Introduction

Lettuce (Lactuca sativa) is an important food crop belonging to the Asteraceae family that is grown worldwide and widely cultivated in home gardens1. It is primarily consumed in salads, sandwiches, and other dishes due to its mild flavor and high fiber content1. This leafy green vegetable is also a valuable source of vitamin A, iron, and calcium2. The value added products of L. sativa range from fresh-cut, packaged salads to nutraceuticals and supplements. The important value added products include lettuce based beverages and smoothies3,4 and functional food products5.

miRNAs are non-coding RNA molecules that typically contain 18–26 nucleotides6. They play crucial roles in regulating gene expression at the post-transcriptional level7. They negatively regulate gene expression in various tissues and at different times and originate from fold-back stem loops known as pre-miRNAs. The pre-miRNA loop is cleaved, resulting in the formation of a short double-stranded RNA (dsRNA). One strand of this dsRNA subsequently associates with the RISC (RNA-induced silencing complex) to form the mature miRNA8. Depending on the degree of complementarity between the miRNA and its target messenger RNA (mRNA), the RISC complex either hinders translation or triggers mRNA degradation, thereby negatively regulating mRNA production5,9.

The diverse and essential functions of miRNAs have been extensively studied in both animals and plants10,11. This class of small RNAs is now recognized as being widely distributed in animals, plants, and viruses and is involved in numerous biological and developmental processes, including cell differentiation, signaling pathways, stem cell maintenance, regulatory mechanisms, and responses to diseases and environmental stresses3,11,12. The discovery of animal miRNAs prompted researchers to investigate the presence of miRNAs in plant cells as well. Using direct cloning technology in 2002, several researchers independently identified over 100 miRNAs in the model plant Arabidopsis thaliana13. miRNAs have also been reported in tobacco, wheat, rice, corn, and other plants14,15. Most of these miRNAs are found to be highly conserved across different plant species. This conservation can be utilized to identify new homologs in various other species. After survey of miRBase (a database for published miRNA sequences with annotations) it was realized that there isn’t even a sigle miRNA sequences reported in miRBase. However15, have identified 21 miRNAs in lettuce, so this research gap and the substantial number of available lettuce ESTs (205,472) encouraged further exploration for new conserved miRNAs in lettuce. Identification of conserved miRNAs in L. sativa can serve many practical purposes such as understanding gene regulatory networks, improving crop yield and pinpointing genes which are valuable in breeding and genetic engineering strategies.

Methodology

The bioinformatic prediction method, a widely used method for identifying conserved miRNAs through a comparative genomic approach. Following the methodology of14,16,17 with minor modifications new conserved miRNAs were predicted and characterized in lettuce. This involved utilizing several bioinformatics resources and tools, including miRBase, BLASTn, BLASTX, Mfold, psRNA Target, Clustal W, WebLogo, Primer 3, NGphylogeny and Quick GO.

Retrieval of candidate miRNA sequences

For the prediction of probable candidate miRNA sequences, known miRNAs from different plants were sought in well-known miRNA repositories, such as miRBase (https://mirbase.org) and the literature on miRNAs. To conduct a homology/similarity search, previously available mature and pre-miRNA sequences from various plant species such as Cynara cardunculus, Glycine max, Helianthus annuus, Helianthus paradoxus, Helianthus argophyllus, Hevea brasiliensis, Populus trichocarpa, Panax ginseng, Paeonia lactiflora, Salicornia europaea, and Oryza sativa were retrieved from miRBase, and subjected to Blastn against L. sativa Expressed Sequence Tags and Nucleotide databases to predict new conserved miRNAs in lettuce. The potential miRNA candidates were further subjected to BLASTX to remove the protein coding sequences. These candidate sequences were saved for downstream analyses (Table 1).

Table 1 The Lactuca sativa predicted miRNAs are characterized in terms of source miRNAs, Precursor length (PL), Minimum free energy (MFE), Mature sequence (MS), Mature sequence arm (MSA), Mature sequence length (MSL), Number of mismatches (NM), Source EST/Nucleotide (SE/SNT), Data Base (DB).
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Prediction of potential miRNA candidates’ hairpin structures

Prediction of potential miRNA candidates’ hairpin structures was carried out using Mfold (Version 2.3) (http://www.unafold.org/mfold/applications/rna-folding-form.php) to generate miRNA secondary structures with minimum folding energy. The mature miRNA sequences were manually examined, applying specified thresholds for miRNA selection, as used by12,4.

Analysis of identified miRNAs’ conservation and evolutionary relationships

Analysis of recently identified miRNAs’ evolutionary relationships and conservation involved comparing the conservation of a recently identified conserved lettuce miRNA (lsa-miR167) to its orthologs in other plant species. The WebLogo program (https://weblogo.berkeley.edu/logo.cgi) was employed for this purpose, and the output was stored for further examination. For intraspecific phylogenetic studies a circle tree of all 27 miRNAs (predicted in lettuce) was generated to understand the functional clustering and sequences conservation by using publically available phylogeny analysis tool (https://ngphylogeny.fr/).

RT-PCR endorsement

In the RT-PCR endorsement, seven randomly chosen newly analyzed lettuce miRNAs, along with a control sample, were submitted to RT-PCR expressional analysis. Primers were constructed for randomly chosen pre-miRNAs using the Primer 3 algorithm (http://bioinfo.ut.ee/primer3-0.4.0) and primer BLAST as demonstrated in Table 2. Total RNA was extracted from lettuce leaves (Iceberg variety) using a Qiagen plant RNA kit, and cDNA was synthesized using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas). Gradient PCR was performed using a 60 μg cDNA template: initial denaturation for 3 min at 95℃, for 35 cycles; annealing temperature was selected for 35 s at 56℃ to 62℃; extension at 72 for 30 s; and final elongation step at 72℃ for 10 min. PCR products were separated on a 1.7% (w/v) agarose gel.

Table 2 The Lettuce pre-miRNAs primer sequences for RT-PCR experimental validation.
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Predicting miRNA-targeted genes

To predict miRNA-targeted genes, mature miRNA sequences were subjected to psRNATarget (https://www.zhaolab.org/psRNATarget/) to determine miRNA targets. Quick Go was then utilized for functional and enrichment analysis on the anticipated potential lettuce miRNA targets7,15.

Results

The newly predicted lettuce miRNAs

The conserved nature of plant miRNAs plays a significant role in the plant world, allowing the use of known miRNAs from one plant to identify miRNAs in another. A collection of 6,868 angiospermic plant pre-miRNAs from miRBase was utilized, excluding previously identified lettuce miRNAs to prevent redundancy. Bioinformatics prediction resulted in the identification of 27 newly conserved miRNAs belonging to various miRNA families such as MIR160, 165, 166, 167, 168, 319, 394, 403, 408, 862, 1446, 2086, 2118, 3630, 4413, 5523, 5368, 5538, 6111, 6113, 6167, 6255, 6482, 6485, 10,194, 11,602 and 11,604. The experimental approach used by {Baloch, 2018 #18} was employed to validate these miRNAs, confirming their credibility.

Properties and features of lettuce miRNAs

The properties and features of the newly identified lettuce miRNAs were analyzed with parameters such as source database, strand orientation, length of precursors and mature miRNAs, minimum folding energy, mature sequence arm, and number of mismatches as shown in Table 1. These miRNAs demonstrated similarities with various plant families, highlighting their conserved nature. The newly identified pre miRNA secondary structures were also predicted by using MFOLD as shown in Figure Supplementary File S.1. Hence, the MFE of the recently identified lettuce pre-miRNAs stands as a pivotal feature in the characterization of these miRNAs. As prescribed by Mfold the average MFE of the newly identified lettuce miRNAs spanned from-73.30 kcal mol ₋1 to ₋8 kcal mol₋1 with an average of ₋33 kcal mol₋1. Pre-miRNAs with MFE values between ₋74 and ₋64 kcal mol₋1 (3) made up 11% of the total pre-miRNAs based on class boundaries, whereas those in the range of ₋63 to ₋53 (2) made up 7%, ₋52 to ₋42 (2) 7%, ₋41 to ₋31 (5) 19% and ₋19 to ₋9 (7) 26%. Approximately 30% of pre-miRNAs had MFE values between -30 and -20 (8), which is the bulk of pre-miRNAs. The lengths of the preserved lettuce pre-miRNAs ranged from 46 to 211 nucleotides, with an average length of 103 nucleotides. According to classification based on class boundaries the precursor miRNAs ranged from 46–73 nucleotides (9 out of 27) 33%, 74–101 nucleotides (8 out of 27) 30%, 102–129 nucleotides (4 out of 27) 15%, 130–157, 158–185 nucleotide (3 out of 27) 11% and 186–213 nucleotides (3 out of 27) 11%.

The lengths of the newly identified conserved mature lettuce miRNAs been found range from 19–22 nucleotides with an average of 21. Furthermore, class boundaries were falling as 19 nucleotides (1 out of 27) 4%, 20 nucleotides (5 out of 27) 18%, 21 nucleotides (10 out of 27) 37% and 22 nucleotides (11 out of 27) 41% of aggregate. The range of mature sequence lengths seen in lettuce is consistent with that of other plant species’ miRNAs. The current study found that 22 of the 27 newly analyzed miRNAs were found on the sense strand, accounting for 81% of all miRNAs found. 5 of the 27 miRNAs, or 19% of the total miRNA population, are found in the anti-sense strand orientation. 15 mature sequences, or 56% of the total, are found on the stem-loop secondary structure’s 5’ arm, while the remaining 12 mature sequences, or 44% of the total are found on the 3’ arm. The average GC% of the recently predicted lettuce miRNAs ranges from 7 to 41% on the whole.

Amplification and validation of lettuce miRNAs

Experimental validation through RT-PCR was performed for seven randomly selected lettuce miRNAs as illustrated in (Fig. 1). Where six miRNAs were successfully validated, one (lsa-MIR-408) did not show expression. Discrepancies in expression could be attributed to various developmental phases and environmental variables, as observed by other researchers studying different plant species16,18.

Fig. 1
figure 1

Lettuce miRNAs RT-PCR Validation. One control sample and seven Lettuce miRNAs. 1 Control (sample prepared with PCR mix and a set of reverse and forward primers, excluding cDNA template), 2 (lsa-MIR-160), 3 (lsa-MIR-168), 4 (lsa-MIR319), 5 (lsa-MIR403), 6 (lsa-MIR408), 7 (lsa-MIR1446) and 8 (lsa-MIR6111) were chosen and analyzed for RT-PCR expression. On an agarose gel with a 1.7% (w/v) concentration and a 100 base pair DNA leader, the product of each sample was separated.

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Phylogeny and conservation analysis in lettuce miRNAs

The lsa-MIR167 Conservation WebLogo analysis revealed that this newly identified conserved mature miRNA sequence, demonstrates a high degree of conservation across various plant species. This includes Cynara cardanculus, Arabidopsis thaliana, Oryza sativa malus domestica, and cucumis melo (Fig. 2). This suggests that lsa-MIR167 plays an important role in plant biology and is well-preserved throughout these species.

Fig. 2
figure 2

Alignment of the Lettuce pre-miRNAs with their orthologue pre-miRNAs for sequence conservation analyses using WebLogo: a sequence logo generator, showing lsa-miR398 mature miRNA conservation with Cynara cardanculus, Arabidopsis thaliana, Oryza sativa malus domestica, and cucumis melo highlighted in box.

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The circle tree of all 27 miRNAs (Fig. 3). provides insights into the evolutionary insights of conservation and divergence in gene families within same species.

Fig. 3
figure 3

Circle tree showing all predicted miRNAs and their closeness with one another generated with NGphylogeny. The all identified lactuca miRNAs are clustered together based on their similarities. This suggests that miRNAs within the same cluster likely share a common evolutionary origin and may have similar functions. The branches are indicating the evolutionary distance between the miRNAs.

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Prediction of potential targets for Lettuce miRNAs

The prediction of potential targets for lettuce miRNAs is a crucial step in characterizing and annotating these newly discovered miRNAs. A total of 74 targets were predicted, and enrichment analysis using g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) revealed 97 GO terms associated with key processes such as biotic and abiotic stress, growth and development, transcription factors, transporters, and metabolism as described in Table 3.

Table 3 Enrichment GO analysis of putative lettuce miRNA targets within three GO domains, Molecular Function (MF), Biological Process (BP), and Cellular Component (CC).
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The most enriched molecular function terms were purine ribonucleotide binding (GO:0,032,555), oxidoreductase activity (GO:0,016,491), protein kinase activity (GO:0,004,672), hydrolase activity (GO:0,004,672), acyltransferase activity (GO:0,016,746), and glycosyltransferase activity (GO:0,016,757). These results suggest that the miRNAs involved in these processes are likely to play important roles in the cell.

The most enriched biological process terms were organonitrogen compound metabolic process (GO:1,901,564), carbohydrate metabolic process (GO:0,005,975), protein targeting (GO:0,006,605), response to auxin (GO:0,009,733), cellular response to toxic substance (GO:0,097,237), electron transport chain (GO:0,022,900), and isoprenoid metabolic process (GO:0,006,605). These results suggest that the miRNAs involved in these processes are likely to play important roles in cellular metabolism, signaling, and stress response.

The most enriched cellular component terms were plasma membrane (GO:0,005,886), RNA polymerase complex (GO:0,030,880), oxidoreductase complex (GO:1,990,204), microbody (GO:0,042,579), plant-type cell wall (GO:0,009,505), TIM23 mitochondrial import inner membrane translocation complex (GO:0,005,744), and endopeptidase Clp complex (GO:0,009,368). These results suggest that the miRNAs involved in these processes are likely to play important roles in cellular structure and function. (Fig. 4) depicts the enrichment analysis Lettuce miRNA 2086 (one of the predicted miRNAs in this study).

Fig. 4
figure 4

Lettuce miRNA 2086 is associated with the regulation of target gene transcriptional attenuation, impacting various molecular functions (MF), biological processes (BP), and cellular components (CC) as analyzed through QuickGO.

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Discussion

The newly predicted lettuce miRNAs exhibit conserved features, aligning with known miRNA families across diverse plant species. The experimental validation through RT-PCR supports their existence and expression. The analysis of miRNA properties, including length, folding energy, and strand orientation, provides valuable insights into their structural and functional characteristics. Additionally, the prediction of potential targets and their association with important biological processes enhances our understanding of the roles these miRNAs may play in lettuce physiology. The comprehensive approach combining bioinformatics prediction coupled with experimental validation, and functional annotation contributes to the knowledge of miRNA biology in lettuce. Similar results have been reported by many researchers for the miRNAs in different plants and animals17,19,20.

The average MFE of the newly identified lettuce miRNAs was -33 kcal mol-1, which is within the range of MFEs reported for miRNAs from other plant species8,17,21. This suggests that the lettuce miRNAs are likely to be stable in vivo. The lengths of the newly identified lettuce miRNAs ranged from 19 to 22 nucleotides, which is also consistent with the lengths of miRNAs from other plant species2,12,16,22. The majority of the newly identified lettuce miRNAs were found on the sense strand and some of these are also found on antisense strand as well, which is also consistent with the findings of other studies of plant miRNAs3,23.

The identification of miRNA clusters in our study aligns with previous findings, emphasizing their coordinated regulatory significance across diverse cellular processes. The presence of three precursor miRNAs, lsa-MIR165, lsa-MIR168 and lsa-MIR3630 in a cluster with two mature sequences underscores the intricate regulatory potential encoded within these genomic regions, same as described by10,11.

A total of 74 targets were predicted for the newly identified lettuce miRNAs. This suggests that these miRNAs have the potential to regulate a wide range of cellular processes. The identified GO Molecular Function (MF) terms highlight diverse roles played by miRNAs in lettuce, ranging from fundamental cellular activities to specialized biochemical functions. Notably, the conserved miRNAs exhibit a broad spectrum of binding activities, including purine ribonucleotide binding, oxidoreductase activity, and protein kinase activity. This implies their involvement in key cellular processes such as nucleotide metabolism, redox reactions, and signal transduction through protein phosphorylation. The same results were also obtained by4,10,18. Moreover, the enrichment analysis elucidates the participation of miRNAs in various biological processes (BP) within lettuce. These encompass organ nitrogen compound metabolic processes, carbohydrate metabolic processes, and responses to external stimuli like auxin and toxic substances. The conserved miRNAs also play a role in intricate cellular events such as electron transport chain and protein targeting, suggesting their involvement in essential pathways related to energy generation and intracellular trafficking.The analysis further highlights the cellular components (CC) associated with lettuce miRNAs, providing insights into their subcellular localization. These include the plasma membrane, RNA polymerase complex, microbody, and plant-type cell wall, emphasizing the diverse cellular compartments where these miRNAs may exert their regulatory effects.

Conclusion

In summary, this study enhances our understanding of the intricate regulatory networks orchestrated by miRNAs in lettuce, laying the foundation for future investigations into the molecular mechanisms governing these essential regulatory molecules in this important crop species.