miR858a-encoded peptide, miPEP858a, interacts with the miR858a promoter and requires the C-terminus for associated functions

Abstract

MicroRNAs (miRNAs) are key regulators of gene expression and typically processed from primary transcripts (pri-miRNAs). Recent discoveries highlight that certain pri-miRNAs also encode miRNA-encoded peptides (miPEPs), which influence miRNA function. However, the molecular mechanisms underlying miPEP activity, including the specific domains or essential amino acid residues required for their function, remain largely unexplored. In this study, we elucidated that the pri-miR858a-derived peptide, miPEP858a, directly interacts with the promoter of the MIR858 gene in Arabidopsis (Arabidopsis thaliana). Notably, the C-terminal region of miPEP858a, composed of 14 amino acid residues, is critical for its functionality. Through DNA–protein interaction assays, including yeast 1-hybrid, chromatin immunoprecipitation (ChIP-qPCR), electrophoretic mobility shift assay, and promoter–reporter analyses, we demonstrated that miPEP858a binds to a specific region within the MIR858 promoter. Exogenous application of a synthetic peptide corresponding to the C-terminal region of miPEP858a resulted in enhanced MIR858 expression, leading to phenotypic changes similar to those observed with the full-length miPEP858a. Moreover, the truncated C-terminal peptide was able to complement mutant plants lacking endogenous miPEP858a, emphasizing its role in regulating miR858a expression and downstream target genes involved in flavonoid biosynthesis and plant development. These findings suggest that the full-length miPEP858a may not be necessary for its biological function, with the C-terminal region being sufficient to modulate miRNA expression. This discovery reveals opportunities for identifying functional domains in other miPEPs, potentially reducing peptide synthesis costs, and offering a more efficient strategy for enhancing agronomic traits in crop plants without the need for complex biotechnological interventions.

Introduction

The increasing world population is challenging and always concerned with food safety and security. Due to poor yield, drastic changes in environmental conditions affect crop production, and food security is debatable nowadays. Thus, modern science, especially biotechnology, is paving the way for this emerging issue to ensure food security and higher agricultural production with lesser yield losses (Chakraborty and Newton 2011; Zhang et al. 2020; Moore et al. 2021; Li et al. 2022). It has been estimated that about one-third of agricultural production depends upon the use of pesticides, impacting human health, and the rest is affected by climate change (Tudi et al. 2021). Thus, crop improvement with novel, easy, and safe methods is the utmost requirement to meet the demands of the population (Ormancey et al. 2021; Ormancey et al. 2023; Sharma et al. 2024). Advancements in computational approaches, peptidomics, and transcriptomics allow us to identify the various regulatory peptides in plants that can be potent tools to improve agronomic traits (Tavormina et al. 2015; Fabre et al. 2021).

It has been well demonstrated that primary transcripts of miRNA encode regulatory peptides called microRNA-encoded peptides (miPEPs) (Lauressergues et al. 2015). In soybean (Glycine max), it has been observed that miPEP172c increases the expression of miR172c, thus ultimately increasing nodule number (Couzigou et al. 2016). Functional characterization of small peptide encoded by miR171d, vvi-miPEP171d, promotes the adventitious root development by activating the expression of vvi-miR171d (Chen et al. 2020). Exogenous application of miPEP164c is also known to be involved in inhibiting the proanthocyanidin content through acting on its pathway and facilitating the accumulation of anthocyanin biosynthesis (Vale et al. 2021). Another fully characterized miR858a-encoded peptide, miPEP858, regulates the expression of miR858 and target genes, resulting in modulated levels of flavonoids due to changes in the expression of genes associated with phenylpropanoid pathway, auxin signaling, and regulated by light-dependent transcription factor, ELONGATED HYPOCOTYL 5 (HY5) (Sharma et al. 2020; Sharma et al. 2022). Another report suggests the involvement of miR408, which targets a glutathione S-transferase GSTU25, and exogenous application of miR408-encoded peptide, miPEP408, plays a role in sulfur assimilation and detoxifying the environmental pollutant (Kumar et al. 2023). The external application of synthetic Vvi-miPEP172b and Vvi-miPEP3635b to the grape plantlets enhances cold tolerance compared with control conditions (Chen et al. 2022). Till now, little is known about the mechanism of action of miPEPs; however, the molecular basis of miPEP specificity has been deciphered that suggests the miPEP activity relies on its own miORF (Lauressergues et al. 2022). Altogether, these studies suggest the potential role of these miPEPs in enhancing desired traits in several crop plants. In addition, previous studies on miPEPs confined to identification, characterizing their function and effects of exogenous treatment for enhancing the phenotype and other associated functions.

Major bottlenecks regarding the synthesis of peptides, even in smaller concentration, are quite expensive and difficult to adopt for agricultural produce (Feng et al. 2023). The majority of small signaling peptides undergo C-terminal processing to produce mature signals (Matsubayashi 2011; Matsubayashi 2014). A study revealed that AtPEP3 contains a minimal functional fragment capable of enhancing tolerance to salinity stress (Nakaminami et al. 2018). In a recent study, Ormancey et al. (2024) demonstrated that truncated miPEPs are active in increasing the expression of their nascent pri-miRNA. Our earlier study showed that exogenous application of miPEP858 affects flavonoid biosynthesis in addition to plant growth and development. Therefore, we hypothesized whether a functional minimal fragment in miPEP858 is cost-effective and functions similarly to miPEP858.

We report that miPEP858 interacts with the promoter of the MIR858 gene, with the C-terminal region of miPEP858 being crucial for regulating both miR858 and its target gene expression. Previous studies have demonstrated that the miR858-encoded peptide, miPEP858, plays a role in regulating the expression of key R2R3-MYB transcription factors (MYB111, MYB11, MYB12) and their associated gene functions (Sharma et al. 2016; Piya et al. 2017; Tirumalai et al. 2019; Sharma et al. 2020). In our study, we dissected full-length miPEP858 into distinct fragments and individually analyzed their functions. Our results on internalization efficiency and promoter interaction reveal that although the N-terminal region of miPEP858 is internalized by plant cells, it does not interact with the promoter to regulate MIR858 expression. In contrast, the C-terminal region of miPEP858 exhibited the highest responsiveness, as demonstrated through exogenous application of truncated peptides. Promoter–reporter analysis following the application of these truncated peptides showed that the C-terminal peptides induced GUS expression at levels comparable to full-length miPEP858a. Complementation of miPEP858a^CR lines with the C-terminal peptide also restored growth-related phenotypes, further confirming the functional importance of this region. Moreover, we observed significant effects on CHS and MYB12 protein levels, as the C-terminal peptide led to increased expression of mature and pre-miR858, which in turn reduced the accumulation of target proteins. This study highlights the critical role of the C-terminal region of miPEP858 in modulating gene expression and underscores the potential of miPEPs in agriculture. Identifying minimal functional fragments of miPEPs in other crops could pave the way for utilizing these peptides to enhance agronomic traits across a variety of species.

Results

C terminus of miPEP858a interacts on miR858a promoter and regulates expression

Several reports have been published on the characterization of plant miPEPs; however, the molecular mechanism underlying miPEP action remains elusive. The application of exogenous miPEP to promoter–reporter lines has been shown to enhance reporter expression (Sharma et al. 2020; Kumar et al. 2023). Notably, recent evidence in animals indicates the regulation of miR-31 expression through its encoded peptide, miPEP31 (Zhou et al. 2022). MiPEP31 functions as a transcriptional repressor, negatively influencing miR-31 expression by binding to its promoter region. In Arabidopsis, promoter/reporter studies of miPEP858a also support enhanced GUS gene activity and expression upon peptide supplementation. This implies that miPEP858a can bind to the promoter region of miR858a, thereby regulating its promoter activity. To unravel the molecular mechanism of miPEP function, various approaches were employed, including the exploration of possible mechanisms such as the modeling of 3D structures and its binding on the promoter. Bioinformatics analysis suggests 3 putative binding sites (R1, R2, and R3) for miPEP858a on the miR858a promoter (Fig. 1A and Supplementary Fig. S1).

To further substantiate the interaction between miR858 promoter and miPEP858a, we conducted a ChIP assay using miPEP858a overexpression (miPEP858aOX) and miPEP858a knockout (miPEP858a^CR) lines. In this study, the miPEP858a^CR line possesses a truncated miPEP858 of 26 amino acids, resulting from a single-nucleotide deletion that introduced a stop codon (Sharma et al. 2020). To confirm the presence of the truncated miPEP858a protein in planta, a western blot analysis was performed using both N-terminal- and C-terminal-specific antibodies (Supplementary Fig. S2). The results confirmed the accumulation of the truncated N-terminal peptide in the mutant lines. Despite the presence of this truncated peptide, the miPEP858a^CR plants exhibited significant alterations at both phenotypic and molecular levels (Sharma et al. 2020). ChIP-qPCR analysis of the wild-type (WT) and miPEP858aOX line revealed a significant enrichment of DNA fragments containing the R1 binding site, whereas no such enrichment was observed for fragments containing the other 2 putative binding sites (R2 and R3) (Fig. 1, B and C). In contrast, none of the fragments (R1, R2, or R3) showed enhanced enrichment in the miPEP858a^CR line (Fig. 1D), indicating that the R1 site plays a crucial role in miPEP858a-mediated promoter interaction. These findings also suggest that the C-terminal region of miPEP858a likely might play a critical role in its functionality, potentially through its interaction with the promoter, which may be essential for proper regulatory activity.

To elucidate the in vivo molecular mechanism underlying miPEP858a activity, a yeast 1-hybrid (Y1H) assay was performed to assess the interaction between the PromiR858a sequence (used as bait) and the 44-amino acid miPEP858a protein (prey). The Y1H assay demonstrated a strong and specific interaction between PromiR858a and miPEP858a, suggesting direct involvement of miPEP858a in promoter regulation (Fig. 1E). To confirm the functional importance of the R1 binding site, we generated a promoter construct with the R1 sequence deleted (ΔR1PromiR858a) and performed a Y1H assay with miPEP858a. The results showed a complete loss of interaction between ΔR1PromiR858a and miPEP858a, affirming that the R1 binding site is indispensable for the binding of miPEP858a to its promoter (Fig. 1E). To confirm the binding specificity of miPEP858a to the miR858a promoter, a Y1H assay was performed using the PromiR858a sequence alongside another miPEP (miPEP-X). The results revealed a lack of interaction between PromiR858a and miPEP-X, thereby validating the specific binding of miPEP858a to the miR858a promoter (Supplementary Fig. S3).

To determine whether the full-length miPEP858a or a specific region, particularly the C-terminal amino acids, is required for promoter binding, interaction studies were conducted using truncated fragments of miPEP858a (Fig. 1F). The fragments included the first 22 amino acids from the N terminus (1H) and the remaining 22 amino acids from the C terminus (2H) (Supplementary Fig. S4). Y1H assay results demonstrated that the C-terminal fragment (2H) of miPEP858a successfully interacted with the promoter, while the N-terminal fragment (1H) failed to do so (Fig. 1F). Additionally, a shorter fragment from the C-terminal region (3F), consisting of 14 amino acids, was also tested in the interaction study. Remarkably, the 3F fragment maintained its ability to bind PromiR858a, suggesting that a minimal C-terminal region is sufficient for interaction. In contrast, when the Y1H assay was performed using ΔR1PromiR858a (promoter lacking the R1 binding site) with full-length miPEP858a or the truncated fragments (1H, 2H, and 3F), no interaction was detected (Fig. 1F and Supplementary Fig. S5), reinforcing the critical role of the R1 site in binding. These findings, in combination with the ChIP assay results using the miPEP858a^CR line, which retains the intact N-terminal sequence, confirm that the C-terminal region of miPEP858a is essential for promoter interaction and functionality.

To confirm the direct binding of miPEP858a to the miR858a promoter, an in vitro electrophoretic mobility shift assay (EMSA) was performed. The results demonstrated a significant shift when miPEP858a was incubated with the R1 probe, whereas no shift was observed with the R2 or R3 probes under similar conditions. This result indicates that miPEP858a specifically binds to the miR858a promoter (Fig. 2A). This finding was further corroborated using a transient expression assay with various promoter–reporter constructs (Fig. 2B and Supplementary Fig. S6). Histochemical analysis revealed that the PromiR858a:miPEP::GUS construct exhibited strong GUS activity, which was significantly enhanced when coinfiltrated with miPEP858aOX, similar to the observed GUS expression in the stable miPEP858aOX line (Fig. 2, C and D). In contrast, the ΔR1PromiR858a:miPEP::GUS construct, which lacks the R1 binding site, showed a complete loss of GUS activity. Notably, this loss of activity could not be restored even after coinfiltration with miPEP858aOX, underscoring the essential role of the R1 binding site in mediating the biological function of the miRNA promoter (Fig. 2, C and D).

This finding was further validated using a transient expression assay with miPEP858a^CR lines and the PromiR858a:miPEP::GUS construct, which exhibited negligible GUS activity. Based on these results, it was hypothesized that if the C-terminal domain of miPEP858a is crucial for promoter binding, GUS activity should be restored when coinfiltrated with miPEP858aOX. As anticipated, coinfiltration of miPEP858aOX with the PromiR858a:miPEP::GUS construct in the miPEP858a^CR line led to a significant restoration of GUS activity, confirming that the C-terminal region is indispensable for promoter interaction and functional activity. These results collectively demonstrate that miPEP858a has the capacity to bind to its own promoter, with the C-terminal region playing a key role in this binding. Moreover, this interaction is crucial for miR858a's function, suggesting that miPEP858a binding regulates the expression of miR858a by influencing promoter activity.

C terminus of miPEP858a is required for miPEP858 function

The primary transcript of miRNA has been found to encode miRNA-encoded peptides (miPEPs), which enhance the transcription of their corresponding miRNAs (Lauressergues et al. 2015; Gautam et al. 2023). However, specific regions or domains on miPEPs responsible for their activities have not been identified as yet. To identify functional domains within miPEPs or their potential as full-length peptides, we focused on miPEP858a, known to modulate the expression of miR858 and its target genes. Two truncated peptides, 1H (N-terminal 22 amino acids) and 2H (C-terminal 22 amino acids), were designed (Fig. 3A). Exogenous application of miPEP858a enhances primary root length (Sharma et al. 2020), leading us to hypothesize that these truncated peptides would exhibit similar functionality if active. Root length data revealed that 1H had no significant effect, while 2H significantly increased root length in a concentration-dependent manner compared with control seedlings (Fig. 3, B to E).

Additionally, smaller truncated peptides were designed by fragmenting the full-length peptide into 3 segments: 1F (N terminus, 15 amino acids), 2F (middle region, 15 amino acids), and 3F (C terminus, 14 amino acids) (Fig. 3A). Growing WT seeds on media supplemented with these peptides at 2 concentrations indicated that 3F had the potential to enhance root length, similar to miPEP858a, while 1F and 2F showed no significant changes in root length (Fig. 3, F to I).

To address the lack of observed effects from the truncated peptides 1H, 1F, and 2F, 2 potential explanations were identified: either the peptide concentrations used were insufficient to modulate miR858a expression and its related phenotypes, or the peptides were not efficiently internalized by plant cells. To explore these possibilities, WT seeds were grown on media supplemented with higher concentrations of 1H, 1F, and 2F peptides (ranging from 0.1 to 100 µM). Despite this, no significant changes were observed in root length or in the expression levels of miR858a and its target genes (Supplementary Figs. S7 and S8). To assess peptide uptake, fluorescent carboxyfluorescein (FAM)-labeled truncated peptides were used, followed by confocal microscopy. The imaging results confirmed that the 1H peptide fragment could enter plant cells, similar to the truncated functional peptides 2H and 3F (Fig. 3J and Supplementary Fig. S9). To investigate whether the N-terminal (1H) or C-terminal (2H) peptides localize to the nucleus, we incubated seedlings with FAM-labeled peptides (both N terminus and C terminus) and subsequently stained them with DAPI to visualize the nucleus. Interestingly, we observed that both peptides were present within the nucleus, indicating their nuclear localization (Supplementary Fig. S10). These findings suggest that while 1H can be internalized, its inability to influence miR858a-related phenotypes may be due to factors other than internalization. In contrast, peptides 2H and 3F, which retain key C-terminal amino acids, were more effective in modulating root length and expression patterns similar to miPEP858a.

Modulation of miR858 and associated gene expression by truncated peptides

Previously, full-length miPEP858 was recognized for enhancing miR858 expression and subsequently downregulating its target and associated genes (Sharma et al. 2020). Building on our initial findings, we hypothesized that if 2H and 3F can enhance root length, they may similarly impact miR858 expression. Expression analysis revealed a significant increase in mature miR858a expression when treated with 2H and 3F, while other truncated peptides showed no significant difference compared with control seedlings (Fig. 4A). Additionally, analysis of pre-miR858, target, and associated genes demonstrated elevated pre-miR858 expression and significant downregulation of target genes (Fig. 4B). To validate these results, we examined MYB12 and CHS protein accumulation using specific antibodies. Functional truncated peptides (2H and 3F) led to a significant decrease in protein levels, akin to full-length miPEP858, compared with other truncated peptides (Fig. 4C). Since miR858 targets genes in the phenylpropanoid pathway (Sharma et al. 2016), our results indicated the downregulation of genes encoding MYB12 and CHS upon supplementation with 2H and 3F truncated peptides. Additionally, we analyzed the expression of other pathway genes with all truncated peptides. The results suggested significant downregulation with 2H and 3F, and no significant modulation with 1H, 1F, and 2F truncated peptides (Supplementary Fig. S11). In summary, these findings highlight the crucial role of the minimal region 2H and 3F truncated peptides in downregulating the expression of genes involved in the phenylpropanoid pathway.

Functional truncated peptides modulate metabolite content

Exogenous application of C-terminal truncated peptides enhanced the expression of key genes in the phenylpropanoid pathway, prompting an analysis of metabolite accumulation. Total flavonol content analysis indicated a significant decrease in seedlings treated with functional truncated peptides (2H and 3F) compared with those supplemented with nonfunctional truncated peptides (Fig. 4D). In-depth HPLC analysis quantifying kaempferol and quercetin levels revealed a significant decrease in seedlings treated with functional truncated peptides compared with the control (Fig. 4, E and F and Supplementary Fig. S12). Total anthocyanin analysis suggested reduced accumulation in seedlings grown on media supplemented with the C-terminal peptide (Fig. 4G). In line with our previous study indicating increased flavonol and anthocyanin levels at the expense of lignin production, we analyzed lignin levels under different growth conditions. Cross-sectional staining of WT plants treated twice with truncated peptide confirmed significantly enhanced lignification in interfascicular and vascular tissues (Fig. 4H). Additionally, the length of interfascicular fibers and vascular bundles increased when supplemented with 2H and 3F truncated peptides (Fig. 4, I and J). These results underscore the potential of the functional region (2H and 3F) of miPEP858 to significantly alter metabolite levels.

Clathrin-mediated endocytosis is required for truncated peptides for internalization

Functional truncated peptides (2H and 3F) have demonstrated significant effects on key genes in the phenylpropanoid pathway, influencing plant growth and development. Previous studies have indicated the necessity of clathrin-mediated internalization for miPEP entry into plant cells (Ormancey et al. 2020; Sharma et al. 2020; Badola et al. 2022). To investigate the entry mode of truncated peptides, FAM-labeled 2H and 3F peptides were synthesized. WT and clathrin mutant (chc1, chc1.2, chc2, chc2.2) seedlings were incubated with these peptides, and microscopic observation revealed fluorescence within plant cells in WT seedlings, while clathrin mutants showed no internal fluorescence (Fig. 4K and Supplementary Figs. S13 and S14). Furthermore, clathrin mutants were grown on media with and without truncated peptides. Root length analysis indicated no effect on the root length of clathrin mutants with the exogenous application of C-terminal peptide and other nonfunctional truncated peptides (Supplementary Figs. S15 and S16). In conclusion, our results confirm clathrin-mediated endocytosis of FAM-labeled 2H and 3F peptides, facilitating internalization into plant cells, similar to previous findings with full-length miPEP858a.

C-terminal peptides restore miPEP858a function in miPEP858^CR plants

The exogenous application of miPEP858 is known to complement the phenotype and molecular function of miPEP858^CR lines (Sharma et al. 2020). To study whether truncated peptides can restore the phenotype of miPEP858^CR seedlings, miPEP858^CR and WT seedlings were grown on media supplemented with nonfunctional and functional truncated peptides. The results suggested the increase in root length and overall development of the miPEP858^CR seedling supplemented with functional truncated peptides (2H and 3F) along with full-length miPEP858 as a positive control (Fig. 5, A to D). To further determine that functional truncated peptides affect miPEP858^CR seedlings via miR858-associated pathway, miR858^CR seedlings were grown on media supplemented with functional truncated peptides. No modulation in the root length of miR858^CR lines was observed when supplemented with truncated peptides along with miPEP858 (Supplementary Fig. S17). This suggests that truncated peptides modulate the phenotype through miR858-associated pathway.

The effect of truncated peptides on root length and development of miPEP858^CR further prompted us to demonstrate the effect of these functional truncated peptides on the mature WT and miPEP858^CR plants (Fig. 5E). Exogenous application of truncated peptides suggested the early bolting phenotype in plants treated with truncated peptides than the plants treated with water (Fig. 5F). Since the overall growth was enhanced after external application of truncated peptides, we also analyze the rosette diameter of the Col-0 and miPEP858^CR plants treated with the functional truncated peptides. Analysis suggested the overall increase in rosette diameter of peptide-treated WT as well as miPEP858^CR plants (Fig. 5G). Together, these results suggest that the exogenous application of functional truncated peptides has the potential to complement miPEP858^CR at the phenotypic level.

To investigate the molecular impact of functional truncated peptides on miPEP^CR lines, we analyzed the expression of miR858 and its target and associated genes in miPEP858^CR seedlings supplemented with 2H and 3F peptides. The expression analysis suggests the increase in expression of miR858 and decrease in expression of its target and associated genes (MYB12, CHS) in miPEP858^CR seedlings when supplemented with the 2H and 3F truncated peptides (Fig. 6A). We also studied the MYB12 and CHS protein accumulation in response to exogenous supplementation of functional truncated peptides, and it suggested the lesser protein accumulation in miPEP858^CR seedlings treated with 2H and 3F peptide compared with control (water), miPEP858^CR, and WT seedlings (Fig. 6B). Lignin staining of cross sections of 35-d-old mature stem under the microscope revealed lesser lignification in the vascular tissues and interfascicular fibers of lignin in miPEP858^CR control plants compared with the plants treated with functional 2H and 3F truncated peptides (Fig. 6C). Measurement of length of vascular bundle and interfascicular fiber suggested an increase in length of vascular bundle and interfascicular bundle when treated with functional truncated peptide compared with control plants (Fig. 6, D and E). Estimation of total flavonols and anthocyanins content also suggested that the exogenous supplementation of functional truncated peptide led to a decrease in flavonols and anthocyanins content in miPEP858^CR seedlings compared with water-treated miPEP858^CR seedlings (Fig. 6, F and G). These results suggest that the exogenous application of C-terminal peptide has the potential to complement the function of miR858 in miPEP858^CR lines and also lead to modulation in gene expression associated with the phenylpropanoid pathway.

C-terminal truncated peptides regulate PSK4 expression and associated phenotypes

miPEP858 is recognized for upregulating miR858 transcription, and the exogenous application of miPEP858 to promoter lines enhances the expression of the reporter gene (Sharma et al. 2020). Expanding our investigation, we examined the impact of truncated peptides on miPEP858 promoter lines. GUS histochemical staining of miPEP858 promoter lines (Pro:ATG1::GUS and Pro:ORF1::GUS) supplemented with truncated peptides indicated increased GUS expression in promoter/reporter lines treated with functional 2H and 3F peptides. Conversely, other truncated peptides showed no significant change in histochemical GUS staining (Fig. 7, A and C). These findings were further corroborated by expression analysis of the GUS gene, revealing increased expression in ATG1 and ORF promoter seedlings treated with 2H and 3F truncated peptides (Fig. 7, B and D).

It has been reported that synthetic miPEP858 enhances the expression of the PSK4 transcript (Badola et al. 2022). To assess the impact of truncated peptides on PSK4 expression, WT seedlings were treated with truncated peptides, and the analysis revealed an increased PSK4 transcript only in response to the supplementation of 2H and 3F functional truncated peptides. In contrast, other truncated peptides failed to modulate the expression of the PSK4 gene (Fig. 7E). The analysis of auxin-related genes indicated a significant increase in their expression in seedlings treated with 2H and 3F peptides. Conversely, nonfunctional truncated peptides did not induce changes in auxin-related gene expression (Fig. 7F and Supplementary Fig. S18A). Considering that PSK4 overexpression lines lead to increased expression of growth-related expansins (Badola et al. 2022), we analyzed the expression of these expansins in truncated peptide-treated seedlings. The analysis suggested an increase in the expression of growth-related expansins in WT seedlings supplemented with 2H and 3F functional truncated peptides (Fig. 7G and Supplementary Fig. S18B). Exogenous application of synthetic miPEP858 increases GUS expression in PSK4 promoter seedlings; therefore, we performed histochemical GUS staining and expression analysis of the GUS gene in PSK4 promoter lines. Both histochemical GUS staining and expression analysis indicated an increase in GUS gene expression in PSK4 promoter seedlings supplemented with 2H and 3F functional truncated peptides (Fig. 7, H and I). Overall, these results suggest the potential of truncated functional peptides to enhance miPEP858 and PSK4 expression, along with various growth-related genes (auxin- and growth-related expansins), thereby influencing plant growth and development.

Discussion

Small signaling peptides fall into 2 categories: secreted and nonsecreted peptides, serving as vital signaling molecules for cell-to-cell communication. Many of these peptides are translated as precursor proteins, giving rise to mature peptides that function in diverse developmental and physiological processes (Matsubayashi and Sakagami 2006; Murphy et al. 2012; Anyatama et al. 2024). Small peptides, such as PHYTOSULFOKINE (PSK), CLAVATA (CLE), INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), C-TERMININALLY ENCODED PEPTIDE (CEP), and ROOT MERISTEM GROWTH FACTOR (RGF), play crucial roles in root development, stomatal conductance, abscission processes, stem cell maintenance, root elongation, and stress response (Aalen et al. 2013; Qian et al. 2018; Takahashi et al. 2018; Olsson et al. 2019; Datta et al. 2024). Typically, small signaling peptides, whether secreted or nonsecreted, undergo proteolytic processing to generate a mature signal peptide, which is then posttranslationally modified. The N-terminal segment processes small signaling region, with their functional C-terminal regions responsible for their biological functions. In the case of nonsecreted peptides, these molecules are released directly from cells, executing their functions without the need for processing and maturation (Matsubayashi 2011). One example is AtPEP1, a small peptide that triggers signals for the innate immune response against pathogen attacks (Yamaguchi et al. 2006). Similarly, AtPEP3, another peptide from the same family, is involved in salinity stress tolerance. Its C-terminal peptide is activated in response to salinity, inhibiting salinity-induced chlorophyll bleaching during stress conditions (Nakaminami et al. 2018). In summary, small peptides play significant roles in plant developmental processes and responses to stress.

miRNA858 has the potential to regulate the phenylpropanoid pathway by targeting MYB transcription factors (MYB111, MYB11, MYB12) (Sharma et al. 2016; Singh et al. 2025). The primary sequence of miR858 encodes a regulatory peptide, miPEP858. Exogenous application of miPEP858a not only affects plant growth and development but also modulates secondary plant products (Sharma et al. 2020). While miR858 plays a role in the phenylpropanoid pathway, major growth changes depend on downstream molecular components, including the small signaling peptide PSK4. The crosstalk between AtPSK4 and miR858/MYB3 is crucial for plant growth and development, underscoring the significance of these small peptides. Despite this, exogenous application of miPEP858a significantly induces the expression of the reporter gene GUS in Pro:PSK4 lines, highlighting the importance of miPEP858 in controlling the PSK4 peptide (Badola et al. 2022). In summary, small peptides effectively regulate various developmental and physiological processes. In our previous study (Sharma et al. 2020), CRISPR–Cas9-based mutants were developed, resulting in edited lines with altered growth patterns and secondary metabolite levels. Sequence analysis of miPEP858-edited lines revealed a truncated amino acid sequence at the C terminus while maintaining the intact N terminus, emphasizing the essential role of C-terminal amino acids in the functionality of miPEP858a.

While some miPEPs play known roles in various developmental processes, their exogenous application for controlling desired traits in plants can be prohibitively expensive. Custom-made synthetic peptides are costly, and achieving an optimal concentration for a functional phenotype often necessitates a higher peptide amount for exogenous treatment. To address the cost concern, a minimal 14-amino-acid fragment of miPEP858 has been identified, reducing synthesis expenses while providing a similar effect as the full-length 44-amino-acid miPEP858. This minimal fragment has been demonstrated to exhibit a comparable phenotype and modulation in the accumulation of flavonols/anthocyanins, similar to full-length miPEP858. To identify functional truncated peptides, several designs were explored from the N- and C-terminal ends of miPEP858, including 1st half, 2nd half, 1st fragment, 2nd fragment, and 3rd fragment (Fig. 3A). Our ChIP and Y1H analyses revealed that peptides from the C-terminal end (2H and 3F) provided a response similar to miPEP858 through binding to specific region in the promoter. Hence, it was hypothesized that 2H and 3F fragments could serve as functional truncated peptides, influencing the phenotype and modulating the expression of flavonoid biosynthesis pathway genes. Expression analysis of miR858 indicated an increase in expression in response to 2H and 3F. Additionally, significant changes in the expression of MYB12 and CHS were observed in response to functional truncated peptides compared with the other 3 nonfunctional peptides, with lower protein accumulation in the case of functional truncated peptides (Fig. 4, A to C and Supplementary Fig. S11). Based on this, we hypothesized that if the C terminus is functional in modulating phenotypes and regulating the expression of key genes, it would lead to an increase in GUS expression. As expected, enhanced GUS staining was observed when promoter seedlings were treated with 2H and 3F peptides in combination with miPEP858 (Fig. 7, A to D). This result suggests that the C terminus exhibits functionality similar to the full-length miPEP858.

This research underscores the significance of the C-terminal end of miPEP858, housing crucial functional amino acids essential for the phenotype and modulation of secondary plant products, particularly flavonols and anthocyanins. Clathrin-mediated endocytosis is a prominent internalization pathway in plants (Dhonukshe et al. 2007; Kitakura et al. 2011), with evidence suggesting that miPEP165 and miPEP858 are internalized via this mechanism. To explore the uptake of truncated peptides, FAM-labeled nonfunctional (1H) and functional peptides (3F and 2H) were incubated with plant cells. The results revealed an uptake pattern similar to that observed for miPEP165 and miPEP858 (Ormancey et al. 2020; Badola et al. 2022). This also suggested that nonfunctional N-terminal peptide (1H) is taken up by the plants, but does not function as C-terminal peptides (2H and 3F).

Earlier report hypothesized that miPEP can enhance and regulate the activity of its own promoter (Waterhouse and Hellens 2015). Consistent with this hypothesis, exogenous application of miPEP858 on PromiR858a:ATG1::GUS and PromiR858a:ORF::GUS effectively revealed an increase in GUS activity (Sharma et al. 2020). Furthermore, miPEP858a^CR-edited lines exhibited a significant reduction in miR858 expression. Interestingly, complementing the miPEP858a^CR-edited lines with exogenous miPEP or coinfiltrating with miPEP restored miPEP activity. However, by contrast, Lauressergues et al. (2022) demonstrated that the miORF (the miPEP open-reading frame present in the transcript) constitutes the miPEP responsive element required for miPEP-mediated activation. Reporter constructs without the miORF, either the promoter alone or targeted deletion of miORF, remained incentive to miPEP activation. This suggests that miPEPs can use different mechanisms to activate their cognate miRNA. Similarly, Ormancey et al. (2024) showed that a truncated peptide (consisting 10 amino acids) from miPEP156a, regardless of its N-terminal or C-terminal origin, enhanced miR156a expression. In contrast, the miR858 expression was enhanced only with C-terminal amino acids, highlighting a possible divergence in the regulatory pathways of these miPEPs.

Our earlier study indicates that full-length miPEP858a regulates plant growth and development by modulating the expression of PSK4, enhancing the expression of growth-related auxin and expansin genes (Badola et al. 2022). To further validate the function of truncated peptides, the expression of PSK4 was analyzed in Arabidopsis seedlings supplemented with all truncated peptides. The analysis suggested increased expression in response to supplementation with peptides derived from the C-terminal end. The effect of functional truncated peptides was also analyzed on Pro:PSK4 seedlings supplemented with functional truncated peptides, showing a significant increase similar to full-length miPEP858. Like miPEP858, C-terminal truncated peptides exhibited a significant increase in the expression of auxin and expansins, key genes required for growth and development (Fig. 7, F and G).

Based on our study, we propose a model illustrating the role of the C-terminal region of miPEP858, functioning almost similarly to full-length miPEP858 in modulating phenylpropanoid pathway genes as well as plant growth and development. Consequently, we conclude that C-terminal peptides offer a cost-effective approach to enhancing agronomic traits without the need for cumbersome biotechnological methods (Fig. 8). Ultimately, our results underscore the importance of identifying minimal functional fragments as an alternative for exogenous use, providing not only cost-effectiveness but also the potential to enhance agronomic traits. This research establishes a groundwork for identifying more functional peptides across various species and using them exogenously to improve desired agronomic traits. Synthetic peptides can effectively be employed to enhance agronomic traits in crop plants, contributing to advancements in food safety and security in the future.

Materials and methods

Plant materials and growth conditions

Arabidopsis thaliana (Col-0) was used as the WT throughout the study. Firstly, seeds were surface-sterilized and then placed on half-strength MS medium (Hi-Media) containing 1.5% sucrose, pH ∼5.72. After 2 d of stratification in the dark at 4 °C, the plates were transferred to a growth chamber (Percival) set at a 16-h light/8-h dark photoperiod cycle, 180 µmolm⁻² s⁻¹ light intensity, 22 to 24 °C temperature, and 60% to 70% relative humidity. Vertically grown 10-d-old seedlings were used for the root length measurements. The miR858^CR, miPEP858^CR, ATG-GUS, ORF-GUS, and Pro:PSK4 lines were previously generated by our group (14, 30) and chc1 AT3G11130 SALK_112213, chc1.2, SALK_103252, chc2 AT3G08530 SALK_042321, and chc2.1 SALK_028826 mutants were obtained from ABRC used in this study for analysis. To study the effect of all truncated peptides, Col-0 seedlings were grown on media supplemented with water (control) and all the truncated peptides along with miPEP858 as a positive control throughout the study.

Synthetic peptide assay

The synthetic peptides having purity >95% were synthesized through Link Biotech http://www.linkbiotech.com/. All peptides were dissolved in water (stock concentration, 5 mm). The seedlings were treated with different concentrations, ranging from 0.1 to 0.5 μM peptide, diluted in the agar medium or in water for spraying on the mature plants. For mature plants grown in soilrite, 14- and 21-d-old plants were treated with peptide (0.5 μM) diluted in water. The following peptides were used for the study:

• miPEP858a: MGGIESLLFTIVRDIGRYGTVCVVYNIKCVYTTRTKASTRTSHP,

• 1st half [1H]: MGGIESLLFTIVRDIGRYGTVC,

• 2nd half [2H]: VVYNIKCVYTTRTKASTRTSHP,

• 1st fragment [1F]: MGGIESLLFTIVRDI,

• 2nd fragment [2F]: GRYGTVCVVYNIKCV,

• 3rd fragment [3F]: YTTRTKASTRTSHP.

5′ RLM-RACE analysis

To map transcription start site (TSS) of miR858a, modified 5′ RLM-RACE was performed using a First Choice RLM-RACE Kit (Invitrogen) as per manufacture instructions. Total plants’ RNA was isolated, and 2 µg of RNA was treated with CIP (calf intestinal phosphatase) to remove the 5′-phosphate from all RNA molecules that contain free 5′-phosphates. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from the full-length mRNA leaving a 5′-monophosphate. A synthetic RNA adapter was ligated to the RNA containing a 5′-phosphate. cDNA was synthesized using random primer, by reverse transcriptase provided in kit. RACE and nested-RACE PCR were subsequently carried out with adaptor-specific forward primers and gene-specific reverse primers. The PCR products were cloned into the cloning vector (pTZ57R/T Fermentas) and sequenced to locate the TSS of miR858.

miPEP858a structure prediction (folding) and molecular docking

The miRNA858a location in Arabidopsis was retrieved from the miRBase database (https://www.mirbase.org/browse/results/?organism=ath). The promoter and miRNA precursor regions were extracted based on the provided locations for the miRNA. Functional open-reading frames (miPEP858a) were then translated for their protein-coding strands. Subsequently, the interaction between miPEP858a and the promoter sequence of miPEP858a was investigated. The identified miRNA peptide was structurally predicted using the PEP-FOLD server (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/). The 3D structure of the promoter region was predicted through the make-na server (https://web.archive.org/web/20170430174556/http:/structure.usc.edu/make-na/server.html). Docking analysis was performed to examine the interaction between the promoter (DNA) and miPEP858a using the NPDock server (https://genesilico.pl/NPDock/).

Chromatin immunoprecipitation

ChIP assays were conducted using 3-wk-old seedlings. Six grams of seedling tissues was weighed and cross-linked in 37 ml of 1% formaldehyde mixed with 50 ml extraction buffer 1 [0.4 m sucrose, 10 mm Tris–HCl, pH 8, 5 mm beta-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail; sigma]. Cross-linking was performed under vacuum for 10 min, followed by the addition of 2.5 ml of 2 m glycine to stop the process. After rinsing the seedlings twice with cold autoclaved water, excess water was removed, and the samples were stored at −80 °C. The next day, chromatin isolation was initiated by grinding the tissue to a fine powder using a precooled mortar and pestle. The powder was resuspended in 20 ml of extraction buffer 1, filtered through a cell strainer, and centrifuged. The pellet was successively resuspended in extraction buffers 2 (0.25 m sucrose, 10 mm Tris–HCl, pH 8, 10 mm MgCl₂, 1% Triton X-100, 5 mm beta-mercaptoethanol, 0.1 mm PMSF, and protease inhibitor cocktail; Sigma) and 3 (1.7 m sucrose, 10 mm Tris–HCl, pH 8, 0.15% Triton X-100, 2 mm MgCl₂, 5 mm beta-mercaptoethanol, 0.1 mm PMSF, and protease inhibitor cocktail; Sigma), followed by centrifugation. The crude pellet was suspended in nuclei lysis buffer, sonicated to achieve an average fragment size of 0.3 to 1.0 kb, and checked for sonication efficiency. Magnetic beads (A/G) were dissolved in ChIP dilution buffer and used to preclear the sonicated chromatin. The sonicated chromatin was then incubated overnight with both N-terminal and C-terminal, miPEP858-specific antibodies. After washing, the elution buffer was added to the beads, and the solution underwent an elution process. The eluate was subjected to a reverse cross-linking step, and DNA was extracted, precipitated, washed, and resuspended in TE buffer. A small aliquot of the extracted DNA was used for qPCR analysis.

Y1H assay

For the Y1H assay, the coding sequence of miPEP858a and truncated miPEP (1H, 2H, and 3F) was amplified and cloned into the pGADT7AD vector to generate a fusion with the activation domain. The promoter sequence of miPEP858a and without R1 binding site (∼1,500 bp upstream of the start codon) was amplified and cloned into the pAbAi vector to create the DNA bait sequence. To investigate the interaction between the miPEP858a peptide and the miPEP858a promoter, the pAbAi vector containing the miPEP858a promoter was linearized, transformed into the Y1HGold yeast strain, and allowed to grow on SD/-Ura media. Positive transformant colonies were confirmed by PCR, followed by selection on aureobasidin A (AbA). In the next step, the pGADT7 vector containing the miPEP858a coding sequence was transformed into the Y1HGold [Bait] strain and allowed to grow on SD/-Leu/AbA media.

Electrophoretic mobility shift assay

The second-generation DIG Gel Shift EMSA kit (Roche, United States) was used to label the probes with digoxigenin as per the manufacturer's instructions. Labeled probes were incubated at 21 °C for 30 min in binding buffer (100 mm HEPES [pH 7.6], 5 mm EDTA, 50 mm (NH₄)₂SO₄, 5 mm DTT, Tween 20, 1% [w/v], 150 mm KCl) with or without miPEP858a (1,000 nmol). The binding reaction was resolved on a 12% polyacrylamide gel in 0.5× TBE buffer and semi-dry blotted (Transblot, BIO-RAD, United States) onto a positively charged nylon membrane (Amersham Pharmacia Biotech) followed by UV cross-linking. The membrane was finally incubated with disodium 3- (4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.13,7] decan}-4-yl)phenyl phosphate (CSPD) chemiluminescent solution and visualize by using ChemiDoc (MP System, Bio-Rad). The EMSA blot images were captured using Image Lab version 5.2.1 build 11 (Bio-Rad Laboratories).

Plasmid constructs

For transient assay, the promoter region containing complete ORF of miPEP858a was taken from our earlier study (Sharma et al. 2020) and promoter region without R1 binding site was PCR-amplified using Q5 high-fidelity DNA polymerase (NEB) from genomic DNA. The amplicon was cloned into pTZ57R/T, then transferred into plant expression vector pBI121 using the HindIII and BamHI restriction sites, and transformed into Agrobacterium GV3101. For the overexpression of miPEP858a, the ORF was amplified using cDNA of Col-0, which was also taken from our earlier study (Sharma et al. 2020).

Transient expression assay

All the constructs, including the empty vector (positive control, where GUS gene is under control of CaMV35S promoter), were transformed into Agrobacterium tumefaciens (strain GV3101) and used for agro-infiltration in 5-wk-old Nicotiana tabacum and 3-wk-old A. thaliana leaves. A. tumefaciens strains were cultured in 5 ml of LB (Luria broth with antibiotics) after 24 h of incubation at 28 °C, 160 rpm. The secondary inoculations were performed in 50 ml of solution (49 ml of LB and 1 ml of 0.1 m MES), and the cultures were incubated at 28 °C at 160 rpm overnight in induction medium (10 mm MgCl₂, 10 mm MES (pH 5.6), 150 μM acetosyringone) and incubated for 3 h at 28 °C. The cultures were then diluted to an absorbance at 600 nm of 0.5 and injected into N. tabacum and A. thaliana leaves using a blunt-end syringe. The plants were placed under constant light (∼70 μmol photons per m² per seconds) for 72 h before the GUS assays were performed in infiltrated leaves.

Confocal microscopy imaging

For peptide uptake assays in Arabidopsis roots, fluorescent carboxyfluorescein (5-FAM)-labeled peptides were purchased from Link Biotech (95% to 98% purity). Three-day-old Arabidopsis WT and chc mutant seedlings were incubated with 5-FAM-labeled peptides (50 µM) in MG buffer (10-mM MgCl₂ buffer, pH 5.8) at 22 °C for 12 h. After incubation with labeled peptide, the seedlings were washed 3 times by gentle shaking for 5 min in MG buffer, and the roots were analyzed under confocal microscope at an excitation of 495 nm/emission of 545 nm. For nucleus localization DAPI staining was performed by incubating the roots in phosphate-buffered saline (PBS) containing 1 μg/ml of DAPI for 3 to 5 min at room temperature in the dark. After 2 washes with PBS buffer, the root was analyzed under a confocal microscope, at an excitation of 358 nm/emission 461 nm (Zeiss LSM710, Zeiss LSM Image Examiner version 4.2.0.121, CarlZeiss), solid-state laser (10% of 10 mW) for FAM, Propidium iodide (PI), and DAPI.

Gene expression analysis

The gene expression was analyzed through qRT-PCR (quantitative real-time PCR). Total RNA was extracted from different samples, treated with DNAase (Thermo), and (1 µg) reverse-transcribed using the Revert AidH Minus First Strand cDNA Synthesis Kit (Thermo) as per the manufacturer's instructions. The cDNA was diluted 20 times with nuclease-free water, and 2 µl was used as a template for qRT-PCR performed using Fast SYBR Green Mix (Applied Biosystems) in a Fast 7500 Thermal Cycler instrument (Applied Biosystems). The expression was normalized using tubulin and analyzed through the comparative ΔΔ^CT method (Schmittgen and Livak 2008). For the expression analysis of mature miR858a, TaqMan PCR assays were used following the manufacturer's protocol (Applied Biosystems). Small nuclear RNA (snoR41Y) was used as a normalization control. The primer sequences used in the study are listed in Supplementary Table S1.

Histochemical GUS staining

The histochemical GUS staining was performed using a previously described method (Jeferson et al. 1989). Promoter/reporter seedlings of the Pro:miPEP858:ATG:GUS, Pro:miPEP858:ORF:GUS and ProPSK4:GUS transgenic lines were dipped in a solution containing 100-mM sodium phosphate buffer (pH 7.2), 10- mM EDTA, 0.1% Triton X-100, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, and 1 mg mL^–1 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-GluC) at 37 °C for 4 to 6 h. After staining, chlorophyll was removed by incubation and multiple washes using 70% ethanol. The seedlings were observed under a Leica microscope (LAS version 4.12.0; Leica Microsystems, Wetzlar, Germany) for the GUS staining.

Total flavonol and anthocyanin quantification

Five-day-old seedlings were extracted in 1 ml of 80% methanol at 4 °C for 2 h with shaking. The mixture was centrifuged at 12,000 g for 12 min. The supernatants (0.5 ml) were taken to 2 ml with methanol and subsequently mixed with 0.1 ml of aluminum chloride (10% water solution), 0.1 ml of potassium acetate (1 m), and 2.8 ml of MQ water. After 30 min of incubation, the absorbance was taken at 415 nm. The calibration curve was developed using rutin as the standard. The total flavonol content was calculated as the equivalents of rutin used as the standard (Loyola et al. 2016). Total anthocyanin content was quantified according to the previously described method (Li et al. 2016). Briefly, the 5-d-old seedlings (300 mg) were crushed in liquid N₂ and transferred in the extraction solution (isopropanol/HCl/H₂O::18:1:81). The samples were then heated at 95 °C for 3 min, followed by incubation at room temperature in the dark for 2 h. After centrifugation, the absorbance of the supernatants was measured at A₅₃₅ and A₆₅₀. Total anthocyanin content was calculated as (A₅₃₅−2.2A₆₅₀)/g FW.

Extraction and quantification of flavonols

For the extraction of flavonols, the seedlings (300 mg) were ground in liquid nitrogen and extracted in 80% methanol overnight at room temperature. Total extracts were hydrolyzed in an equal amount of 6N HCl at 70 °C for 40 min. This was further preceded by the addition of an equal amount of methanol to prevent the precipitation of the aglycones. The extracts were filtered through 0.2-µm filters (Millipore) before the metabolite analysis using HPLC. All the samples were analyzed by HPLC-PDA with a Waters 1525 Binary HPLC Pump system comprising PDA detector following the method developed by Niranjan et al. (2011). Breeze 2 software (Waters) was used for the quantification of various metabolites through HPLC.

Lignin staining

To visualize lignified cells in stems, hand-cut sections from 35-d-old mature plants were stained using phloroglucinol (Sigma-Aldrich) for 1 min and visualized on a Leica DM2500 microscope.

Total protein extraction and western blot analysis

Total protein extraction and western blot analysis were essentially carried out as per the methods described in Sharma et al. (2020). The western blot images were captured using Image Lab version 5.2.1 build 11 (Bio-Rad Laboratories). The commercial antibodies used in the analysis were anti-actin antibody (A0480, Sigma-Aldrich) and anti-CHS (AS122615, Agrisera). N-terminal and C-terminal antibodies for miPEP858a were generated against peptide (GIESLLFTIVRDIGRY) and (CVYTTRTKASTRTSHP), respectively. The polyclonal antibodies were generated in rabbit against a peptide (CEGSDNNLWHEKENP) in the MYB12 protein sequence and were affinity-purified against the peptide (Eurogenetec).

Statistical analysis

The statistical tests and n numbers, including sample sizes or biological replications, are described in the figure legends. All the statistical analyses were performed using 2-tailed Student's t-tests using GraphPad Prism version 9.0 software. All the experiments were repeated at least 3 times independently, with similar results.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: AtHY5 (AT5G11260), AtCHS (AT5G13930), AtFLS (AT5G08640), AtMYB11 (AT3G62610), AtMYB12 (AT2G47460), AtMYB111 (AT5G49330), AtCHC (AT3G11130), AtmiR858a (AT1G71002), AtPSK4 (AT3G49780), AtAUX1 (AT2G38120), AtPIN1 (AT1G73590), AtPIN2 (AT5G57090), AtABCB19 (AT3G28860), AtYUC1 (AT4G32540), AtEXPA2 (AT5G05290), AtEXPA11 (AT1G20190), and AtEXPA15 (AT2G03090).

Acknowledgments

The authors also acknowledge Dr. Manju Singh from Central Instrumentation Facility, CSIR-CIMAP, for phytochemical analysis and Dr. Sanchita, CSIR-CIMAP, and Dr. Mehar Hasan Asif, CSIR-NBRI, for bioinformatics analysis. The images in Figures 1A, 2D, and 8 were created with BioRender.com. CSIR-CIMAP publication number is CIMAP/Pub/2025/23.

Author contributions

P.K.T., H.G., and A.S. conceived and designed the study. H.G., A.S., A.A., and H.S. participated in the execution of all the experiments. H.G., A.S., A.A., H.S., and P.K.T. interpreted and discussed the data and wrote the manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Binding of miPEP858a to the MIR858 promoter.

Supplementary Figure S2. Details of the CRISPR-edited miPEP858a line and antibodies used to confirm truncated miPEP858a.

Supplementary Figure S3. Y1H assay using the PromiR858a promoter and miPEP-X.

Supplementary Figure S4. Design of truncated miPEP858a fragments.

Supplementary Figure S5. Y1H assay using the ΔR1PromiR858a promoter with miPEP858a or truncated miPEP (1H, 2H, 3F).

Supplementary Figure S6. Mapping of the TSS of miR858a and the constructs used in this study.

Supplementary Figure S7. Effect of higher concentrations of 1H, 1F, and 2F on root length.

Supplementary Figure S8. Effect of higher concentrations of 1H, 1F, and 2F on expression of miR858a and its target genes.

Supplementary Figure S9. Internalization of FAM-labeled 1H truncated peptide.

Supplementary Figure S10. Internalization of FAM-labeled C-terminal and N-terminal truncated peptide in the nucleus.

Supplementary Figure S11. Effect of functional and nonfunctional peptides on phenylpropanoid pathway genes.

Supplementary Figure S12. Quantification of flavonols.

Supplementary Figure S13. Internalization of FAM-labeled truncated peptide (2H) in clathrin mutants.

Supplementary Figure S14. Internalization of FAM-labeled truncated peptide (3F) in clathrin mutants.

Supplementary Figure S15. Effect of functional truncated peptides on the growth of clathrin mutants.

Supplementary Figure S16. Effect of nonfunctional truncated peptides on the growth of clathrin mutants.

Supplementary Figure S17. Exogenous application of functional truncated peptides fails to complement miR858^CR lines.

Supplementary Figure S18. Effect of nonfunctional peptides on expression of auxin and expansin genes.

Supplementary Table S1. List of primers used in this study.

Funding

This research was supported by the Council of Scientific and Industrial Research (CSIR-India), New Delhi, in the form of NCP project no. MLP006. P.K.T. also acknowledges Science and Engineering Research Board (SERB), New Delhi, India, for JC Bose National Fellowship (JCB/2021/000036). H.G., A.A., and H.S. acknowledge the Council of Scientific and Industrial Research (CSIR-India) and Department of Biotechnology (DBT) New Delhi, India, for a Senior Research Fellowship. A.S. acknowledges the Department of Science and Technology (DST-India) for the DST-Inspire Faculty Project (GAP509).

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• LAS Gramene: AT1G55580

• LAS Araport: AT1G55580

• MYB12 Gramene: AT2G47460

• MYB12 Araport: AT2G47460

• MYB111 Gramene: AT3G46130

• MYB111 Araport: AT3G46130

• AtPSK4 Gramene: AT3G49780

• AtPSK4 Araport: AT3G49780

• chc1 Gramene: AT5G14170

• chc1 Araport: AT5G14170

• DAPI CHEBI: CHEBI:51231

• root AmiGo: PO:0009005

• AUX1 Gramene: At2G38120

• AUX1 Araport: At2G38120

References

Author notes