the psRNATarget ( http://plantgrn.noble.org/psRNATarget http://plantgrn.noble.org/psRNATarget ( Dai and Zhao, 2011 Dai and Zhao, 2011 ), and TAPIR ( http://bioinformatics.psb.ugent.be/webtools/tapir http://bioinformatics.psb.ugent.be/webtools/tapir ( Bonnet et al., 2010) Bonnet et al., 2010) ) programs with default parameters. Unique mature miR156 sequences were chosen to predict their targets in rice, Brachypodium , sorghum, maize, and foxtail millet. To gain high-confidence predictions, no more than three mismatches were allowed between miR156 and its targets. A G:U wobble was regarded as a 0.5 mismatch, and no mismatches were allowed at positions 10 and 11 (from the 5' end of the sequence), which are the canonical cleavage sites in plants ( Franco-Zorrilla et al., 2007 Franco-Zorrilla et al., 2007 ). 2.5 Phylogenetic analysis of SPL s and MIR156 s 2 Because neither the gene sequences nor the protein sequences of SPLs could be aligned exactly, only the amino acid sequences of the conserved SBP domains were used for phylogenetic analysis. SBP sequences (119) were extracted from Arabidopsis, rice, Brachypodium , foxtail millet, sorghum, and maize, and were aligned with the MUSCLE algorithm with manual modifications ( Edgar, 2004 Edgar, 2004 ). Evolutionary histories were inferred using the neighbor-joining method. A bootstrap test with 1000 replicates was used to build the tree, and the evolutionary distances were computed using the Poisson correction method and were expressed as the number of amino acid substitutions per site in MEGA X ( https://www.megasoftware.net https://www.megasoftware.net ) ( Kumar et al., 2018 Kumar et al., 2018 ). To construct the phylogenetic tree of MIR156 s, stem-loop sequences were aligned by Clustal W with manual modifications ( Larkin et al., 2007 Larkin et al., 2007 ). MEGA X ( Kumar et al., 2018 Kumar et al., 2018 ) was then used to construct a maximum-likelihood phylogenetic tree based on the Kimura-2-parameter model and a bootstrap test with 1000 replicates ( Kimura, 1980 Kimura, 1980 ). 2.6 Expression profile of SPL s in rice 2 To obtain the hierarchical clustering of OsSPL expression profiles among diverse tissues, expression data for OsSPL s were obtained from the Gene Expression Omnibus (GEO) ( Barrett et al., 2011 Barrett et al., 2011 ) database under accession numbers GSE19602 and GSE19824, and RNAseq data were obtained from the Rice Genome Annotation Project ( http://rice.plantbiology.msu.edu/index.shtml http://rice.plantbiology.msu.edu/index.shtml ). The data were transformed into a matrix to satisfy the condition of MultiExperiment Viewer ( http://mev.tm4.org http://mev.tm4.org ) ( Saeed et al., 2006 Saeed et al., 2006 ) and log 2 -transformed for better visualization. Hierarchical clustering of the genes was assessed using a Euclidean distance metric in MultiExperiment Viewer. 2.7 DNA methylation patterns of SPL s and MIR156 s in rice 2 To examine methylation of MIR156 s and OsSPL s in rice, we retrieved sequences covering a region 2 kb upstream from the start codon, the gene body, and the 3'-untranslated region (3'-UTR) of OsSPL s, and about 900 bp upstream and downstream of O. sativa-MIR156 ( osa-MIR156 ) sequences (2 kb in total including the stem-loop sequence) from genome databases. Bisulfite sequencing data were downloaded from GEO under accession number GSE22591. DNA methylation patterns of OsSPL s and osa-MIR156 s were explored according to Zemach et al. (2010) Zemach et al. (2010) with minor modifications. Briefly, (1) extracted sequences were converted to FASTQ format; (2) low-quality reads were removed; (3) adaptors were trimmed, and sequences of < 18 nt were discarded; (4) custom Perl scripts were used to convert all Cs to Ts and Gs to As for subsequent alignment with OsSPL s using Bowtie ( Langmead et al., 2009 Langmead et al., 2009 ), allowing up to two mismatches; (5) duplicate simple reads were filtered to single reads; and (6) methylcytosines in the context of CG, CHG, and CHH were counted to calculate the methylation rate of OsSPL s and osa-MIR156 s in the four tissues. 3 Results 1 3.1 Identification and analysis of miR156 in the grass family 2 The miRBase database includes 12 miR156 sequences from rice, 10 from Brachypodium , 9 from sorghum, and 12 from maize ( Table 1 Table 1 ). These miR156 precursor sequences were used to identify ten in foxtail millet ( Table 1 Table 1 and Fig. S1). The chromosomal distribution of miR156 orthologs is shown in Table S1. Only one of the two identical tandem copies of osa - MIR156f was used for further analysis. In addition, one of the Brachypodium miR156 sequences, bdi-MIR156a (MI0011566), appeared to be misannotated in miRBase since it does not contain the canonical precursor stem-loop structure and appears to be a fragment of Bradi4g33770 (an SPL ). The folding free energy of all the newly identified miR156 members was lower than -50 kcal/mol, and they were all located in intergenic regions, both typical features of miRNAs in plants ( Nozawa et al., 2012 Nozawa et al., 2012 ). The orthologous MIR156 gene copies are listed within a single row. The names of previously identified MIR156 s are those used in miRBase, and the newly identified MIR156 s were named according to the syntenic analysis with bold fonts. As maize is allotetraploid, the two homeologous chromosomes were named Maize 1 and Maize 2. Rice Brachypodium Foxtail millet Sorghum Maize 1 Maize 2 osa-MIR156a sit-MIR156a osa-MIR156b bdi-MIR156e sit-MIR156b sbi-MIR156b zma-MIR156b osa-MIR156c bdi-MIR156g sit-MIR156c sbi-MIR156c zma-MIR156c osa-MIR156d bdi-MIR156c sit-MIR156d sbi-MIR156a zma-MIR156d osa-MIR156e bdi-MIR156d sit-MIR156e sbi-MIR156h zma-MIR156h zma-MIR156e osa-MIR156f bdi-MIR156b sit-MIR156f sbi-MIR156i zma-MIR156a osa-MIR156g bdi-MIR156i sit-MIR156g sbi-MIR156f zma-MIR156g osa-MIR156h_j sbi-MIR156e zma-MIR156k osa-MIR156i bdi-MIR156h sit-MIR156i sbi-MIR156g zma-MIR156l zma-MIR156i osa-MIR156k bdi-MIR156j sit-MIR156h sbi-MIR156d zma-MIR156j osa-MIR156l sit-MIR156j zma-MIR156f Alignment of the MIR156 orthologous regions (130–1500 kb) from rice, Brachypodium , foxtail millet, sorghum, and maize revealed that all but osa-MIR156l , sit-MIR156j , and zma-MIR156f are located within syntenic regions ( Figs. 1 Figs. 1 and S2). We found that the rice locus osa-MIR156a has a corresponding copy only in foxtail millet, not in Brachypodium , sorghum, or maize; and osa-MIR156h - j has orthologous copies in sorghum and maize, but not in Brachypodium or foxtail millet ( Figs. 1 Figs. 1 and S2). Alignment of all 52 miR156 precursor sequences from the five grass species showed that their sequences were not similar outside of the stem-loop sequence. There was little conservation of precursor sequences, but the mature miR156 sequences were extremely well conserved. Therefore, phylogenetic analysis was performed using only the stem-loop sequences. All miR156 genes could be divided into five groups and ten subgroups, with nearly all the orthologous MIR156 copies clustered in the same subgroup ( Fig. 2 Fig. 2 ). Three species-specific genes, osa-MIR156l , sit-MIR156j , and zma-MIR156f clustered with the osa-MIR156a , sit-MIR156f , and zma-MIR156g subgroups, respectively, suggesting that the former set was duplicated from the latter ( Fig. 2 Fig. 2 ). Furthermore, two homologous MIR156 copies were present at the osa-MIR156i ( zma-MIR156i and zma-MIR156l ) and osa-MIR156e ( zma-MIR156e and zma-MIR156h ) loci in maize, supporting previous findings that maize is allotetraploid ( Gaut et al., 2000 Gaut et al., 2000 ). 3.2 Predicted targets of miR156 2 Putative targets of miR156 were predicted by psRNATarget ( Dai and Zhao, 2011 Dai and Zhao, 2011 ) and TAPIR ( Bonnet et al., 2010 Bonnet et al., 2010 ), both of which predicted that most targets would be SPL s ( Fig. 3 Fig. 3 a). A total of 12 SPL s were predicted in rice, 9 in Brachypodium , 10 in sorghum, 19 in maize, and 10 in foxtail millet ( Figs. 3 Figs. 3 b and S3). Further analysis showed that the miRNA responsive elements (MREs) in most of these SPL s were located in the last exon of the gene. In contrast, the MREs of OsSPL4 , OsSPL13 , BdSPL11 , SiSPL12 , ZmSPL12a , and ZmSPL12b were located in the 3'-UTR (Table S2), apparently because of the insertion of an upstream terminator UAA codon. 3.3 Identification and analysis of SPL s in grass genomes 2 Previous studies revealed 19 SPL s in the rice genome ( Xie et al., 2006 Xie et al., 2006 ), which we used as reference sequences to identify 17 SPL s in Brachypodium , 18 in sorghum, 31 in maize, and 18 in foxtail millet ( Table 2 Table 2 ). The BdSPL17 identified in this study is annotated in the Brachypodium database as two genes, Bradi4g18890 and Bradi4g18900 . However, further analysis showed it to be one continuous gene that is orthologous to OsSPL19 . Alignment of orthologous regions of all the SPL s revealed that only OsSPL11 and ZmSPL19 have no orthologous copies in other species and that all other SPL s are present in the syntenic regions (Fig. S4). Furthermore, we did not find an ortholog of OsSPL12 in Brachypodium , and a tandem duplication event appears to have occurred at the ZmSPL15 ( ZmSPL15a and ZmSPL15b ) locus in maize ( Table 2 Table 2 and Fig. S4). Rice SQUAMOSA promoter-binding protein-like ( SPL ) genes ( Xie et al., 2006 Xie et al., 2006 ) were used as reference genes to search for orthologs in other grass genomes. The two homeologous chromosomes were named Maize 1 and Maize 2. 2 No. 2 Rice 2 Brachypodium 2 Foxtail millet Standard SPL gene name 2 Gene name from the respective genome database Standard SPL gene name 2 Gene name from the respective genome database Standard SPL gene name Gene name from the respective genome database 1 OsSPL1 2 LOC_Os01g18850 BdSPL1 2 Bradi2g11240 SiSPL1 Si000259m.g 2 OsSPL2 2 LOC_Os01g69830 BdSPL2 2 Bradi2g59110 SiSPL2 Si001804m.g 3 OsSPL3 2 LOC_Os02g04680 BdSPL3 2 Bradi3g03510 SiSPL3 Si017520m.g 4 OsSPL4 2 LOC_Os02g07780 BdSPL4 2 Bradi3g05510 SiSPL4 Si017749m.g 5 OsSPL5 2 LOC_Os02g08070 BdSPL5 2 Bradi3g05720 SiSPL5 Si017338m.g 6 OsSPL6 2 LOC_Os03g61760 BdSPL6 2 Bradi1g02760 SiSPL6 Si034095m.g 7 OsSPL7 2 LOC_Os04g46580 BdSPL7 2 Bradi5g17720 SiSPL7 Si010262m.g 8 OsSPL8 2 LOC_Os04g56170 BdSPL8 2 Bradi5g24670 SiSPL8 Si022315m.g 9 OsSPL9 2 LOC_Os05g33810 BdSPL9 2 Bradi2g25580 SiSPL9 Si021754m.g 10 OsSPL10 2 LOC_Os06g44860 BdSPL10 2 Bradi1g31390 SiSPL10 Si006559m.g 11 OsSPL11 2 LOC_Os06g45310 2 12 OsSPL12 2 LOC_Os06g49010 2 SiSPL11 Si006472m.g 13 OsSPL13 2 LOC_Os07g32170 BdSPL11 2 Bradi1g26720 SiSPL12 Si031154m.g 14 OsSPL14 2 LOC_Os08g39890 BdSPL12 2 Bradi3g40030 SiSPL13 Si013870m.g 15 OsSPL15 2 LOC_Os08g40260 BdSPL13 2 Bradi3g40240 SiSPL14 Si013151m.g 16 OsSPL16 2 LOC_Os08g41940 BdSPL14 2 Bradi3g41250 SiSPL15 Si013747m.g 17 OsSPL17 2 LOC_Os09g31438 BdSPL15 2 Bradi4g33770 SiSPL16 Si030195m.g 18 OsSPL18 2 LOC_Os09g32944 BdSPL16 2 Bradi4g34667 SiSPL17 Si030892m.g 19 OsSPL19 2 LOC_Os11g30370 BdSPL17 2 Bradi4g18900 SiSPL18 Si026656m.g 20 2 2 2 No. 2 Sorghum 2 Maize 1 2 Maize 2 Standard SPL gene name 2 Gene name from the respective genome database Standard SPL gene name 2 Gene name from the respective genome database Standard SPL gene name Gene name from the respective genome database 1 SbSPL1 2 Sb03g011920 ZmSPL1a 2 GRMZM2G133646 ZmSPL1b GRMZM2G081127 2 SbSPL2 2 Sb03g044160 ZmSPL2a 2 GRMZM5G878561 ZmSPL2b GRMZM2G371033 3 SbSPL3 2 Sb04g003175 ZmSPL3a 2 GRMZM2G065451 ZmSPL3b GRMZM2G097275 4 SbSPL4 2 Sb04g004940 ZmSPL4a 2 GRMZM2G163813 ZmSPL4b GRMZM2G126827 5 SbSPL5 2 Sb04g005180 ZmSPL5a 2 GRMZM2G168229 ZmSPL5b GRMZM2G111136 6 SbSPL6 2 Sb01g002530 ZmSPL6a 2 GRMZM2G156756 ZmSPL6b GRMZM2G138421 7 SbSPL7 2 Sb06g024630 ZmSPL7 2 GRMZM2G148467 8 SbSPL8 2 Sb06g031290 ZmSPL8a 2 GRMZM2G036297 ZmSPL8b GRMZM2G058588 9 SbSPL9 2 Sb09g020110 ZmSPL9 2 GRMZM2G109354 10 SbSPL10 2 Sb10g026200 ZmSPL10 2 GRMZM2G101499 11 2 2 12 SbSPL11 2 Sb10g029190 ZmSPL11 2 GRMZM2G414805 13 SbSPL12 2 Sb02g034180 ZmSPL12a 2 GRMZM2G113779 ZmSPL12b GRMZM2G067624 14 SbSPL13 2 Sb07g027740 ZmSPL13a 2 GRMZM2G460544 ZmSPL13b GRMZM2G160917 15 SbSPL14 2 Sb07g027420 ZmSPL14a 2 GRMZM2G098557 ZmSPL14b GRMZM2G169270 16 SbSPL15 2 Sb07g026220 ZmSPL15a 2 GRMZM2G101511 ZmSPL15b AC233751.1_FGT002 17 SbSPL16 2 Sb02g028420 ZmSPL16a 2 GRMZM2G126018 ZmSPL16b GRMZM2G307588 18 SbSPL17 2 Sb02g029300 ZmSPL17 2 GRMZM2G061734 19 SbSPL18 2 Sb05g017510 ZmSPL18 2 GRMZM2G106798 20 2 ZmSPL19 2 GRMZM2G156621 The SBP domain of all the SPLs was encoded by the first and second exons despite considerable variation in the gene lengths (from 648 bp to > 10 kb; Table S2). Alignment of the nucleotide and protein sequences showed large variation overall, but there was greater conservation within the 80-amino-acid SBP domain. In addition, 14 SPL s were identified in moss ( Physcomitrella patens ) and 24 in green algae ( Chlamydomonas reinhardtii ). Therefore, a phylogenetic tree was constructed from the SBP domains of 102 SPL sequences from grasses, 17 from Arabidopsis, 14 from moss, and 1 from green algae (as the out-group; Fig. 4 Fig. 4 a). We found that the SPL s fell into eight groups (G1‒G8). All of the non-miR156-targeted SPL s clustered in groups G2, G3, G4, and G8, and all of the miR156-targeted SPL s clustered in groups G1, G5, G6, and G7 ( Fig. 4 Fig. 4 a). G8 contained SPL s from all species, including green algae, suggesting that this group may represent the ancestral SPL . Furthermore, gene structure analysis showed that members within each group had a similar exon-intron structure and that all non-miR156-targeted SPL s had more than 10 exons (except for SPL s from angiosperms in G3), whereas miR156-targeted SPL s contained only 2‒4 exons ( Fig. 4 Fig. 4 b). Two species-specific genes, OsSPL11 and ZmSPL19 , clustered in the same clade with OsSPL4 and ZmSPL4 , respectively ( Fig. 4 Fig. 4 a), suggesting that they were duplicated from OsSPL4 and ZmSPL4 , respectively, after their divergence from other species. We also observed that a common homologous region was not present in seven loci ( OsSPL7 , OsSPL10 , OsSPL11 , OsSPL12 , OsSPL16 , OsSPL18 , and OsSPL19 ), and one orthologous gene was deleted at the OsSPL9 locus in the maize genome ( Figs. 4 Figs. 4 a and S4). The SBP domains of the SPL s were rich in cysteine, arginine, and histidine residues, which suggests the presence of two Zn 2+ -binding sites ( Birkenbihl et al., 2005 Birkenbihl et al., 2005 ; Kropat et al., 2005 Kropat et al., 2005 ), and a putative nuclear localization signal (NLS) that partly overlaps with the second Zn 2+ -binding site was located in the C-terminus of the SBP domain ( Fig. 5 Fig. 5 ). Intriguingly, the conserved SBP domains of some SPLs, such as BdSPL7, ZmSPL2a, and ZmSPL15b, were separated by additional exon or sequence insertions, which presumably come together in the three-dimensional (3D) conformation of the protein to form one active DNA-binding site. 3.4 Expression profiles of SPL s in different rice tissues and at different developmental stages 2 Because rice miR156 expression is higher in the juvenile phase than in adult tissues ( Sunkar et al., 2008 Sunkar et al., 2008 ; Paterson et al., 2009 Paterson et al., 2009 ), we hypothesized that miR156-targeted SPL s would show a converse trend. Expression data for SPL s in 11 rice tissues/developmental stages were retrieved from the GEO database. The OsSPL expression patterns were classified into four types ( Fig. 6 Fig. 6 and Table S3). The non-miR156-targeted genes OsSPL1 , OsSPL6 , OsSPL9 , and OsSPL15 showed relatively stable and high expression throughout all plant developmental phases ( Fig. 6 Fig. 6 ). In particular, OsSPL15 was expressed at a very high level in all 11 tissues/developmental stages, suggesting that it probably acts as a general housekeeping gene in plant growth and development. In contrast, the miR156-targeted genes OsSPL2 , OsSPL3 , OsSPL12 , and OsSPL13 were more highly expressed in early stages (from shoots to post-emergence inflorescence) than in later stages (anther to 25-DAP (day after pollination) endosperm; Fig. 6 Fig. 6 ). Eight genes, including five miR156-targeted genes ( OsSPL4 , OsSPL11 , OsSPL14 , OsSPL16 , and OsSPL18 ) and three non-miR156-targeted genes ( OsSPL5 , OsSPL8 , and OsSPL10 ) were more highly expressed in pistils and in pre- and post-emergence inflorescence stages than in other tissues/developmental stages. The remaining miR156-targeted genes ( OsSPL7 , OsSPL17 , and OsSPL19 ) were expressed at low levels in all 11 tissues/developmental stages. Expression levels of almost all the SPL s peaked at the pre-emergence inflorescence stage, which strongly correlates with flowering in rice. They gradually decreased through the post-emergence inflorescence stage and then dropped dramatically ( Fig. 6 Fig. 6 ). As expected, miR156-targeted OsSPL s were more highly expressed in the adult reproductive phase than in the vegetative phase, and OsSPL s that contain MREs had similar expression patterns ( Fig. 6 Fig. 6 ). 3.5 Methylation patterns of SPL s and MIR156 s in rice tissues 2 To investigate whether DNA methylation correlates with the expression of MIR156 s and SPL s, we examined the DNA methylation status of the promoter, gene body, and 3'-UTRs of all MIR156 s and SPL s in four rice tissues (root, shoot, embryo, and endosperm) using data from GEO (Tables S4 and S5). The promoters (2 kb upstream from the start codon) of SPL s exhibited variable patterns of methylation. Almost no ( < 0.5%) methylation was seen in the promoters of some genes ( OsSPL3 , OsSPL7 , OsSPL12 , and OsSPL18 ), slight methylation (1%‒10%) in others ( OsSPL1 , OsSPL2 , OsSPL6 , OsSPL8 , OsSPL10 , OsSPL13 , OsSPL15 , OsSPL16 , and OsSPL19 ), and a high degree of methylation ( > 15%) in the remaining genes ( OsSPL4 , OsSPL5 , OsSPL9 , OsSPL11 , OsSPL14 , and OsSPL17 ). More than 50% of the methylation in the highly methylated promoters occurred within distinct methylation blocks ( Fig. 7 Fig. 7 a, Tables S4 and S6). Methylation levels of all SPL promoters were lower in endosperm than in other tissues except for OsSPL2 and the genes with little or no promoter methylation. Furthermore, overall promoter methylation at CG sites was 2-fold higher than at CHG sites and 3-fold higher than at CHH sites. However, CG-site methylation was 5-fold higher than at CHG sites and 12-fold higher than at CHH sites in the endosperm (Table S6), which suggested a more pronounced hypomethylation in CHG and CHH contexts in the endosperm. In terms of gene body methylation, the SPL s fell into two groups: those that were highly methylated ( > 5%; OsSPL1 , OsSPL3 , OsSPL4 , OsSPL6 , OsSPL9 , OsSPL12 , OsSPL14 , OsSPL15 , OsSPL17 , and OsSPL19 ) and those with little or no methylation ( < 5%; Fig. 7 Fig. 7 b, Tables S4 and S7). About 40% of the gene body methylation in the highly methylated group was at CG sites, whereas CHG and CHH methylation made up < 1%. We also found that no cytosines were methylated in MREs. In contrast to the methylation patterns in promoters and gene bodies, there was almost no methylcytosine in the 3'-UTR of the SPL s, except for three methylated CG sites in OsSPL4 , five in OsSPL14 , and one in OsSPL15 (Table S4). In the case of MIR156 , the methylation frequency in the putative promoter regions (1 kb upstream of the stem-loop structure) of osa-MIR156a / f / g / h / j / l was > 10% ( Fig. 7 Fig. 7 c and Table S5), and methylation at CG sites was 2-fold higher than that at CHG sites and 3-fold higher than that at CHH sites (Tables S5 and S8), similar to the SPL promoters. In contrast, there were almost no methylcytosines in the gene bodies (stem-loop sequences) except in osa-MIR156d , in which gene body methylation reached 75% in the endosperm (Table S5). However, because all mature miR156 sequences are nearly identical, we were unable to assess the effect of DNA methylation on their expression. 4 Discussion 1 4.1 Origin and evolution of SPL s 2 In plants, miRNAs tend to facilitate the cleavage of their target mRNA, rather than inhibit translation, as is more common in animals ( Llave et al., 2002 Llave et al., 2002 ; Palatnik et al., 2003 Palatnik et al., 2003 ). SPL s, which encode a conserved SBP domain, are the primary targets of miR156 ( Figs. 3 Figs. 3 b and S3). SBP-box genes have been identified in moss and green algae but not in other algae, which indicates that they are plant-specific and likely originated before the divergence of green plants ( Cardon et al., 1999 Cardon et al., 1999 ; Riese et al., 2007 Riese et al., 2007 ; Guo et al., 2008 Guo et al., 2008 ; Chang et al., 2016 Chang et al., 2016 ; Lei and Liu, 2016 Lei and Liu, 2016 ; Morea et al., 2016 Morea et al., 2016 ; Peng et al., 2016 Peng et al., 2016 ; Wang et al., 2018 Wang et al., 2018 ; Song et al., 2020 Song et al., 2020 ). Our phylogenetic analysis showed that most CrSPL s clustered together as an out-group (data not shown), but some clustered with the non-miR156-targeted SPL s ( Fig. 4 Fig. 4 a). We therefore hypothesize that the original SPL was a non-miR156-targeted gene that then diversified into non-miR156-targeted and miR156-targeted SPL s that contained MREs after the divergence of green algae from the progenitor of land plants, but before land plants diverged. Similar phylogenetic histories are thought to have occurred in other plant-specific transcription factors such as DNA-binding with one finger (DOF) and APETALA2 (AP2) ( Shigyo et al., 2006 Shigyo et al., 2006 ; Moreno-Risueno et al., 2007 Moreno-Risueno et al., 2007 ; Agarwal et al., 2016 Agarwal et al., 2016 ; Jin et al., 2018 Jin et al., 2018 ). Our analysis suggests that only four SPL s were present before the divergence of land plants, three of which were non-miR156-targeted and one of which was miR156-targeted. The SPL s then expanded to at least 7 copies before the divergence of monocots and dicots, and to 18 copies in the grass family ( Fig. 4 Fig. 4 a). Moreover, we found that the miR156-targeted and non-miR156-targeted SPL s had distinguishable gene structures (compare G1 and G5‒G7 with G2‒G4 and G8, respectively; Fig. 4 Fig. 4 ). However, the gene structure of one group of the non-miR156-targeted genes (G3) showed similarities to that of the miR156-targeted genes ( Fig. 4 Fig. 4 b). Further analysis showed that SPL8 in this group had a putative MRE in its 3'-UTR (Fig. S5), suggesting that it could be converted into an miR156-targted gene by alternative splicing. We also found that three genes ( OsSPL5 , OsSPL8 , and OsSPL10 ) in this group had the same expression pattern as miR156-targeted SPL s ( Fig. 6 Fig. 6 ). Our data suggested that non-miR156-targeted genes could diversify into miR156-targeted genes by simultaneous evolution toward fewer exons and the formation of MREs ( Fig. 8 Fig. 8 ). 4.2 Origin and evolution of MIR156 in grass genomes 2 One proposed mechanism for the evolutionary origins of miRNAs is that they were initially generated from the inverted duplication of a target gene. That no miR156 sequence was identified in green algae suggests that MIR156 arose from its target SPL after the divergence of green algae, but before land plants emerged ( Tang, 2010 Tang, 2010 ). This raises the intriguing possibility that the co-evolution of MIR156 s and its target SPLs may have been an important regulatory mechanism during evolution. Mature miR156 sequences are highly conserved in grasses and Arabidopsis ( Fig. 4 Fig. 4 a), but their gene sequences are extremely diverse. This indicates that there may have been strong selective pressure to maintain the secondary structure of miR156 species, further suggesting that miR156 has served a critical role in the development of plants throughout their evolutionary history ( Axtell et al., 2007 Axtell et al., 2007 ). Moreover, our results demonstrated that tandem, segmental, and whole-genome duplications appear to have played important roles in the expansion of the MIR156 gene family in grass genomes. Almost all orthologous MIR156 genes clustered together in our phylogenetic analysis, and the original MIR156 had expanded to at least ten genes before the divergence of the grasses. However, the MIR156 s from Arabidopsis clustered together as an out-group (data not shown), suggesting that no orthologous MIR156 genes are present in monocots and dicots and that miRNAs in this branch may have resulted from a different evolutionary pathway. Interestingly, we observed that miR529 and miR156 were very similar, differing only at nucleotides 10 and 14 (Fig. S6). Furthermore, some SPL s (such as OsSPL14 and OsSPL17 ) are also targeted by miR529 ( Jeong et al., 2011 Jeong et al., 2011 ; Yue et al., 2017 Yue et al., 2017 ). In contrast to the decline in miR156 after seedling development, miR529 expression is sustained throughout the growth and development of plants, and OsSPL14 is predominantly cleaved by miR529 in panicles ( Jeong et al., 2011 Jeong et al., 2011 ). miR156 is highly expressed in various plants and tissues ( Sunkar et al., 2008 Sunkar et al., 2008 ; Gou et al., 2011 Gou et al., 2011 ; Jiao et al., 2011 Jiao et al., 2011 ; Schreiber et al., 2011 Schreiber et al., 2011 ), and in some cases accounts for nearly half of the total miRNA expression ( Barrett et al., 2011 Barrett et al., 2011 ), suggesting that it may be a housekeeping miRNA. Furthermore, MIR156 and MIR529 are both present in the common ancestor of embryophytes, whereas MIR529 is not present in the common ancestor of Eudicots-Asterids ( Cuperus et al., 2011 Cuperus et al., 2011 ; Axtell and Meyers, 2018 Axtell and Meyers, 2018 ), which suggests that these two genes originated from the same ancestor but had different evolutionary fates. 4.3 Correlation of DNA methylation with gene expression 2 A large number of OsSPL s were methylated within their gene bodies, primarily at CG sites. DNA methylation was markedly less prevalent in the 5' and 3' regions of the transcription unit, where it could affect transcription factor binding ( Zilberman et al., 2007 Zilberman et al., 2007 ). In addition, there was a positive correlation between gene body methylation and promoter hypomethylation and transcription (Figs. 6 6 and 7 7 , Tables S4‒S6) ( Zilberman et al., 2007 Zilberman et al., 2007 ). OsSPL 9 was highly methylated in certain regions upstream of the start site, but it had a very high expression level, suggesting that the core promoter could lie outside of these methylation regions. Another six OsSPL s showed higher promoter methylation but no difference in their levels of expression compared with genes that were less frequently methylated in their promoters ( Fig. 6 Fig. 6 and Table S4). We hypothesized that the methylation within these upstream regions may not be in functional motifs and/or promoter regions. Furthermore, we observed methylation patterns that were indistinguishable between some miR156-targeted OsSPL s and the rest of the non-miR156-targeted OsSPL s ( OsSPL5 , OsSPL8 , and OsSPL10 ). This confirmed our assumption that DNA methylation functions as a control mechanism at a pre-transcriptional level, whereas miRNAs are involved at a post-transcriptional level. These mechanisms may thus provide complementary gene regulation. Non-CG methylation was reduced in all OsSPL s in the endosperm compared with the other tissues (shoot, root, and embryo), especially within the promoter, whereas CG hypomethylation was localized ( Fig. 7 Fig. 7 , Tables S4 and S6). This is consistent with a previous study ( Zemach et al., 2010 Zemach et al., 2010 ) and suggests that hypomethylation in rice endosperm is a mechanism for activating genes. References Agarwal G , Garg V , Kudapa H , et al. , 2016 . Genome-wide dissection of AP2/ERF and HSP90 gene families in five legumes and expression profiles in chickpea and pigeonpea . Plant Biotechnol J , 14 ( 7 ): 1563 - 1577 . https://doi.org/10.1111/pbi.12520 https://doi.org/10.1111/pbi.12520 Genome-wide dissection of AP2/ERF and HSP90 gene families in five legumes and expression profiles in chickpea and pigeonpea Axtell MJ , Meyers BC , 2018 . Revisiting criteria for plant microRNA annotation in the era of big data . Plant Cell , 30 ( 2 ): 272 - 284 . https://doi.org/10.1105/tpc.17.00851 https://doi.org/10.1105/tpc.17.00851 Revisiting criteria for plant microRNA annotation in the era of big data Axtell MJ , Snyder JA , Bartel DP , 2007 . Common functions for diverse small RNAs of land plants . Plant Cell , 19 ( 6 ): 1750 - 1769 . https://doi.org/10.1105/tpc.107.051706 https://doi.org/10.1105/tpc.107.051706 Common functions for diverse small RNAs of land plants Barrett T , Troup DB , Wilhite SE , et al. , 2011 . NCBI GEO: archive for functional genomics data sets—10 years on . Nucleic Acids Res , 39 ( S1 ): D1005 - D1010 . https://doi.org/10.1093/nar/gkq1184 https://doi.org/10.1093/nar/gkq1184 NCBI GEO: archive for functional genomics data sets—10 years on Bartel DP , 2004 . MicroRNAs: genomics, biogenesis, mechanism, and function . Cell , 116 ( 2 ): 281 - 297 . https://doi.org/10.1016/S0092-8674(04)00045-5 https://doi.org/10.1016/S0092-8674(04)00045-5 MicroRNAs: genomics, biogenesis, mechanism, and function Bennetzen JL , Schmutz J , Wang H , et al. , 2012 . Reference genome sequence of the model plant setaria . Nat Biotechnol , 30 ( 6 ): 555 - 561 . https://doi.org/10.1038/nbt.2196 https://doi.org/10.1038/nbt.2196 Reference genome sequence of the model plant setaria Birkenbihl RP , Jach G , Saedler H , et al. , 2005 . Functional dissection of the plant-specific SBP-domain: overlap of the DNA-binding and nuclear localization domains . J Mol Biol , 352 ( 3 ): 585 - 596 . https://doi.org/10.1016/j.jmb.2005.07.013 https://doi.org/10.1016/j.jmb.2005.07.013 Functional dissection of the plant-specific SBP-domain: overlap of the DNA-binding and nuclear localization domains Bonnet E , He Y , Billiau K , et al. , 2010 . TAPIR, a web server for the prediction of plant microRNA targets, including target mimics . Bioinformatics , 26 ( 12 ): 1566 - 1568 . https://doi.org/10.1093/bioinformatics/btq233 https://doi.org/10.1093/bioinformatics/btq233 TAPIR, a web server for the prediction of plant microRNA targets, including target mimics Cardon G , Höhmann S , Klein J , et al. , 1999 . Molecular characterisation of the Arabidopsis SBP-box genes . Gene , 237 ( 1 ): 91 - 104 . https://doi.org/10.1016/S0378-1119(99)00308-X https://doi.org/10.1016/S0378-1119(99)00308-X Molecular characterisation of the Arabidopsis SBP-box genes Chang JZ , Yan FX , Qiao LY , et al. , 2016 . Genome-wide identification and expression analysis of SBP-box gene family in Sorghum bicolor L . Hereditas (Beijing) , 38 ( 6 ): 569 - 580 (in Chinese) . https://doi.org/10.16288/j.yczz.16-008 https://doi.org/10.16288/j.yczz.16-008 Genome-wide identification and expression analysis of SBP-box gene family in Sorghum bicolor L Cuperus JT , Fahlgren N , Carrington JC , 2011 . Evolution and functional diversification of MIRNA genes . Plant Cell , 23 ( 2 ): 431 - 442 . https://doi.org/10.1105/tpc.110.082784 https://doi.org/10.1105/tpc.110.082784 Evolution and functional diversification of MIRNA genes Dai XB , Zhao PX , 2011 . PsRNATarget: a plant small RNA target analysis server . Nucleic Acids Res , 39 ( S2 ): W155 - W159 . https://doi.org/10.1093/nar/gkr319 https://doi.org/10.1093/nar/gkr319 PsRNATarget: a plant small RNA target analysis server Edgar RC , 2004 . MUSCLE: multiple sequence alignment with high accuracy and high throughput . Nucleic Acids Res , 32 ( 5 ): 1792 - 1797 . https://doi.org/10.1093/nar/gkh340 https://doi.org/10.1093/nar/gkh340 MUSCLE: multiple sequence alignment with high accuracy and high throughput Feng SH , Jacobsen SE , Reik W , 2010 . Epigenetic reprogramming in plant and animal development . Science , 330 ( 6004 ): 622 - 627 . https://doi.org/10.1126/science.1190614 https://doi.org/10.1126/science.1190614 Epigenetic reprogramming in plant and animal development Fornara F , Coupland G , 2009 . Plant phase transitions make a SPLash . Cell , 138 ( 4 ): 625 - 627 . https://doi.org/10.1016/j.cell.2009.08.011 https://doi.org/10.1016/j.cell.2009.08.011 Plant phase transitions make a SPLash Franco-Zorrilla JM , Valli A , Todesco M , et al. , 2007 . Target mimicry provides a new mechanism for regulation of microRNA activity . Nat Genet , 39 ( 8 ): 1033 - 1037 . https://doi.org/10.1038/ng2079 https://doi.org/10.1038/ng2079 Target mimicry provides a new mechanism for regulation of microRNA activity Gaut BS , le Thierry D'Ennequin M , Peek AS , et al. , 2000 . Maize as a model for the evolution of plant nuclear genomes . Proc Natl Acad Sci USA , 97 ( 13 ): 7008 - 7015 . https://doi.org/10.1073/pnas.97.13.7008 https://doi.org/10.1073/pnas.97.13.7008 Maize as a model for the evolution of plant nuclear genomes Gielen H , Remans T , Vangronsveld J , et al. , 2016 . Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7 . BMC Plant Biol , 16 : 145 . https://doi.org/10.1186/s12870-016-0830-4 https://doi.org/10.1186/s12870-016-0830-4 Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7 Gou JY , Felippes FF , Liu CJ , et al. , 2011 . Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor . Plant Cell , 23 ( 4 ): 1512 - 1522 . https://doi.org/10.1105/tpc.111.084525 https://doi.org/10.1105/tpc.111.084525 Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor Guo AY , He K , Liu D , et al. , 2005 . DATF: a database of Arabidopsis transcription factors . Bioinformatics , 21 ( 10 ): 2568 - 2569 . https://doi.org/10.1093/bioinformatics/bti334 https://doi.org/10.1093/bioinformatics/bti334 DATF: a database of Arabidopsis transcription factors Guo AY , Zhu QH , Gu XC , et al. , 2008 . Genome-wide identification and evolutionary analysis of the plant specific SBP-box transcription factor family . Gene , 418 ( 1-2 ): 1 - 8 . https://doi.org/10.1016/j.gene.2008.03.016 https://doi.org/10.1016/j.gene.2008.03.016 Genome-wide identification and evolutionary analysis of the plant specific SBP-box transcription factor family Huijser P , Schmid M , 2011 . The control of developmental phase transitions in plants . Development , 138 ( 19 ): 4117 - 4129 . https://doi.org/10.1242/dev.063511 https://doi.org/10.1242/dev.063511 The control of developmental phase transitions in plants International Rice Genome Sequencing Project , Sasaki T , 2005 . The map-based sequence of the rice genome . Nature , 436 ( 7052 ): 793 - 800 . https://doi.org/10.1038/nature03895 https://doi.org/10.1038/nature03895 The map-based sequence of the rice genome Jeong DH , Park S , Zhai JX , et al. , 2011 . Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage . Plant Cell , 23 ( 12 ): 4185 - 4207 . https://doi.org/10.1105/tpc.111.089045 https://doi.org/10.1105/tpc.111.089045 Massive analysis of rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential target RNA cleavage Jiao YP , Song WB , Zhang M , et al. , 2011 . Identification of novel maize miRNAs by measuring the precision of precursor processing . BMC Plant Biol , 11 : 141 . https://doi.org/10.1186/1471-2229-11-141 https://doi.org/10.1186/1471-2229-11-141 Identification of novel maize miRNAs by measuring the precision of precursor processing Jiao YQ , Wang YH , Xue DW , et al. , 2010 . Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice . Nat Genet , 42 ( 6 ): 541 - 544 . https://doi.org/10.1038/ng.591 https://doi.org/10.1038/ng.591 Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice Jin JH , Wang M , Zhang HX , et al. , 2018 . Genome-wide identification of the AP2/ERF transcription factor family in pepper ( Capsicum annuum L .). Genome , 61 ( 9 ): 663 - 674 . https://doi.org/10.1139/gen-2018-0036 https://doi.org/10.1139/gen-2018-0036 Genome-wide identification of the AP2/ERF transcription factor family in pepper (Capsicum annuum L Kimura M , 1980 . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences . J Mol Evol , 16 ( 2 ): 111 - 120 . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences Kozomara A , Griffiths-Jones S , 2011 . miRBase: integrating microRNA annotation and deep-sequencing data . Nucleic Acids Res , 39 ( S1 ): D152 - D157 . https://doi.org/10.1093/nar/gkq1027 https://doi.org/10.1093/nar/gkq1027 miRBase: integrating microRNA annotation and deep-sequencing data Kropat J , Tottey S , Birkenbihl RP , et al. , 2005 . A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element . Proc Natl Acad Sci USA , 102 ( 51 ): 18730 - 18735 . https://doi.org/10.1073/pnas.0507693102 https://doi.org/10.1073/pnas.0507693102 A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element Kumar S , Stecher G , Li M , et al. , 2018 . MEGA X: molecular evolutionary genetics analysis across computing platforms . Mol Biol Evol , 35 ( 6 ): 1547 - 1549 . https://doi.org/10.1093/molbev/msy096 https://doi.org/10.1093/molbev/msy096 MEGA X: molecular evolutionary genetics analysis across computing platforms Lal S , Pacis LB , Smith HMS , 2011 . Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes / microRNA156 module by the homeodomain proteins PENNYWISE and POUND-FOOLISH in Arabidopsis . Mol Plant , 4 ( 6 ): 1123 - 1132 . https://doi.org/10.1093/mp/ssr041 https://doi.org/10.1093/mp/ssr041 Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA156 module by the homeodomain proteins PENNYWISE and POUND-FOOLISH in Arabidopsis Langmead B , Trapnell C , Pop M , et al. , 2009 . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome . Genome Biol , 10 ( 3 ): R25 . https://doi.org/10.1186/gb-2009-10-3-r25 https://doi.org/10.1186/gb-2009-10-3-r25 Ultrafast and memory-efficient alignment of short DNA sequences to the human genome Larkin MA , Blackshields G , Brown NP , et al. , 2007 . Clustal W and Clustal X version 2 . 0 . Bioinformatics , 23 ( 21 ): 2947 - 2948 . https://doi.org/10.1093/bioinformatics/btm404 https://doi.org/10.1093/bioinformatics/btm404 Clustal W and Clustal X version 2 Law JA , Jacobsen SE , 2010 . Establishing, maintaining and modifying DNA methylation patterns in plants and animals . Nat Rev Genet , 11 ( 3 ): 204 - 220 . https://doi.org/10.1038/nrg2719 https://doi.org/10.1038/nrg2719 Establishing, maintaining and modifying DNA methylation patterns in plants and animals Lei KJ , Liu H , 2016 . Research advances in plant regulatory hub miR156 and targeted SPL family . Chem Life , 36 ( 1 ): 13 - 20 (in Chinese) . https://doi.org/10.13488/j.smhx.20160103 https://doi.org/10.13488/j.smhx.20160103 Research advances in plant regulatory hub miR156 and targeted SPL family Liu MM , Shi ZY , Zhang XH , et al. , 2019 . Inducible overexpression of Ideal Plant Architecture1 improves both yield and disease resistance in rice . Nat Plants , 5 ( 4 ): 389 - 400 . https://doi.org/10.1038/s41477-019-0383-2 https://doi.org/10.1038/s41477-019-0383-2 Inducible overexpression of Ideal PlantArchitecture1 improves both yield and disease resistance in rice Llave C , Xie ZX , Kasschau KD , et al. , 2002 . Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA . Science , 297 ( 5589 ): 2053 - 2056 . https://doi.org/10.1126/science.1076311 https://doi.org/10.1126/science.1076311 Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA Manning K , Tör M , Poole M , et al. , 2006 . A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening . Nat Genet , 38 ( 8 ): 948 - 952 . https://doi.org/10.1038/ng1841 https://doi.org/10.1038/ng1841 A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening Martin RC , Liu PP , Goloviznina NA , et al. , 2010 . microRNA, seeds, and darwin?: diverse function of miRNA in seed biology and plant responses to stress . J Exp Bot , 61 ( 9 ): 2229 - 2234 . https://doi.org/10.1093/jxb/erq063 https://doi.org/10.1093/jxb/erq063 microRNA, seeds, and darwin?: diverse function of miRNA in seed biology and plant responses to stress Meyers BC , Axtell MJ , Bartel B , et al. , 2008 . Criteria for annotation of plant microRNAs . Plant Cell , 20 ( 12 ): 3186 - 3190 . https://doi.org/10.1105/tpc.108.064311 https://doi.org/10.1105/tpc.108.064311 Criteria for annotation of plant microRNAs Miao CB , Wang Z , Zhang L , et al. , 2019 . The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice . Nat Commun , 10 : 3822 . https://doi.org/10.1038/s41467-019-11830-5 https://doi.org/10.1038/s41467-019-11830-5 The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice Miura K , Ikeda M , Matsubara A , et al. , 2010 . OsSPL14 promotes panicle branching and higher grain productivity in rice . Nat Genet , 42 ( 6 ): 545 - 549 . https://doi.org/10.1038/ng.592 https://doi.org/10.1038/ng.592 OsSPL14 promotes panicle branching and higher grain productivity in rice Morea EGO , da Silva EM , Silva GFFE , et al. , 2016 . Functional and evolutionary analyses of the miR156 and miR529 families in land plants . BMC Plant Biol , 16 : 40 . https://doi.org/10.1186/s12870-016-0716-5 https://doi.org/10.1186/s12870-016-0716-5 Functional and evolutionary analyses of the miR156 and miR529 families in land plants Moreno-Risueno MA , Martínez M , Vicente-Carbajosa J , et al. , 2007 . The family of DOF transcription factors: from green unicellular algae to vascular plants . Mol Genet Genomics , 277 ( 4 ): 379 - 390 . https://doi.org/10.1007/s00438-006-0186-9 https://doi.org/10.1007/s00438-006-0186-9 The family of DOF transcription factors: from green unicellular algae to vascular plants Nozawa M , Miura S , Nei M , 2012 . Origins and evolution of microRNA genes in plant species . Genome Biol Evol , 4 ( 3 ): 230 - 239 . https://doi.org/10.1093/gbe/evs002 https://doi.org/10.1093/gbe/evs002 Origins and evolution of microRNA genes in plant species Palatnik JF , Allen E , Wu XL , et al. , 2003 . Control of leaf morphogenesis by microRNAs . Nature , 425 ( 6955 ): 257 - 263 . https://doi.org/10.1038/nature01958 https://doi.org/10.1038/nature01958 Control of leaf morphogenesis by microRNAs Paterson AH , Bowers JE , Bruggmann R , et al. , 2009 . The Sorghum bicolor genome and the diversification of grasses . Nature , 457 ( 7229 ): 551 - 556 . https://doi.org/10.1038/nature07723 https://doi.org/10.1038/nature07723 The Sorghum bicolor genome and the diversification of grasses Peng H , He XJ , Gao J , et al. , 2016 . Genome-wide identification and function analysis of SBP gene family in maize . Acta Agron Sin , 42 ( 2 ): 201 - 211 (in Chinese) . https://doi.org/10.3724/SP.J.1006.2016.00201 https://doi.org/10.3724/SP.J.1006.2016.00201 Genome-wide identification and function analysis of SBP gene family in maize Ramamurthy RK , Xiang QY , Hsieh EJ , et al. , 2018 . New aspects of iron-copper crosstalk uncovered by transcriptomic characterization of Col-0 and the copper uptake mutant spl7 in Arabidopsis thaliana . Metallomics , 10 ( 12 ): 1824 - 1840 . https://doi.org/10.1039/c8mt00287h https://doi.org/10.1039/c8mt00287h New aspects of iron-copper crosstalk uncovered by transcriptomic characterization of Col-0 and the copper uptake mutant spl7 in Arabidopsis thaliana Reinhart BJ , Weinstein EG , Rhoades MW , et al. , 2002 . MicroRNAs in plants . Genes Dev , 16 ( 13 ): 1616 - 1626 . https://doi.org/10.1101/gad.1004402 https://doi.org/10.1101/gad.1004402 MicroRNAs in plants Riese M , Höhmann S , Saedler H , et al. , 2007 . Comparative analysis of the SBP-box gene families in P . patens and seed plants . Gene , 401 ( 1-2 ): 28 - 37 . https://doi.org/10.1016/j.gene.2007.06.018 https://doi.org/10.1016/j.gene.2007.06.018 Comparative analysis of the SBP-box gene families in P. patens and seed plants Saeed AI , Bhagabati NK , Braisted JC , et al. , 2006 . TM4 microarray software suite . Methods Enzymol , 411 : 134 - 193 . https://doi.org/10.1016/S0076-6879(06)11009-5 https://doi.org/10.1016/S0076-6879(06)11009-5 TM4 microarray software suite Schnable PS , Ware D , Fulton RS , et al. , 2009 . The B73 maize genome: complexity, diversity, and dynamics . Science , 326 ( 5956 ): 1112 - 1115 . https://doi.org/10.1126/science.1178534 https://doi.org/10.1126/science.1178534 The B73 maize genome: complexity, diversity, and dynamics Schreiber AW , Shi BJ , Huang CY , et al. , 2011 . Discovery of barley miRNAs through deep sequencing of short reads . BMC Genomics , 12 : 129 . https://doi.org/10.1186/1471-2164-12-129 https://doi.org/10.1186/1471-2164-12-129 Discovery of barley miRNAs through deep sequencing of short reads Shigyo M , Hasebe M , Ito M , 2006 . Molecular evolution of the AP2 subfamily . Gene , 366 ( 2 ): 256 - 265 . https://doi.org/10.1016/j.gene.2005.08.009 https://doi.org/10.1016/j.gene.2005.08.009 Molecular evolution of the AP2 subfamily Sollome J , Martin E , Sethupathy P , et al. , 2016 . Environmental contaminants and microRNA regulation: transcription factors as regulators of toxicant-altered microRNA expression . Toxicol Appl Pharmacol , 312 : 61 - 66 . https://doi.org/10.1016/j.taap.2016.06.009 https://doi.org/10.1016/j.taap.2016.06.009 Environmental contaminants and microRNA regulation: transcription factors as regulators of toxicant-altered microRNA expression Song J , Cao XN , Wang HL , et al. , 2020 . Genome wide identification and expression analysis of the SBP gene family in foxtail millet . J Nucl Agric Sci , 34 ( 7 ): 1409 - 1420 (in Chinese) . https://doi.org/10.11869/j.issn.100-8551.2020.07.1409 https://doi.org/10.11869/j.issn.100-8551.2020.07.1409 Genome wide identification and expression analysis of the SBP gene family in foxtail millet Sunkar R , Zhou XF , Zheng Y , et al. , 2008 . Identification of novel and candidate miRNAs in rice by high throughput sequencing . BMC Plant Biol , 8 : 25 . https://doi.org/10.1186/1471-2229-8-25 https://doi.org/10.1186/1471-2229-8-25 Identification of novel and candidate miRNAs in rice by high throughput sequencing Tang GL , 2010 . Plant microRNAs: an insight into their gene structures and evolution . Semin Cell Dev Biol , 21 ( 8 ): 782 - 789 . https://doi.org/10.1016/j.semcdb.2010.07.009 https://doi.org/10.1016/j.semcdb.2010.07.009 Plant microRNAs: an insight into their gene structures and evolution Teixeira FK , Colot V , 2010 . Repeat elements and the Arabidopsis DNA methylation landscape . Heredity (Edinb) , 105 ( 1 ): 14 - 23 . https://doi.org/10.1038/hdy.2010.52 https://doi.org/10.1038/hdy.2010.52 Repeat elements and the Arabidopsis DNA methylation landscape The International Brachypodium Initiative , 2010 . Genome sequencing and analysis of the model grass Brachypodium distachyon . Nature , 463 ( 7282 ): 763 - 768 . https://doi.org/10.1038/nature08747 https://doi.org/10.1038/nature08747 Genome sequencing and analysis of the model grass Brachypodium distachyon Voinnet O , 2009 . Origin, biogenesis, and activity of plant microRNAs . Cell , 136 ( 4 ): 669 - 687 . https://doi.org/10.1016/j.cell.2009.01.046 https://doi.org/10.1016/j.cell.2009.01.046 Origin, biogenesis, and activity of plant microRNAs Wang J , Zhou L , Shi H , et al. , 2018 . A single transcription factor promotes both yield and immunity in rice . Science , 361 ( 6406 ): 1026 - 1028 . https://doi.org/10.1126/science.aat7675 https://doi.org/10.1126/science.aat7675 A single transcription factor promotes both yield and immunity in rice Willmann MR , Poethig RS , 2007 . Conservation and evolution of miRNA regulatory programs in plant development . Curr Opin Plant Biol , 10 ( 5 ): 503 - 511 . https://doi.org/10.1016/j.pbi.2007.07.004 https://doi.org/10.1016/j.pbi.2007.07.004 Conservation and evolution of miRNA regulatory programs in plant development Wu L , Zhang QQ , Zhou HY , et al. , 2009 . Rice microRNA effector complexes and targets . Plant Cell , 21 ( 11 ): 3421 - 3435 . https://doi.org/10.1105/tpc.109.070938 https://doi.org/10.1105/tpc.109.070938 Rice microRNA effector complexes and targets Xie KB , Wu CQ , Xiong LZ , 2006 . Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice . Plant Physiol , 142 ( 1 ): 280 - 293 . https://doi.org/10.1104/pp.106.084475 https://doi.org/10.1104/pp.106.084475 Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice Xie YR , Liu Y , Wang H , et al. , 2017 . Phytochrome-interacting factors directly suppress MIR156 expression to enhance shade-avoidance syndrome in Arabidopsis . Nat Commun , 8 : 348 . https://doi.org/10.1038/s41467-017-00404-y https://doi.org/10.1038/s41467-017-00404-y Phytochrome-interacting factors directly suppress MIR156 expression to enhance shade-avoidance syndrome in Arabidopsis Xu L , Yuan K , Yuan M , et al. , 2020 . Regulation of rice tillering by RNA-directed DNA methylation at miniature inverted-repeat transposable elements . Mol Plant , 13 ( 6 ): 851 - 863 . https://doi.org/10.1016/j.molp.2020.02.009 https://doi.org/10.1016/j.molp.2020.02.009 Regulation of rice tillering by RNA-directed DNA methylation at miniature inverted-repeat transposable elements Yan JP , Chia JC , Sheng HJ , et al. , 2017 . Arabidopsis pollen fertility requires the transcription factors CITF1 and SPL7 that regulate copper delivery to anthers and jasmonic acid synthesis . Plant Cell , 29 ( 12 ): 3012 - 3029 . https://doi.org/10.1105/tpc.17.00363 https://doi.org/10.1105/tpc.17.00363 Arabidopsis pollen fertility requires the transcription factors CITF1 and SPL7 that regulate copper delivery to anthers and jasmonic acid synthesis Yang RX , Li PC , Mei HL , et al. , 2019 . Fine-tuning of miR528 accumulation modulates flowering time in rice . Mol Plant , 12 ( 8 ): 1103 - 1113 . https://doi.org/10.1016/j.molp.2019.04.009 https://doi.org/10.1016/j.molp.2019.04.009 Fine-tuning of miR528 accumulation modulates flowering time in rice Yao SZ , Yang ZR , Yang RX , et al. , 2019 . Transcriptional regulation of miR528 by OsSPL9 orchestrates antiviral response in rice . Mol Plant , 12 ( 8 ): 1114 - 1122 . https://doi.org/10.1016/j.molp.2019.04.010 https://doi.org/10.1016/j.molp.2019.04.010 Transcriptional regulation of miR528 by OsSPL9 orchestrates antiviral response in rice Yu N , Cai WJ , Wang SC , et al. , 2010 . Temporal control of trichome distribution by microRNA156-targeted SPL genes in Arabidopsis thaliana . Plant Cell , 22 ( 7 ): 2322 - 2335 . https://doi.org/10.1105/tpc.109.072579 https://doi.org/10.1105/tpc.109.072579 Temporal control of trichome distribution by microRNA156-targeted SPL genes in Arabidopsis thaliana Yue EK , Li C , Li Y , et al. , 2017 . MiR529a modulates panicle architecture through regulating SQUAMOSA PROMOTER BINDING-LIKE genes in rice ( Oryza sativa ) . Plant Mol Biol , 94 ( 4-5 ): 469 - 480 . https://doi.org/10.1007/s11103-017-0618-4 https://doi.org/10.1007/s11103-017-0618-4 MiR529a modulates panicle architecture through regulating SQUAMOSA PROMOTER BINDING-LIKE genes in rice (Oryza sativa) Zemach A , Kim MY , Silva P , et al. , 2010 . Local DNA hypomethylation activates genes in rice endosperm . Proc Natl Acad Sci USA , 107 ( 43 ): 18729 - 18734 . https://doi.org/10.1073/pnas.1009695107 https://doi.org/10.1073/pnas.1009695107 Local DNA hypomethylation activates genes in rice endosperm Zhang BH , Pan XP , Cobb GP , et al. , 2006 . Plant microRNA: a small regulatory molecule with big impact . Dev Biol , 289 ( 1 ): 3 - 16 . https://doi.org/10.1016/j.ydbio.2005.10.036 https://doi.org/10.1016/j.ydbio.2005.10.036 Plant microRNA: a small regulatory molecule with big impact Zhang GY , Liu X , Quan ZW , et al. , 2012 . Genome sequence of foxtail millet ( Setaria italica ) provides insights into grass evolution and biofuel potential . Nat Biotechnol , 30 ( 6 ): 549 - 554 . https://doi.org/10.1038/nbt.2195 https://doi.org/10.1038/nbt.2195 Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential Zhang Y , Schwarz S , Saedler H , et al. , 2007 . SPL8, a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis . Plant Mol Biol , 63 ( 3 ): 429 - 439 . https://doi.org/10.1007/s11103-006-9099-6 https://doi.org/10.1007/s11103-006-9099-6 SPL8, a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis Zilberman D , Gehring M , Tran RK , et al. , 2007 . Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription . Nat Genet , 39 ( 1 ): 61 - 69 . https://doi.org/10.1038/ng1929 https://doi.org/10.1038/ng1929 Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription Zuker M , 2003 . Mfold web server for nucleic acid folding and hybridization prediction . Nucleic Acids Res , 31 ( 13 ): 3406 - 3415 . https://doi.org/10.1093/nar/gkg595 https://doi.org/10.1093/nar/gkg595 Mfold web server for nucleic acid folding and hybridization prediction Jianhong XU designed the study, wrote and edited the manuscript. Erkui YUE performed the experiments, analyzed the data, and wrote the manuscript. Hua TAO performed the experiments and analyzed the data. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (No. 31901515). 10.1631/jzus.B2000519 1673-1581（2021）05-0366-17 2020-09-01 2020-11-02 2021-05-15 2022-05-09 2021-03-05 © Zhejiang University Press 2021 Erkui YUE, Hua TAO, and Jianhong XU declare that they have no conflict of interests. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 22