Introduction

Cultivated oat (Avena sativa L.; 2n = 6x = 42, AACCDD) has a large (12.5 Gb) hexaploid (6x), repetitive genome with nonhomologous chromosomal exchange and functional divergence of duplicated genes that present significant challenges in the identification and characterization of novel genes1. Oat research has been accelerated recently with the release of a comprehensive annotated genome assembly that deciphered large-scale ancestral chromosome rearrangements including translocations and inversions2. The instability in the genomic structure of oats may be attributed to the absence of an orthologue equivalent to TaZIP4-B2 (located in the Ph1 locus) found in wheat. This orthologue in wheat plays a crucial role in stabilizing the genome structure by inhibiting crossing over among homeologous chromosomes during meiosis.3,4,5. Despite the aforementioned challenges, the recent availability of this high-quality genome assembly acted as a pivotal catalyst to pursue this study.

Transcription factors (TFs) are a huge group of regulators responsible for governing gene expression and act as on–off switches in controlling various developmental processes6. SQUAMOSA promoter binding like (SPL) proteins are plant-specific TFs responsible for the regulation of growth phase transitions including inflorescence development and architecture7. They have a conserved SQUAMOSA promoter binding protein domain (SBP) that comprises 76 amino acids8. The SBP domain binds to a sequence i.e., TNCGTACAA, where GTAC is the crucial core sequence found in the promoter region of the target gene9. The SBP domain consists of three essential motifs i.e., two zinc-binding fingers, Cys3His1 (Zn1) and Cys2HisCys (Zn2), and a bipartite nuclear localization signal (NLS) that moderately protrude over the Zn2 motif, which lies at the C-terminal of the domain8,10. So far, 16 AtSPLs in Arabidopsis thaliana9, 56 TaSPLs in wheat11, 19 OsSPLs in rice12, 17 HvSPLs in barley13 and 18 BdSBPs in Brachypodium distachyon14 have been reported. SPL genes regulate various developmental processes, including panicle architecture15, vegetative to reproductive phase change16, embryogenesis17, fertility18, hormonal signalling19, flowering20, copper homeostasis21 and germination and starch debranching22.

The advancements in the understanding of microRNAs (miRNAs) have introduced an additional layer of gene regulation. The miRNAs are a large group of small (20–24 nucleotides) non-coding RNAs that are initially reported to be involved in the larval differentiation of Caenorhabditis elegans23. Research findings suggest that plant miRNAs play a significant role in the gene regulation network of different developmental stages (review by Wang and Wang)24. MiRNAs target various transcriptional factor-encoding genes, making them a pivotal hub that regulates protein-DNA and protein–protein interactions25. More than half of the SPL transcriptional factors are targets of miRNA156, where antagonistic expression of SPL and miR156 have been observed24. In Arabidopsis thaliana, 10 of the 16 AtSPLs are targets of AtmiR15620, whilst 11 of 19 OsSPLs are targeted by OsmiR156 in rice12. MiR156 is involved in repressing flowering in Populus canadensis26, Chinese cabbage27, Arabis alpina28, and Cardamine flexuosa29. In rice, the overexpression of OsmiR156b delays flowering12, while the transition from the vegetative to the reproductive stage in barley is governed by the antagonistic expression of miR156 and HvSPL genes13. Despite various pathways (photoperiod, gibberellic acid [GA], autonomous, and vernalization) being reported for flowering regulation, the miR156/SPLs describe a new age-dependent pathway. We report the first comprehensive study of the SPL/miR156 regulatory hub in oat in which 28 SPLs (AsSPLs) and 21 novel precursors of miR156 (AsmiR156) were identified. This work includes phylogeny, gene structure analysis, conserved motifs, and AsmiR156 target site prediction. Furthermore, the differential expression of AsSPLs and AsmiR156 at different growth stages reveals their putative role in oat development. Elucidating the SPL/miR156 regulatory hub that governs reproductive transition in oat will aid in engineering heading date and improve oat adaptation to changing environments.

Results

Genome-wide analysis of SQUOMOSA promoter binding protein-like (SPL) gene family in oat

Based on similarity with the barley orthologs (HvSPLs) and the existence of the SBP domain, 28 putative SPLs were found in oat and were classified as AsSPLs. Twenty-five SPLs were identified in the latest Avena sativa cv. Sang genome V1 and three additional SPLs (AsSPL1D, AsSPL6D, AsSPL17D) were identified in the Avena sativa cv. OT3098 V2 genome. As expected, all AsSPLs had three copies in the hexaploid oat genome, except for AsSPL23, which had a single copy in the D genome. The genes were distributed across all 21 chromosomes except for chromosomes 4C and 6D. Interestingly, AsSPL3s, AsSPL6s, AsSPL11s, and AsSPL15s were not situated on homeologous chromosomes, likely due to ancestral chromosome rearrangements. The number of exons ranged from two to eleven, whilst the deduced length of the amino acid sequences varied between 179 and 1114. The majority of the SPLs were potentially localized in the nucleus, three in the chloroplast, two in the endoplasmic reticulum, and one each in vacuoles and extracellular regions (Table 1).

Table 1 Characterization of identified SPL genes in Avena sativa.
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Gene structure of AsSPLs

Examination of the genetic structure diversity of AsSPLs, which includes parameters like gene length, number, and distribution of exons and introns, revealed that at least one intron was found in the centre of the coding region of each AsSPL, and the number of exons in a gene ranged from 2 to 11 (Table 1). AsSPL9C had the longest gene size at 11.8 kb, while AsSPL13D had the shortest at 2.8 kb (Fig. 1A). The motif search in the AsSPL full-length protein sequences reported 10 conserved motifs. Motifs 1, 3, and 4 are present in each AsSPL and constitute the typical SBP domain; however, other motifs are undetermined (Fig. 1B). The AsSPL13s, AsSPL17s, and AsSPL23D contained no motif except those constituting the crucial SBP domain. As expected, the motifs were conserved amongst the homoeologues AsSPLs.

Figure 1
figure 1

Gene structure of AsSPLs and conserved motifs in AsSPL protein sequences. (A) 5′ and 3′ UTRs are represented by dark red boxes; exons and introns are depicted using pink boxes and lines, respectively. The bar scale at the bottom corresponds to the gene size. (B) Distribution of conserved motifs in AsSPLs.

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Phylogenetic analysis and syntenic relationship of AsSPLs

Characteristics of amino acid sequences and evolutionary relationships categorized SPL proteins were into eight groups with the Arabidopsis thaliana protein (AtSPL4) as an outlier (Fig. 2). The absence of any AsSPL members in group 3 and group 7 led to the division of the AsSPL gene family into 6 distinct subfamilies. The subfamilies with the smallest number of AsSPL members were Group 2 and Group 8, which had three members each, followed by Group 5, with four members, whereas the remaining subfamilies had six AsSPLs each. The oat AsSPL11A was closely related to the rice OsSPL4 as compared to its homoeologues AsSPL11C and AsSPL11D.

Figure 2
figure 2

Phylogenetic analysis of SPL protein sequences. The neighbor-joining (NJ) tree of SPL proteins from Arabidopsis thaliana (AtSPL), rice (OsSPL), barely (HvSPL), wheat (TaSPL), sorghum (SbSPL), maize (ZmSPL), Brachypodium distachyon (BdSBP), and oat (AsSPLs). AsSPLs are highlighted using a star.

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Two highly conserved zinc fingers (Zn-1 and Zn-2) and a nuclear localization signal (NLS) were found in the SBP domain (Fig. 3). Interestingly, SPL9A/C/D has a single amino acid mutation in the Zn-1(Cys-Cys-Cys-His), where the fourth histidine is mutated to cysteine. In addition to this, the oat SBP domain also displays significant conservation of specific sequences, namely CQQC, SCR, and RRR, suggesting their importance in the domain’s functionality. Synteny analysis of oat SPL proteins demonstrated synteny amongst most of the AsSPLs except AsSPL1C, AsSPL16A, and AsSPL23D, which were non-syntenic (Fig. 4).

Figure 3
figure 3

Multisequence alignment of SBP domain from AsSPL protein sequences. Two signature Zn fingers (Zn1, Cys3His1; Zn-2, Cys2HisCys) and one NLS region are marked on the top.

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Figure 4
figure 4

Synteny analysis of oat SPL sequences. The chromosomes on A, C, and D genomes are represented by blue, red, and green blocks, respectively. Syntenic relationships amongst all the annotated genes in the oat genome are depicted using grey lines, while synteny between the AsSPLs is shown using blue lines. Non syntenic AsSPLs are highlighted with a red box.

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The cis-regulatory elements in AsSPLs promoter regions

The promoter region (1-kb upstream) of AsSPL genes contained 83 cis-acting elements (Table S1). These elements were further divided into seven groups, named development-related elements, light-response elements, environment stress-related elements, promoter-related elements, hormone-responsive elements, site-binding-related elements, and other elements. Amongst these, light-response-related elements (21) and other elements (22) covered a significant proportion, followed by development-related elements (12). Most AsSPLs were found to contain regulatory elements responsive to abscisic acid (ABRE) and methyl jasmonate (TGACG-motif and CGTCA-motif). However, a small number of genes contained regulatory elements related to other plant hormones, such as gibberellin (P-box, TATC-Box, and GAREmotif), auxin (AuxRR-core and TGA-element), ethylene (ERE), and salicylic acid (TCA-element). Light-response-related element: G-box was the most abundant, while Sp1 was identified in almost half of the AsSPLs. Amongst the development-related elements, a wide range of elements were found to be involved in the regulation of zein metabolism (O2-site), meristem (CAT-box and dOCT), endosperm (GCN4_motif, and AAGAA-motif), seed-specific (RY-element), root-specific (as-1, motif I), and phloem-specific (AC-I, II) expression. Four defence-related elements were identified in a few AsSPLs, including wounding and pathogen responsiveness (W box, box S, WUN-motif, and TC-rich repeats). Besides this, essential elements for anaerobic induction (ARE), anoxic-specific (GC-motif, and LTR) and drought inducibility (MBS) were widely distributed across the AsSPLs. The presence of transcription initiation related promoter elements (TATA-box, and CAAT-box) in all the AsSPLs validates the reliability of our promoter analysis. Finally, 22 other elements with unknown functions were also found out of which STRE and MYB are present in around 94% of the AsSPLs.

To gain a profound understanding of the importance of these cis-acting elements, a Venn diagram analysis was conducted specifically on those elements present in more than 10 genes (Fig. 5). A random distribution of light-responsive and development-related elements was observed among the AsSPLs (Fig. 5A,B). Amongst the hormone-responsive elements, 13 AsSPLs contained ABRE (ACGTG), TGACG-motif, and CGTCA-site at the same time (Fig. 5C). On the contrary, environment stress-related elements, i.e., ARE (AAACCA), and GC-motif (CCCCCG) were together present in only 2 AsSPLs (AsSPL11C and AsSPL17A) (Fig. 5D). Interestingly, out of 28 AsSPLs, 14 contained TATA-box and CAAT-box, while the other 14 had all four promoter-related elements, including CCGTCC motif and A-box (Fig. 5E).

Figure 5
figure 5

Venn analysis of cis-acting elements in the promoter regions of AsSPLs. (A) Development-related elements. (B) Light-responsive elements. (C) Hormone-responsive elements. (D) Environmental stress-related elements. (E) Promoter-related elements.

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AsmiR156/AsSPL module in oat

There is abundant evidence demonstrating a conserved role of miR156/SPL module in critical developmental processes in various crops24. However, there is no published report on miR156 in oat. Genome-wide investigation for miR156 precursors and mature sequences identified 21 putative AsmiR156 genomic loci. The length of precursors ranged from 170 to 264 nucleotides (Table 2 and Table S6), falling in the desirable range for plant miRNA precursors30. With few exceptions, the AsmiR156 precursors were found across all the oat chromosomes. As expected, the precursor sequences were distributed into 7 groups (AsmiR156a-AsmiR156g) of three highly conserved copies in each sub-genome (A, C, and D) (Fig. 6A). Intriguingly, group II and III precursors (AsmiR156b and AsmiR156c) on chromosome 3 A/C/D lie very close to each other (196–244 bp) and may act as a single polycistronic unit.

Table 2 Characteristics of putative pre-miRNA156 in oat.
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Figure 6
figure 6

Oat miR156 (AsmiR156) family members and their target site in oat AsSPL genes. (A) Alignment of mature sequences of seven AsmiR156 family members. The mature sequence of AsmiR156 a/b/c/d/e/f and g are annotated in green. (B) miR156 target site in AsSPL3s, 11 s, 13 s, 16 s, 17 s, and 23 genes. The grey box represents CDS, green box SBP domain and line 3′UTR. The miR156 target sites with the nucleotide positions of AsSPL transcripts are marked in red. RNA sequence of each complementary site from 5′ to 3′ and the predicted miRNA sequence from 3′ to 5′ are indicated.

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AsmiR156 target site prediction identified 16 out of 28 AsSPLs as potential targets of AsmiR156 with putative binding sites in the CDS of eleven genes (Fig. 6B, Table S2). While, AsmiR156 targets five AsSPLs (AsSPL3C, AsSPL11A, AsSPL13A, AsSPL13C, and AsSPL13D) in the 3’UTR. In contrast to AsSPL13s, which exhibited 3' UTR target sites across all homoeologues, only the C and A genome copies of AsSPL3s and AsSPL11s, respectively, had an AsmiR156 target in the 3’UTR. These results indicate a probable post-transcriptional regulation of certain AsSPLs by AsmiR156, hence playing a key role in regulating the gene function in oat.

Expression of AsSPLs and AsmiR156 during vegetative to reproductive transition in oat

To gain an insight into the function of the AsSPLs, the transcript abundance of some AsSPLs and mature AsmiR156 was measured in oat at various growth stages using qRT-PCR. The stages include tillering (GS-22), inflorescence emergence (GS-54), milking (GS-75) and mature seed. The selection of AsSPLs was based on including members from different subgroups to ensure comprehensive coverage of gene diversity. A wide range of differential expression patterns were observed in the selected AsSPLs in different tissues (Fig. 7). A relatively high expression of AsSPL9s was observed at the vegetative stage, i.e., tillering (GS-22) (Fig. 7D). On the contrary, AsSPL1s, AsSPL3s, and AsSPL15s showed lower expression in the vegetative stage (GS-22) but higher in the developing inflorescence stage (GS-54) (Fig. 7A,B,F). Intriguingly, AsSPL3s had an extremely high expression (Fig. 7B) in the inflorescence emergence stage (GS-54) as compared to the vegetative tillering stage (GS-22). As expected, AsmiR156 abundance was higher during the vegetative stage (GS-22) which significantly reduced during the inflorescence emergence (GS-54) stage (Fig. 7G), implying the putative role of AsSPL3s/AsmiR156 in the vegetative to reproductive phase change in oat. The expression profiling was consistent in AsSPL6s and AsSPL11s across the different growth stages. Most of the selected AsSPLs showed comparatively low expression in the mature oat panicles.

Figure 7
figure 7

Expression pattern of AsSPL genes and AsmiR156 during growth phase transition in oat. (A–F) Transcript abundance of AsSPL1s, AsSPL3s, AsSPL6s, AsSPL9s, AsSPL11s and AsSPL15s, (G) AsmiR156. Error bars are indicated. The lowercase letter above the bar indicates the significant difference (P ≤ 0.05), while ** denotes (P < 0.01).

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Discussion

This study presents the pioneer comprehensive genome-wide investigation of the SPL/miR156 hub in oat, identifying 28 oat SPLs (AsSPLs). The oat evolution process involving allohexaploidization has expanded the AsSPL gene family, leading to the formation of gene triplets. Polyploidy provides a fertile ground for gene duplication and neo-functionalization to occur. The additional gene copies resulting from whole genome duplications can undergo mutations and divergence, leading to the acquisition of new functions. This process has a significant role in the species’ evolution and contributes to their ability to acclimate to changing environments. The non-homoeologues distribution of AsSPL3s, AsSPL6s, AsSPL11s, and AsSPL15s can be attributed to various large-scale chromosomal rearrangements in the oat genome, affecting the order and distribution of these AsSPLs in the subgenomes (Fig. 4)2. Although different SPLs may possess variable intron and exon numbers, the first and second exon encodes the SBP domain in plants31.

The close association of genes in the phylogenetic tree can be used to anticipate gene functions. Considering the proximate phylogenetic association between AsSPL11s and OsSPL4, oat SPL11s might regulate grain size and grain yield15. Similarly, AsSPL17A/D shares the same phylogenetic group as OsSPL14s and may promote tillering and branching in oat24. The motif composition of members of each subgroup can support the phylogenetic characterization of AsSPL gene members. The motifs were conserved within the AsSPL homeologs and the members of the same phylogenetic subgroup, but considerable variability was seen amongst the different AsSPL subgroups, which highlights the functional diversity of AsSPL gene members (Figs. 1B and 2). For instance, motif 10 is unique to group 4 AsSPLs (AsSPL3s and AsSPL11s), which may confer a distinct role to these AsSPLs that need further investigation. These results are parallel to the SPL gene studies in wheat32, maize33, and soybean34. All the AsSPLs contained the signature SBP domain with highly conserved motifs, namely Zn-1 and Zn-2 fingers, a NLS region, CQQC, SCR, and RRR. These conserved elements were also seen in the SBP domain of SPL genes in various plant species13,32,35,36. A single amino acid replacement of the fourth His residue to Cys in the first Zn finger of AsSPL9s was found in all the members of subgroup 2, suggesting the presence of special Zn-1 finger in this group that includes SPL gene members from a wide range of species, i.e., Arabidopsis thaliana, maize, wheat, rice, sorghum, barley, and Brachypodium distachyon (Fig. S1). This mutation in the zinc finger binding site may confer a special role to SPL9s. A single mutation in the cysteine and histidine residues of zinc fingers can significantly affect the SPLs binding with the target gene due to large structural changes in the protein8. Similar His to Cys mutation has also been reported in foxtail millet35.

Cis-elements primarily regulate gene expression in organisms that help them acclimate to variable environmental conditions and stresses37. Hence, 1000 bp region upstream of the start codon of AsSPLs was critically examined, and numerous cis-regulatory elements were identified (Table S1). Most of the AsSPLs contained light-response-related elements (Sp1, Box 4, and G-box), indicating the involvement of oat SPLs in plant light-response pathways. The GCN4_motif (TGAGTCA) unique to AsSPL11s, and AsSPL17s, demonstrates their role in endosperm expression. Concomitantly, negative regulation of phloem expression is limited to two C genome AsSPLs (AsSPL9C, and AsSPL15C) controlled by AC-I, and AC-II elements. Besides this, the association of AsSPLs in abiotic and biotic stress-related responses can be attributed to the presence of TC-rich repeats, MBS (drought inducibility), and ARE (anaerobic inducibility) elements. An alfalfa study has also reported improved drought tolerance by miR156-mediated silencing of SPL1338.

Interestingly, a few AsSPLs had cis-regulatory elements for pathogen and wounding response i.e., W box and box S. WRKY transcription factors are widely known for binding to W box element found in the promoter of seven AsSPLs, which activates a dynamic chain of signalling via phosphorylation cascades or kinases39. HvSPL23 was found to positively co-express with the receptor kinase gene, HvWAK1, indicating its potential upstream regulator40. Interestingly, most of the AsSPLs contain auxin- associated elements (AuxRR-core, TGA-box). Auxin is important for root regeneration, callus induction and plant growth. Three SPLs (SPL2, SPL10, and SPL11) have been reported to suppress the expression of AP2/ERFs directly, preventing the auxin buildup in the callus, indicating their role in the auxin synthesis pathway41. Gibberellic acid (GA) is an essential hormone for the vegetative to reproductive phase transition pathways involving the SPL/miR156 module24. AsSPLs contained three gibberellin-responsive elements, i.e., P-box (AsSPL13A, AsSPL13D, AsSPL17C), GARE-motif (AsSPL16A), and TATC-box (AsSPL1C) indicating their potential involvement in the flowering pathways.

The miR156/SPL module appears to have a conserved function of flowering regulation in plant species, where its overexpression leads to a delay in flowering time in rice12, maize42, tomato43, and Arabidopsis thaliana44. The present study reports 21 novel AsmiR156 precursors in oat, which were divided into seven subgroups (AsmiR156 a-g) based on the homology among the precursor sequences (Table 2). The whole genome duplication events like allohexaploidization have an apparent role in the expansion of the miR156 family in oat. The mature sequence of miR156 is conserved amongst the subgroups and other monocots like barley and wheat. However, the proximity (196–244 bp) of group II and group III precursors (AsmiR156b-A/C/D and AsmiR156c-A/C/D) on their respective genomes could indicate their polycistronic nature. According to various genome-wide analyses of various miRNA genes, numerous clustered miRNAs might undergo simultaneous transcription forming a single polycistronic unit25,45,46. In Arabidopsis thaliana, 10 of the 16 AtSPLs are targets of AtmiR15620, whilst 11 of 19 OsSPLs are targets of OsmiR156 in rice12. The target site prediction analysis in oat showed 16 out of 28 AsSPLs as targets of AsmiR156 (Fig. 6B). The majority of AsSPLs possess target sites in the coding region, while five AsSPLs are targeted in the 3’UTR. Especially, AsSPL13s have AsmiR156 target sites in the 3’UTR, which are parallel to HvSPL13 and BdSBP13 in barley and Brachypodium distachyon, respectively. This shows the miR156 target sites are conserved within the homologous genes in different species. Moreover, the differences in AsmiR156 and AsSPL target site sequences were mostly detected in the 14th, 20th, and 21st nucleotides, as observed previously in barley13. Hence, these sites have been under immense selection pressure during the course of evolution, thereby highlighting the critical role of AsmiR156 in governing the expression of AsSPLs in oat.

During plant developmental phases, the expression of SPL genes is upregulated due to the decline in miR156 abundance24. A similar pattern has been observed in AsmiR156 targeted AsSPL3s, where its expression markedly increased during the inflorescence emergence stage (GS-54). Whereas, the downregulation of AsSPL3s during the vegetative stage (GS-22) can be attributed to the abundant AsmiR156, implying the mRNA cleavage (Fig. 7B,G). The barley HvSPL3 and Brachypodium distachyon BdSBP3 also expressed differentially between the vegetative and early reproductive stages13,14. Moreover, the knockout mutants of OsSPL3 and OsSPL4 lead to changes in the heading date, suggesting the conserved role of group 4 SPLs in the flowering47. Nevertheless, the expression of AsSPLs (AsSPL6s, AsSPL15s, AsSPL9s) not targeted by AsmiR156 remained almost consistent over the plant growth stages, validating the conserved role of SPL/miR156 module in inflorescence development and reproductive phase change in oat.

Detailed knowledge of molecular mechanisms that regulate panicle and spikelet development could aid in engineering superior novel architectures and higher yield potential. Rising temperature and climate change scenarios will likely affect pollen viability and fertilization which may reduce yield. Studying such mechanisms can ultimately allow deliberate engineering of flowering time to improve adaptation to changing environments. The current study elucidates the critical role of the miR156/AsSPL hub in developmental phase transitions and panicle development in oat.

Materials and methods

Identification of SPL genes in oat

The GrainGenes (https://wheat.pw.usda.gov/jb?data=/ggds/oat-ot3098v2-pepsico) database was used to obtain the coding (Table S4), genomic (Table S3), and protein sequences (Table S5) of oat SPLs (AsSPLs). The barley SBP domain (Pfam: PF03110) was used as a query to perform tBLASTn against the annotated PepsiCo OT3098 Hexaploid Oat v2 pseudomolecules (2021) and the latest Sang genome2. The SMART tool (http://smart.embl-heidelberg.de/) was used to verify the SBP domain in the SPL protein sequences and subcellular localization of SPL proteins was predicted using the WoLF PSORT tool (https://wolfpsort.hgc.jp/). The accession numbers for AsSPL genes were extracted and putative oat SPLs were named based on their evolutionary relationship with barley SPLs.

Gene structure, phylogeny, and synteny of AsSPL genes

The exonic and intronic regions of each AsSPL gene were obtained using the Gene Structure Display Server program (http://gsds.gao-lab.org/) by comparing their genomic and coding sequences. The SPL sequences of Arabidopsis thaliana were obtained from TAIR (https://www.arabidopsis.org/), whilst the SPL sequences of wheat, barley, Brachypodium distachyon, maize and sorghum were obtained from 1113 to 1452, respectively. The complete SPL protein sequences were aligned using MUSCLE, and a neighbor-joining (NJ) phylogenetic tree was constructed using MEGAv11.0 by setting a bootstrap value of 1000. Further annotation of the phylogenetic tree was performed in iTOL (https://itol.embl.de/). The syntenic relationships amongst the AsSPLs were evaluated using the MCScanX49 and visualized using the shinyCircos-V2.050.

MiRNA156 family in oat and their target site prediction in AsSPLs

Previously reported Hv-miR156 sequence in barley was retrieved from13 to conduct a homology-based search. For short query sequences, the BLASTN algorithm was employed with an increased e-value (E = 10) in the Geneious Prime Software (https://www.geneious.com) for comparison of reference HvmiRNA against the Avena sativa cv. Sang genome. Matches that exhibited a similarity of at least 95% were selected for further analysis. Around 80–250 bp upstream to 80–250 bp downstream regions of the mature miRNA were extracted to obtain pre-miRNA sequences51. M fold software was used to predict fold-back secondary structures for pre-miRNAs52. The pre-miRNA structures were selected based on the criteria by Lu and Yang (2010). psRNATarget tool (http://plantgrn.noble.org/psRNATarget/?function) was used to anticipate the AsmiR156 target sites in cDNA sequences of AsSPLs.

Identification of conserved motifs and Cis-acting elements

The conserved motifs in the AsSPL proteins were identified using default settings in MEME 5.4.1 (http://meme-suite.org/tools/meme), while the maximum width was adjusted to 50, the minimum to 6, and motifs were searched to a maximum number of 10. The Geneious Prime software was used for the creation of the sequence logo of the oat SBP domain sequences (https://www.geneious.com). The 1000 bp upstream sequences (promoter region) of the AsSPLs coding regions were searched for cis-regulatory elements using the PlantCARE database54. Further Venn analysis was carried out for the conserved cis-acting elements using InteractiVenn web-based software55.

Plant material, sample preparation and RNA extraction

The oat cultivar Park obtained from PGRC, Saskatoon, Canada was planted in the growth chambers at Macdonald Campus, McGill University. The plants were grown with a 16:8 photoperiod ratio at day and night temperatures of 22 °C and 15 °C, respectively. A 20:20:20 (nitrogen: phosphorus: potassium) fertilizer was applied after sowing and at the tillering stage to promote plant growth. The young leaf (GS-22), immature panicles (GS-54, GS-75), and mature panicle samples from the oat plants were collected and immediately flash-frozen in liquid nitrogen before storing the samples at − 80 °C. Total RNA was extracted using the modified SDS-LiCl as described in56. Following this, RNA was quantified using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA), and gel electrophoresis was performed to check the purity and integrity.

DNAse I digestion, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)

DNA contamination was removed by DNase I treatment of all samples (Promega, USA). For each sample, 15 min of incubation at 23 °C, followed by the addition of 1 µl 25 mM EDTA to every sample, and the final incubation at 65 °C was performed for 10 min to terminate the reaction. From each sample, 500 ng of RNA was taken to synthesize cDNA using the AffinityScript QPCR cDNA Synthesis Kit (Agilent technology, Canada). For the amplification of miR156, stem-loop RT primer was used for cDNA synthesis. Due to high sequence similarity amongst the protein sequences of AsSPLs homoeologues, common primers for each gene were designed from the coding region 57. AsmiR156 was amplified using the AsmiR156 specific forward primer and universal reverse primer (Table S7). Optical strip tubes were used to perform qRT-PCR analysis using the Mx3000 qPCR system (Stratagene, USA) using 1 μl diluted cDNA, 10 μM gene-specific primers, and 10 μl Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent, USA). The above reaction was carried out by running it for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 30 s at 60 °C. Internal controls included the expression of a reference gene EF1A, which was recommended as the most consistent housekeeping gene amongst different growth stages in oat58. Relative gene expression was determined using the 2 − ΔΔCq method59. qRT-PCR was performed in duplicates and triplicates for AsSPLs and AsmiR156, respectively.

Statistical analysis

One-way analysis of variance (ANOVA) and Tukey's test (P ≤ 0.05) were carried out using JMP Pro 16 software to determine statistical significance amongst the AsSPL expression at different growth stages of oat.

Ethical approval

We confirm that the present research adheres to applicable institutional, national, and international standards and regulations for conducting plant experiments, including the collection of plant material.