Differential expression and global analysis of miR156/SQUAMOSA promoter binding-like proteins (SPL) module in oat

SQUAMOSA promoter binding-like proteins (SPLs) are important transcription factors that influence growth phase transition and reproduction in plants. SPLs are targeted by miR156 but the SPL/miR156 module is completely unknown in oat. We identified 28 oat SPL genes (AsSPLs) distributed across all 21 oat chromosomes except for 4C and 6D. The oat- SPL gene family represented six of eight SPL phylogenetic groups, with no AsSPLs in groups 3 and 7. A novel oat miR156 (AsmiR156) family with 21 precursors divided into 7 groups was characterized. A total of 16 AsSPLs were found to be targeted by AsmiR156. Intriguingly, AsSPL3s showed high transcript abundance during early inflorescence (GS-54), as compared to the lower abundance of AsmiR156, indicating their role in reproductive development. Unravelling the SPL/miR156 regulatory hub and alterations in expression patterns of AsSPLs could provide an essential toolbox for genetic improvement in the cultivated oat.


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.
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).

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 CGTCAmotif).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 www.nature.com/scientificreports/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 (AAA CCA ), and GC-motif (CCC CCG ) 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 CCG TCC motif and A-box (Fig. 5E).

AsmiR156/AsSPL module in oat
There is abundant evidence demonstrating a conserved role of miR156/SPL module in critical developmental processes in various crops 24 .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 precursors 30 .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.
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).

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 nonhomoeologues distribution of AsSPL3s, AsSPL6s, AsSPL11s, and AsSPL15s can be attributed to various largescale 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 plants 31 .
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 yield 15 .Similarly, AsSPL17A/D shares the same phylogenetic group as OsSPL14s and may promote tillering and branching in oat 24 .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 wheat 32 , maize 33 , and soybean 34 .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 species 13,32,35,36 .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 protein 8 .Similar His to Cys mutation has also been reported in foxtail millet 35 .
Cis-elements primarily regulate gene expression in organisms that help them acclimate to variable environmental conditions and stresses 37 .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 lightresponse pathways.The GCN4_motif (TGA GTC A) 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 SPL13 38 .
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 kinases 39 .HvSPL23 was found to positively co-express with the receptor kinase gene, HvWAK1, indicating its potential upstream regulator 40 .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 pathway 41 .Gibberellic acid (GA) is an essential hormone for the vegetative to reproductive phase transition pathways involving the SPL/miR156 module 24 .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.www.nature.com/scientificreports/ 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 rice 12 , maize 42 , tomato 43 , and Arabidopsis thaliana 44 .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 unit 25,45,46 .In Arabidopsis thaliana, 10 of the 16 AtSPLs are targets of AtmiR156 20 , whilst 11 of 19 OsSPLs are targets of OsmiR156 in rice 12 .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 14 th , 20 th , and 21 st nucleotides, as observed previously in barley 13 .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 abundance 24 .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 stages 13,14 .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 flowering 47 .Nevertheless, the expression of AsSPLs (AsSPL6s, AsSPL15s, AsSPL9s) not targeted by AsmiR156 remained almost consistent over  Vol:.( 1234567890

Identification of SPL genes in oat
The GrainGenes (https:// wheat.pw.usda.gov/ jb? data=/ ggds/ oat-ot309 8v2-pepsi co) 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 genome 2 .The SMART tool (http:// smart.emblheide lberg.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:// wolfp sort.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.arabi dopsis.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 MCScanX 49 and visualized using the shinyCircos-V2.0 50.

MiRNA156 family in oat and their target site prediction in AsSPLs
Previously reported Hv-miR156 sequence in barley was retrieved from 13 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.genei ous.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 sequences 51 .M fold software was used to predict fold-back secondary structures for pre-miRNAs 52 .The pre-miRNA structures were selected based on the criteria by Lu and Yang (2010).psRNATarget tool (http:// plant grn.noble.org/ psRNA Targe t/? funct ion) 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:// memesuite.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.genei ous.com).The 1000 bp upstream sequences (promoter region) of the AsSPLs coding regions were searched for cis-regulatory elements using the PlantCARE database 54 .Further Venn analysis was carried out for the conserved cis-acting elements using InteractiVenn web-based software 55 .

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 in 56 .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

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.

Figure 4 . 7 Figure 5 .
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.

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.

Table 1 .
Characterization of identified SPL genes in Avena sativa.a Nomenclature of oat SPLs in this study.b Gene accession number in the oat database.c Length of coding sequence.d Domain prediction by SMART tool.e Number of amino acids in the protein sequence.f Chromosomal location of AsSPL genes.g Chromosomal coordinates of AsSPL genes in the oat genome.h Number of exons in AsSPL genes.i Predicted subcellular location of AsSPL genes.*AsSPLs identified in the Avena sativa cv.OT3098 V2 genome.

Table 2 .
Characteristics of putative pre-miRNA156 in oat.Chr chromosome, Str strand, LP length of pre-miRNA, MFE minimum fold energy, NM number of mismatches between predicted miRNA and miRNA.Vol.:(0123456789) Scientific Reports | (2024) 14:9928 | https://doi.org/10.1038/s41598-024-60739-7 ) 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.