microRNA408 and its encoded peptide regulate sulfur assimilation and arsenic stress response in Arabidopsis

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in regulating various developmental and biological processes. The expression of miRNAs is differentially modulated in response to various biotic and abiotic stresses. Recent findings have shown that some pri-miRNAs encode small regulatory peptides known as microRNA-encoded peptides (miPEPs). miPEPs regulate the growth and development of plants by modulating corresponding miRNA expression; however, the role of these peptides under different stress conditions remains unexplored. Here, we report that pri-miR408 encodes a small peptide, miPEP408, that regulates the expression of miR408, its targets, and associated phenotype in Arabidopsis. We also report that miR408, apart from Plantacyanin (ARPN) and Laccase3 (LAC3), targets a glutathione S-transferase (GSTU25) that plays a role in sulfur assimilation and exhibits a range of detoxification activities with the environmental pollutant. Plants overexpressing miR408 showed severe sensitivity under low sulfur (LS), arsenite As(III), and LS + As(III) stress, while miR408 mutants developed using the CRISPR/Cas9 approach showed tolerance. Transgenic lines showed phenotypic alteration and modulation in the expression of genes involved in the sulfur reduction pathway and affect sulfate and glutathione accumulation. Similar to miR408 overexpressing lines, the exogenous application of synthetic miPEP408 and miPEP408OX lines led to sensitivity in plants under LS, As(III), and combined LS + As(III) stress compared to the control. This study suggests the involvement of miR408 and miPEP408 in heavy metal and nutrient deficiency responses through modulation of the sulfur assimilation pathway.

Introduction

MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act on their targets and down-regulate them through translational repression or mRNA cleavage (Meyers and Axtell, 2019; Tiwari et al., 2020; Li and Yu, 2021). These miRNAs control many developmental and physiological processes and, therefore, are key regulators of plant growth and development (Shriram et al., 2016; Singh et al., 2019; Millar, 2020). Several studies have shown that miRNA has an important regulatory role in biotic and abiotic stresses, such as nutrient deficiency conditions, which inhibit plant growth and development (Fujii et al., 2005; Lin et al., 2013; Sharma et al., 2016; Basso et al., 2019; Pagano et al., 2021). Research on miRNA-encoded peptides (miPEPs) began recently since a study revealed that a few pri-miRNAs have one or more open reading frames (ORFs), which regulate the associated miRNAs by enhancing their transcription (Lauressergues et al., 2015; Couzigou et al., 2015; Sharma et al., 2020; Badola et al., 2022; Lauressergues et al., 2022; Gautam et al., 2023). Although the role of these miPEPs has been explored in various plants (Couzigou et al., 2016; Chen et al., 2020; Badola et al., 2022) however, their mode of action under different stresses remains unexplored.

Heavy metal stress and nutrient limitation are major concerns across the globe that drastically impairs plant growth and development. Among the several heavy metals known so far, arsenic (As) is a naturally occurring toxic metalloid found in water, soil, and rocks. Both natural and anthropogenic activities like mining, industrialization, and pesticide rise As contamination in the food chain (Kumar et al., 2015; Abbas et al., 2018). The effects of As include skin cancer in humans whereas, in plants such as rice, it accumulates in the grains, eventually making the crop unsuitable for consumption. Arsenite [As(III)] and arsenate [As(V)] are two predominant inorganic forms of As that are readily interconvertible (Kumar and Trivedi, 2019). As detoxification is most commonly known to be alleviated by sulfur (S)-mediated detoxification in plant cells (Kumar and Trivedi, 2018). sulfur being an important macronutrient not only leads to the synthesis of the two amino acids cysteine and methionine but also produces glutathione (GSH) and phytochelatins (PCs) essential in combating As stress (Dixit et al., 2015a, 2015b, 2016). Glutathione S-transferases (GSTs) are well known enzymes that plays an important role in regulation of abiotic and biotic stresses by catalyzing the conjugation of GSH to any hydrophobic and electrophilic substrates, and regulate the redox state thus, protecting the cell from oxidative burst (Cummins et al., 2011; Ding et al., 2017).

Sulfur is an essential macronutrient, plays important role in growth and development of plants and combating abiotic stresses (Zhao et al., 2008). Sulfur is an important component of the primary structure of many key enzymes, that regulates various metabolic pathways, including sulfur reduction pathways that forms amino acids cysteine (Cys) and methionine (Met) (Gill et al., 2011; Giordano and Raven, 2014). Sulfur uptake and assimilation is tightly regulated at transcriptional level by various transporters and enzymes (Mazid et al., 2011a, 2011b). Sulfate transportation and assimilation are regulated by several modes, including transcriptional and posttranscriptional regulation in plants. Sulfur plays important role to cope up with the different environmental stresses and regulate the antioxidant system of plant by regulating the sulfur assimilation pathway (Mazid et al., 2011a, 2011b; Anjum et al., 2015). Studies also suggest that Glutathione S-transferase TAU 25 (GSTU25) is differentially regulated in many stresses that directly conjugate GSH to target molecule increase the process of detoxification (Gunning et al., 2014). Transcriptional regulation is mediated by factors such as sulfate limitation, light, sugars, heavy metals, etc., whereas sulfate assimilation is post-transcriptionally regulated by miR395 (Yuan et al., 2016; Kumar et al., 2017; Li et al., 2020). Interestingly, a study by Liang et al., (2015) revealed the differential modulation in the expression of various miRNAs, among which miR408 was identified to be suppressed in response to sulfate deficiency along with carbon and nitrogen limitation.

miR408 is a highly conserved miRNA and its expression is controlled by the availability of copper (Axtell and Bowman, 2008; Kozomara and Griffiths-Jones, 2011; Shahbaz and Pilon, 2019). The targets of miR408 include genes encoding copper-binding proteins, which belong to the phytocyanin family and function as electron transfer shuttles between proteins (Zhang et al., 2017), and laccase, another copper-containing protein involved in oxidative polymerization of lignin (Berthet et al., 2012; Tobimatsu and Schuetz, 2019). The expression of miR408 is substantially affected by a variety of developmental and environmental conditions; however, limited information is available related to its biological function. miR408 has been found as an important component of the HY5–SPL7 gene network that mediates the coordinated response to light and copper, further illustrating its central role in the response of plants to their environment (Zhang et al., 2014). Studies also suggest that PIF1-miR408-Plantacyanin Repression Cascade regulates light-dependent seed germination in Arabidopsis (Jiang et al., 2021). Previous studies illustrated the important role of miR408 in governing tolerance toward abiotic factors such as salinity, cold and oxidative stress, and vice versa in case of drought and osmotic stress (Rajwanshi et al., 2014; Hajyzadeh et al., 2015; Ma et al., 2015; Bai et al., 2018). Although the actual mechanism by which miRNA408 regulates different abiotic stresses is not well understood.

This study emphasizes the role of miR408 and the peptide encoded by miR408 (miPEP408) in response to different stresses such as heavy metal and nutrient deficiency. For a better understanding of the role of miR408, overexpressing and CRISPR-edited plants of miR408 were developed. The overexpression plant of miR408 showed a similar phenotype as peptide supplemented plants with increased root length and fresh weight. Although the accumulation of miR408 was substantially decreased and its targets were upregulated in miR408 mutant plants, which did not show a substantially phenotypic difference compared to the WT. Interestingly, under sulfate deficit and As stress conditions, miR408OX lines showed a more sensitive phenotype than the WT. Interestingly, CRISPR-edited miR408 plants exhibited increased root length and fresh weight under stress conditions compared with the WT. Application of synthetic miPEP408 exogenously led to upregulation of miR408 expression that negatively regulates the target genes affecting plant growth in the control condition. Further, miPEP408-supplemented seedlings under different stress conditions showed a highly sensitive phenotype, similar to the miPEP408OX lines. Therefore, this study gives a wide picture of the response of miR408 toward sulfate limitation and As stress.

Results

Differentially regulated miRNAs under low sulfur and arsenic stress

Identification of miRNAs in response to As and sulfur deficiency could be very helpful to decipher the regulatory pathway involving specific miRNAs in these stresses. Thus, to identify miRNAs with modulated expression in response to As stress and sulfur limitation (LS), small RNA sequencing was performed using seedlings grown under C, LS, As(III) and LS + As(III) conditions for 10 days. The analysis of the small RNA sequencing was done on the basis of the total reads obtained for each sample, raw reads sequence length, total data size obtained and raw reads of samples after adapter removal (Supplemental Tables 1 and 2). The total numbers of known miRNAs found in each of the conditions are represented in Supplemental Table 3. Further analysis revealed that 55 miRNAs are differentially regulated under As(III) and LS and LS + As(III) conditions compared to control (Figure 1A and Supplemental Table 4). Out of total differentially expressed miRNAs, 18 miRNAs are commonly differentially expressed under As(III) and LS and LS + As(III) conditions (Figure 1, A and B; Supplemental Table 5). We identified several miRNAs (miRNA398, miR399, miR408 and miR857), which are downregulated under As and LS conditions (Figure 1B), however, some of the miRNAs were differentially expressed under specific stress conditions. This indicates that the regulatory effect of a miRNA can vary under different stress conditions and physiological requirements of plants affected by different stress conditions. Validation of expression of downregulated miRNAs under different stress conditions suggested that miRNA408 was the most responsive toward As and LS conditions (Supplemental Figure 1).

The function of miR408 has been studied under plant development and abiotic stresses; however, its role in nutrient deficiency and heavy metal stress needs to be explored. To functionally dissect the role of miR408 toward LS and As stress, we first examined the transcript abundance of miR408 in response to LS and As(III) and combined stress [LS + As(III)]. The expression analysis revealed that accumulation of pre-miR408 and mature miRNA is reduced under LS and AsIII stress, while the transcript level was lowest under [LS + As(III)] (Figure 1, C and D). Previously, ARPN and LAC3 genes were identified as cleavable targets of miR408 (Abdel-Ghany and Pilon, 2008). We also analysed potential targets of miR408 involved, directly or indirectly, in the S assimilation pathway to combat nutrient deficiency and As stress. Out of several targets, the analysis led to the identification of one putative target, Glutathione S-transferase TAU 25 (GSTU25) (Supplemental Table 6), which is known to involved in many stresses that directly conjugate GSH to target molecule increase the process of detoxification (Gunning et al., 2014; Ma et al., 2015). Through sequencing of 5′ RNA Ligase Rapid Amplification of cDNA Ends (RLM-RACE) products, we validated that miR408 targets GSTU25 and cleave it 65 nucleotides away from cleavage site (Supplemental Figure 2). The expression of ARPN, LAC3 and GSTU25 was analyzed in the above stress condition. Based on expression analysis, it was observed that LS, As(III) and [LS + As(III)] stress conditions influence the ARPN, LAC3 and GSTU25 expression in a pattern contrary to that of miR408 (Figure 1E). These results indicate that miR408 is regulated by LS and As(III) stress and modulates target expression under these conditions.

MIR408 is transcriptionally regulated by LS and as

To investigate the promoter responsiveness toward LS, As and [LS + As(III)] stress, promoter-reporter lines (wild-type seedling expressing promiR408::GUS) and positive control [Empty vector (EV) with GUS gene under CaMV35S] were grown under different stress conditions. The GUS histochemical analysis suggests that compared to the EV, promiR408::GUS lines showed much reduced GUS activity under LS, As(III) and [LS + As(III)] stress conditions (Figure 2A), consistent with the GUS transcript analysis (Figure 2B). To associate the promoter activity with miR408 expression, the miR408 expression was analysed in all promoter-reporter lines. The analysis revealed significantly decreased transcript levels of miRNA408 in LS, As(III) and [LS + As(III)] (Figure 2C). The ARPN gene expression analysis for miR408 showed a higher accumulation under stress conditions than in control (Figure 2D). These results suggest that the promoter activity and transcript level of miRNA408 are significantly affected and tightly associated under LS and AS stress conditions.

Responses of miR408 overexpression and CRISPR/Cas9-based knockout mutants toward as and ls

To study the impact of LS and As stresses on miR408, miR408 overexpressing lines (mi408OX) and mutated plants (miR408^CR) were developed (Supplemental Figure 3, A and B). Three homozygous miR408OX lines were selected on the basis of enhanced accumulation of mature miR408 and decreased expression of target genes (ARPN, LAC3, and GSTU25) compared with WT (Figure 3, A and C). To develop miR408 mutant plants, we used CRISPR/Cas9 approach, in which gRNA was designed from the pre-miRNA region targeting mature miRNA. However, screening of mutants by sequence analysis revealed deletion and insertions in the mature miRNA sequences obtained (Supplemental Figure 4, A and B). Three homozygous and Cas9-free edited plants of the T4 generation were used for further study. These mutated plants showed significantly reduced mature miR408 accumulation and higher accumulation of target genes (ARPN, LAC3 and GSTU25) (Figure 3, B and D). The accumulation of pre-miRNA408 in overexpression and mutant lines of miR408 was also analysed. The expression analysis suggested that pre-miR408 expression resembles with the mature miRNA accumulation pattern (Supplemental Figure 5, A and B).

To functionally validate the role of miRNA408 in LS and As stress, miR408OX lines and miR408^CR plants were grown under LS, As(III) and combined [LS + As(III)] stress. Phenotypic analysis of miR408OX lines revealed better growth and root length compared with the WT under control conditions. However, under LS, As(III) and [LS + As(III)] stress, miR408OX lines exhibited retarded growth and a decrease in primary root length compared to WT (Figure 3, E and F). These stresses affected the plant growth, which finally reduced the fresh weight of miR408OX lines compared with the WT (Figure 3G). On the contrary, compared with WT, the miR408^CR plants were tolerant and exhibited better growth compared with WT under stress conditions (Figure 3H). Moreover, miR408^CR plants have significantly increased primary root length and the fresh weight as compared with the WT plants under stress conditions (Figure 3, I and J).

Together, these results suggest that mutation in miR408 showed tolerance toward LS, As(III) and combine [LS + As(III)] stress. In contrast, the miR408OX lines were sensitive to these stresses compared to the WT. Altogether, a significant change in the phenotypes in miR408OX and miR408^CR suggests that miR408 plays a very important role in nutrient limitation and heavy metal stress.

miR408 elevates ROS production under LS and as toxicity

Plant cellular machinery increases the production of reactive oxygen species (ROS) under environmental cues such as nutrient deficit, temperature, heavy metal stress etc (Huang et al., 2019; Hasanuzzaman et al., 2020a, 2020b). Higher ROS generation leads to oxidative stress in plants and hampers plant growth. Since miR408OX lines were identified to be sensitive under the different stresses as compared to miR408^CR plants and WT, ROS levels were analysed through NBT and DAB staining.

NBT staining revealed that miR408OX lines were accumulating more O²⁻ however, maximum staining was observed under combined [LS + As(III)] stress but the mutated plants showed lesser accumulation compared with the WT, which indicates that mutated lines have decreased oxidative burst as compared with the miR408OX lines thereby showed tolerance against these stress condition (Figure 4A, and Supplemental Figure 6). Furthermore, DAB staining was performed to monitor the production of H₂O₂ under LS, As(III) and [LS + As(III)] conditions. Higher accumulation of H₂O₂ was observed in miR408OX lines marked as higher brown precipitate. Similar to NBT, maximum DAB staining was found in response to [LS + As(III)] stress, however, miR408^CR plants were observed to accumulate lesser H₂O₂ compared with WT (Figure 4B, and Supplemental Figure 7).

Previous studies showed that As(III) induces oxidative stress and generation of free radicals causes lipid peroxidation and tissue damage (Møller et al., 2007; Shri et al., 2009). Malondialdehyde (MDA) content was measured to quantify lipid peroxidation in miR408OX lines and miR408^CR plants after stress treatment. The miR408OX lines showed significantly higher MDA content in LS, As(III) and [LS + As(III)] stress, whereas the mutated plants showed lesser accumulation of MDA compared with WT (Figure 4C, and Supplemental Figure 8, A and B). These results suggest that the miR408^CR plants have stronger antioxidant and detoxification capability than the miR408OX lines and was associated with the phenotypic data, which showed mutated plants are more tolerant compared with miR408OX lines toward LS, As(III), and [LS + As(III)] stress.

Differential regulation of sulfur metabolism affects detoxification mechanism of miR408OX and miR408^CR plants

Synthesis of various sulfur (S) containing metabolites, including GSH and amino acids (Cys and Met) is regulated by enzymes involved in sulfate assimilation in plants. This pathway is tightly regulated by the demand and supply of sulfur by the various key factors (Leustek et al., 2000; Aarabi et al., 2020). Previous studies reported that heavy metal stress affects sulfur assimilation in the plant by regulating the expression of key enzymes in the pathway (Khare et al., 2017). So, to associate the effect of miR408 on sulfur metabolism and As toxicity, we analysed the expression of genes majorly involved in the sulfur reduction in miR408OX as well as miR408^CR plants.

Differential expression of AtAPS1, 2 and 3 were found in miR408OX and miR408^CR plants under LS, As(III) and combined [LS + As(III)] stress comparison to control. Compared with the WT, the miR408OX lines showed a lower accumulation of AtAPS1, AtAPS2 and AtAPS3 transcripts which suggests that there is a significant decrease of flux toward the reduction pathway. Due to this limitation, miR408OX plants might have experienced a limitation of reduced compounds to combat stress (Figure 5A, and Supplemental Figure 9, A and B). In plants, heavy metal stress induces the accumulation of GSH via activating ATPS accumulation (Asgher et al., 2014; Anjum et al., 2015), so this pathway needs to be upregulated to combat stress. Contrary to the miR408OX, miR408^CR plants were observed to have a higher accumulation of AtAPS1, AtAPS2 and AtAPS3 (Figure 5B, and Supplemental Figure 10, A and B), which suggests the increased flux toward the reduction pathway and higher accumulation of GSH which would be responsible for tolerance of mutated plants under different stresses. The expression of AtAPR1, 2 and 3 in the miR408OX lines was reduced compared with the WT (Figure 5C, and Supplemental Figure 9, C and D), while significantly enhanced expression of APR isoforms was observed in miR408^CR plants in response to LS, As(III) and [LS + As(III)] conditions (Figure 5D, and Supplemental 10, C and D). Enhanced APS and APR activities in miR408^CR plants compared with miR408OX lines might lead to higher accumulation of GSH in miR408^CR plants, therefore, providing tolerance toward LS, As(III) and [LS + As(III)] stress.

Sulfur enters into the plant roots in the form of inorganic sulfate via sulfate transporters (SULTRs). However, all the four families of SULTRs plays an important role in transportation and translocation of S, only SULTR1 and SULTR4 transporters are substantially modulated under sulfate deficiency in plants (Takahashi, 2000; Kumar et al., 2011; Gigolashvili and Kopriva, 2014). Therefore, to study the function of miR408 in regulating sulfate transportation, we analysed the expression of SULTR in miR408OX and miR408^CR plants. SULTR1; 1 and SULTR4; 1 was identified to be significantly downregulated in miR408OX plants under control and stress conditions (Supplemental Figure 11, A and B), but mutated plants showed significantly increased accumulation (Supplemental 11, C and D). These results suggest that there must be a higher accumulation of sulfur in the mi408OX plant cells compared with the mutated plant in control and stress conditions.

From the above results, it was clear that the miR408OX plants and miR408^CR plants have differential regulation of S assimilation pathway genes. So, it was very important to measure the initial and the end product of this pathway, which are sulfur and GSH. Interestingly, higher sulfur accumulation was found in the miR408OX plant compared with the WT in LS, As(III) and [LS + As(III)] stress conditions. However, sulfate content in miR408^CR plants was lower as compared with WT (Figure 5E, and Supplemental Figure 12, A and B). This result suggests that mutated plants have better sulfate assimilation than miR408OX plants and therefore are more tolerant to low sulfur and As stress.

We estimated Glutathione (GSH), which acts as damage control after stress-induced oxidative damage in plants (Kumar and Trivedi, 2018; Hasanuzzaman et al., 2019). GSH content was lower in miR408OX plants which indicate that they are more susceptible to stress conditions and are more sensitive. At the same time, increased GSH was observed in miR408^CR plants in control as well as stress condition, which suggests that mutated plants have a better detoxification mechanism than miR408OX plants (Figure 5F, and Supplemental Figure 13, A and B).

miR408 encodes functional peptide miPEP408-35aa

We analysed upstream from the mature miR408 sequence to identify putative ORFs encoding miPEP408. Analysis revealed presence of two putative ORFs (129 and 108 bp) (Supplemental Figure 14). These ORFs are designated as ORF₁ and ORF₂ based on their location with respect to the mature miR408. The ORF immediately upstream of miR408 was designated ATG1 and the next ORF was called ATG2. These ORFs may encode small peptides of 42 aa and 35 aa, respectively, designated as miPEP408-42aa and miPEP408-35aa (Figure 6A). To know the functional ORF, seedlings were grown on media supplemented with exogenous miPEP408-42aa and miPEP408-35aa at different concentrations (0.25 to 1 μM), as it has been well established that the exogenous application of miPEPs enhanced the transcript of pri-miRNA and associated phenotype. The miPEP408-35aa showed a concentration-dependent increase of seedling growth and the accumulation of mature and pre-miR408, while the miPEP408-42aa supplemented seedlings did not show a significant change in the growth of the seedlings and the expression of mature and pre-miR408 compared to the control condition (Figure 6, B–E, and Supplemental Figure 15). The transcript levels of miR408 targets, ARPN, LAC3 and GSTU25, were reduced in seedlings grown on miPEP408-35aa supplemented media compared with control, whereas miPEP408-42aa did not affect the expression of targets genes (Figure 6, F–G). These results suggested that miPEP408-35aa might be the functional miPEP encoded by a pri-miR408 have the potential to enhance the expression of miR408 and modulate associated phenotype. Preliminary dose-dependent experiments represented that 0.50 μM concentration of miPEP408-35aa was optimal and selected for further studies of miPEP408-mediated regulation of miR408.

To provide better information of functional miPEP further, promoter-reporter lines (PromiR408::GUS) were developed to understand the role of miPEP to the responsiveness of the miR408 promoter. The promoter lines were grown on a medium supplemented with or without synthetic miPEP408-42aa and miPEP408-35aa. Histochemical GUS assay revealed enhanced GUS activity in PromiR408::GUS lines supplemented with miPEP408-35aa, not with miPEP408-42aa, compared with control (Figure 6H). Similarly, increased expression of the GUS gene was only observed in the seedlings grown in media supplemented miPEP408-35aa, not with miPEP408-42aa, compared with control (Figure 6I). These results collectively suggest that the pri-miR408 encodes a small functional peptide of 35 amino acids.

We perform full-length RLM-RACE for the identification of the transcription initiation site of the miR408 and to ascertain that miPEP408-35aa and miPEP408-42aa are encoded by the pri-miR408. One major amplicon resulted from the 5′ RLM-RACE PCR were obtained (Supplemental Figure 16A). PCR products were cloned and after DNA sequence analysis we localized the transcription initiation site at 372 bp upstream from the pre-miR408. (Supplemental Figure 16, B and C).

Application of miPEP408 leads to growth inhibition and exhibits miR408OX-associated phenotypes under stress conditions

From the above experiments, it is understandable that miPEP408 transcriptionally regulates the miR408 expression; however, the impact of miPEP on different stress was still not studied. Hence, we added exogenous miPEP408-35aa peptide (0.50 µm) in control and LS, As(III) and [LS + As(III)] media. Intriguingly, the supplementation of the exogenous peptide to the ½ MS media increased the sensitivity of seedlings toward LS, As(III) and [LS + As(III)] stress, which was evident from the greater reduction in primary root length and fresh weight (Figure 7, A–C). This result indicates that the addition of miPEP408 negatively regulates the plant growth in LS, As(III) and [LS + As(III)] stress in Arabidopsis and behave similarly to miR408OX plants.

To briefly elucidate the miPEP408 function in stress conditions, we developed overexpressing miPEP408 overexpressing lines. These lines accumulated miR408 and decreased the accumulation of its target ARPN expression (Supplemental Figure 17, A and B). To functionally validate the role of miPEP408 in LS and As stress, miPEP408OX lines were grown on LS, As(III) and combined [LS + As(III)] conditions. Phenotypic analysis revealed that miPEP408OX plants show better growth represented as root length and fresh weight compared to the WT whereas seedlings grown under LS, As(III) and [LS + As(III)] conditions showed growth inhibition as a decreased primary root length and fresh weight compared with WT (Figure 7, D–F). These results show that exogenous miPEP408 and miPEP408OX plants exhibit similar phenotypes as miR408OX plants in control and stress conditions (Figure 3E–G).

Differential ROS, sulfur accumulations, and higher lipid peroxidation in exogenous miPEP408-supplemented and miPEP408OX plants

To further study the effect of miPEP408 on the antioxidant machinery, miPEP408OX plants and the WT seedlings were grown on media supplemented with miPEP408 analyzed for ROS accumulation under different stresses. NBT staining results suggested a higher accumulation of O²⁻ radical in seedlings grown with exogenous peptide and miR408OX in LS, As(III) and [LS + As(III)] stress. Additionally, no substantial NBT staining was seen in seedlings grown on control media (supplemented with either water or peptide) and miPEP408OX under control conditions (Figure 8, A and B). In stress conditions, there was a higher accumulation of free radicals in seedlings supplemented with the miPEP408, which behaved like the miR408OX plants (Figure 4A). Furthermore, DAB staining showed that there was higher brown precipitate in the peptide supplemented and miPEP408OX seedlings under LS, As(III) and LS + As(III) stress compared with the control (Supplemental Figure 18, A and B). These results suggest that seedlings grown on peptide supplemented media have a higher accumulation of ROS and are more sensitive to the stress compared to the seedlings grown under stress conditions with control (water) as a supplement. From these results, we concluded that miPEP408-supplemented seedlings are behaving like miR408OX plants under LS, As(III), [LS + As(III)] stress.

MDA content was also estimated in with or without peptide supplemented and miPEP408OX seedlings under LS, As(III), [LS + As(III)] stress. Higher accumulation of MDA was observed in the miPEP408OX and seedlings grown on miPEP408-35aa supplemented media along with LS, As(III) and [LS + As(III)] stress compared to the water added growth media (Figure 8, C and D). There was no significant change in the control condition. These results suggest that miPEP408 enhances the sensitivity compared with the control and shows a similar phenotypic effect as miR408OX plant on LS, As(III) and combined [LS + As(III)] stresses.

To observe the sulfur availability inside the cell, we measured the sulfur content of 10-day-old different lines of miPEP408OX and WT seedlings grown on control and peptide supplemented media, under LS, As(III) and [LS + As(III)] stress conditions. Results imply that miPEP408OX plants and exogenous peptide treatment enhances the sulfate content in control and stress conditions in Arabidopsis (Figure 8, E and F). Measurement of GSH content in the miPEP408OX lines and peptides supplemented seedlings suggest that the exogenous application of miPEP408 and miPEP408OX lines have decreased GSH content in control and stress conditions (Figure 8, G and H).

miPEP408 regulates sulfur metabolism and affects plant tolerance to stress

The above results suggested that miR408 negatively regulates the sulfate assimilation pathway. Therefore, to know whether exogenous application of miPEP408 also impacts sulfate reduction, seedlings grown on media having LS, As(III) and [LS + As(III)] stress along with miPEP408 were used for expression analysis of sulfur assimilation related genes (APS and APR). The accumulation of AtAPS1, AtAPS2, and AtAPS3 is negatively affected in all the stress conditions (Supplemental Figure 19A). This result shows that sulfur is not being used in the reduction pathway and eventually not be used for the synthesis of GSH needed for providing tolerance to plants. The expression of AtAPR1, 2 and 3 was also significantly decreased in the peptide supplemented seedlings in control and stress conditions (Supplemental Figure 19B). Analysis of APS and APR genes in control conditions in miPEP408OX lines showed that accumulation of miPEP408 also affects the sulfur assimilation pathway genes and affect sulfur assimilation (Supplemental Figure 20, A and B). From these results now it is confirmed that miPEP408 regulates the sulfur assimilation pathway gene and might affect the stress tolerance mechanism as shown by miR408 overexpressing plants previously.

This result suggests that miPEP408 either regulates S uptake or its transportation in the cell. As mentioned in previous results, apart from S assimilation genes, sulfate transportation mediated by SULTRs also strictly regulates plants’ responses toward S limitation and As stress. Therefore, the expression of SULTR1 and SULTR4 was analysed in presence of miPEP408 in Arabidopsis. Interestingly, expression profiling revealed a decrease in transcripts of the isoforms associated with both the SULTRs on the application of the exogenous miPEP408 under LS, As(III) and [LS + As(III)] stress (Supplemental Figure 19C). These results showed that the application of the miPEP408 increases sensitivity in plants under LS and As stress conditions.

miR408 modulates the arsenic accumulation

Various reports suggest As toxicity is alleviated by sulfur by affecting the accumulations by modulating amino acids and thiol metabolism in rice (Dixit et al., 2015a, 2015b). Therefore, we observed the effect of miR408 on As accumulation in miR408OX and miR408^CR plants and supplemented with miPEP408 under control, LS, As(III) and LS + As(III) conditions. It was observed that miR408OX plants have a significantly lower accumulation of total As under As(III) and [LS + As(III)] compared with miR408^CR plants (Supplemental Figure 21, A and B). The miPEP408-supplemented seedlings grown on As(III) and LS + As(III) also showed a higher accumulation of As, similar to miR408OX plants (Supplemental Figure 21C).

Discussion

MicroRNAs are small RNAs functioning in the regulation of all the processes of plants that include development and responses to abiotic stresses by posttranscriptional inhibition of target transcript (Millar, 2020; Vakilian, 2020; Pagano et al., 2021). Recent studies showed that the small peptide encoded by some of the pri-miRNAs participate in the regulation of miRNA expression (Couzigou et al., 2015; Lauressergues et al., 2015; Prasad et al., 2020; Sharma et al., 2020; Badola et al., 2022). However, detailed insight into the role of these peptides under different stresses is still lacking. Among the known families of miRNA, miR408 is identified to be conserved and has a fundamental function with its diverse response to nutrient limitation, cold, drought, osmotic and oxidative stress (Ma et al., 2015; Song et al., 2018). This study demonstrated that miR408-edited plants showed a higher degree of survival than miR408 overexpressing plants in nutrient deficiency and As stress. Moreover, the function of the peptides encoded by pri-miR408 was studied in detail and the study suggests that miPEP408 regulates tolerance toward abiotic stresses such as sulfate deficiency and As toxicity.

Small RNA sequence analysis concluded that 55 miRNAs are differentially regulated in As and LS conditions, of which only 18 miRNAs were common in these stresses (Figure 1 and Supplemental Tables 5 and 6). Of common differentially expressed miRNAs, miR408 was the most responsive miRNA toward As and LS conditions (Supplemental Figure 1). The expression of miR408 is differentially regulated in various plant systems in response to different abiotic stresses, like drought, As and nutrient deficiencies (Liang et al., 2015; Sharma et al., 2015; Vasupalli et al., 2020). We analysed the involvement of miR408 toward LS, As(III), [LS + As(III)] stress and observed significant downregulation in the expression of miR408 and vice versa of its targets, ARPN, LAC3 and GSTU25 (Figure 1, C–E). The GSTU25 is well known GST that is highly induced under abiotic stresses leading to accumulation of ROS and shows antioxidative function (Gunning et al., 2014; Ma et al., 2015). The target validation by 5′ RLM-RACE indicated that GSTU25 is the target of miR408 and that confirms the direct relation of miR408 with the modifying sulfur assimilation pathway (Supplemental Figure 2). These results showed that miR408 is involved in As toxicity and low sulfur response by targeting GSTU25, that might regulate the S assimilation.

Since sulfate limitation and As toxicity were identified to regulate the expression of miR408, the activity of the promoter of the miR408 was studied under these stresses. Previous studies have reported that environmental constraints, including heavy metal and nutrient alteration, tightly regulate the promoter activity of responsive gene regulation, thereby affecting plant growth and development (Qi et al., 2007; Tiwari et al., 2020). Expectedly, reduced transcript levels and GUS activity in PromiR408::GUS stable lines under stress conditions compared to EV were observed (Figure 2, A and B). Along with the promoter activity, miR408OX lines and CRISPR-edited plants (miR408^CR) of miR408 were developed to have a detailed understanding of the role of this miRNA in stress conditions. Captivatingly, miR408OX lines showed sensitivity toward LS, As(III) and combined [LS + As(III)] stress compared to the WT plants (Figure 3, E–G). Response of miR408OX lines was consistent with previous studies showing reduced growth in plants overexpressing miR408 under drought and osmotic stress in Arabidopsis (Ma et al., 2015). Although the study by (Ma et al., 2015) revealed improved growth of plants overexpressing miR408 under salinity, cold and oxidative stress, the response in our study might be specific to As stress and LS. On the other hand, miR408^CR plants were observed to be tolerant toward sulfate limitation and As stress as the root length and fresh weight of mutated plants was significantly increased under LS, As(III) and [LS + As(III)] stress compared to the WT (Figure 3, H–J). Previously, CRISPR-edited plants of miR408 were developed in rice, however, their response under different abiotic stresses was not defined (Zhou et al., 2017).

Abiotic stresses such as nutrient deficiency and heavy metal stress increases production of reactive oxygen species (ROS) in plants (Hasanuzzaman et al., 2020a, 2020b). ROS, including superoxide radical (O²⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻), etc., acts as signal transducers in plants, however, excess levels lead to membrane damage (also termed as lipid peroxidation) and disrupt nucleic acids including DNA and RNA (Choudhury et al., 2017; Mittler, 2017; Singh et al., 2019). Increased NBT and DAB staining was observed in miR408OX lines which suggests a higher ROS production in the overexpression plants compared to WT (Figure 4, A and B). This might be a plausible reason for the sensitivity of miR408OX under stress conditions. Additionally, miR408^CR plants accumulated lesser of ROS compared with the WT, which gave clear evidence that miR408 negatively regulates tolerance toward sulfate deficiency and As stress (Figure 4, A and B). A significantly enhanced level of MDA was observed in miR408OX lines compared with the WT under LS, As(III) and [LS + As(III)] conditions. miR408^CR plants accumulated lesser MDA compared to the WT (Figure 4C). Decreased ROS accumulation in miR408^CR plants determines enhanced tolerance against the LS, As(III) and combine [LS + As(III)] condition.

Previous studies revealed that an increase in sulfur reduces As toxicity and also improves thiol metabolism in plants (Dixit et al., 2015a, 2015b). miR408OX and miR408^CR plants showed differential expression of APS and APR genes compared to the WT (Figure 5, A–D). The miR408OX plants showed significantly decreased expression of APS, APR isoforms indicating that the sulfur reduction pathway was negatively regulated in the overexpression plants with reduced accumulation of sulfur and GSH compared to the WT. Interestingly, the mutated plants showed an increased accumulation of various isoforms of APS and APR transcripts, which showed that there must be a higher accumulation of GSH and more reduction of sulfate, which will provide tolerance to the plant against As stress and LS. APS and APR are two major enzymes regulating the flux toward S assimilation in plants and are extensively regulated by the availability of sulfur and GSH (Brunold, 1993; Leustek et al., 2000). The expression analysis indicated that GSTU25 is directly regulated by the miR408 (Figure 4, C and D). Analysis also suggested that compared to miR408OX plants, miR408^CR have higher accumulation of GSTU25, that might lead to better detoxification capabilities.

Glutathione (GSH) is a sulfur-containing compound having redox and nucleophilic properties that are regulated by sulfur availability and plays an important role in minimizing the stress-induced oxidative damage in plants (Mendoza-Cózatl and Moreno-Sánchez, 2006; Banerjee and Roychoudhury, 2019; Pei et al., 2019). The estimation of sulfur and GSH in miR408OX and miR408^CR plants indicated that the sulfur reduction pathway is regulated by the miR408 expression. Overexpression of miR408 led to a higher accumulation of sulfur and lower accumulation of GSH, indicating that the sulfur reduction pathway is negatively regulated by miR408 might be one of the reasons for sensitive phenotype against stress. miR408^CR plants had greater sulfur reduction (lesser sulfate content) and high GSH content suggesting that these plants might have highly efficient sulfur reduction metabolism capability and better use of the sulfur for the GSH biosynthesis compared with the WT (Figure 5, E and F). This result is in corroboration with previous studies in which tolerant natural accession was identified to have lesser sulfate and greater GSH content under LS, As(III) and combine [LS + As(III)] stress (Khare et al., 2017). These analyses suggest that the absence of miR408 confers tolerance to the miR408^CR plants toward LS and As stress.

To identify ORFs encoded by miR408, sequence upstream was analysed and ORFs encoding putative peptides of 35aa and 42aa were identified in pri-miR408 (Supplemental Figure 14). Interestingly, exogenous application of miPEP408-35aa, but not miPEP408-42aa, increased primary root growth of the WT plants as well as accumulation of miR408 (Figure 6, A–D). The impact of miPEP408-35aa was also reflected in the expression of miR408 and its targets, ARPN, LAC3 and GSTU25 whose expression got reduced after peptide treatment compared to the control (Figure 6, F and G). This result was in accordance with the previous research where overexpression of miR408 in rice led to increased growth and development (Zhang et al., 2017).

Histochemical GUS assay illustrated increased GUS activity along with increased GUS expression in PromiR408::GUS lines when grown on media supplemented with miPEP408- 35aa compared to control, but not with miPEP408-42aa (Figure 6, H and I). Similar results were observed in the study by Sharma et al., (2020), which showed transcriptional regulation of miR858 via miPEP858a. Various studies suggest that small peptides might play a key role in abiotic stress tolerance like AtPep3 which is a hormone-like peptide that enhances the salinity stress tolerance in Arabidopsis (Nakaminami et al., 2018). Similarly, the C-terminally ENCODED PEPTIDE 5(CEP5) promotes tolerance in Arabidopsis toward osmotic and drought stress by regulating AUX/IAA Equilibrium (Smith et al., 2020). Recently, a few miPEPs have been identified in plants (Lauressergues et al., 2015; Waterhouse and Hellens, 2015) but their role in abiotic stress tolerance is yet to be explored. We demonstrated that the application of miPEP408 on seedlings and miPEP408OX plants grown on the 1/2 MS media grow better in the control condition. Interestingly, when these seedlings were grown on the LS, As(III) and [LS + As(III)] supplemented with water as control or peptide and miPEP408OX lines exhibited contrasting phenotypes. The seedling grown on stress media supplemented with the exogenous miPEP408 as well as miR408OX showed a sensitive phenotype compared to the water supplemented media (Figure 7, A–F). Treatment with the exogenous miPEP408 peptide and miPEP408OX seedlings showed increased sensitivity toward heavy metal and nutrient deficiency which was also reflected by the increased NBT and DAB staining along with elevated MDA content in the WT plants supplemented with the exogenous peptide and miR408OX plants under different stresses (Figure 8, A–D).

Exogenous supplementation of the miPEP408 or overexpressing miPEP408 negatively affects the expression of important sulfur reduction pathway genes, like APS and APR, that are required for the synthesis of GSH which is involved in the detoxification mechanism (Supplemental Figures 19 and 20). Reduced sulfur and GSH content were observed in overexpressing lines and peptide supplemented seedlings in control and stress conditions (Figure 8, E–H). Analysis suggests that miPEP408 enhance the sensitivity response of Arabidopsis in comparison to the normal conditions, thus reducing the tolerant phenotype in stress conditions. Sulfur assimilation is largely regulated by sulfate transporters (SULTRs) in plants. Expression analysis of SULTR1; 1 and SULTR4; 1 revealed that both the genes were downregulated after supplementation of miPEP408 (Supplemental Figure 19C). This suggests that miPEP408-supplemented seedlings possess a higher accumulation of sulfur, thereby restricting the uptake of sulfur by SULTRs under LS, As(III) and [LS + As(III)] conditions. Similar results have been also reported in contrasting Arabidopsis natural variants under LS and As stress (Khare et al., 2017). Moreover, a recent study elucidated the importance of SULTR1; 1 in providing tolerance against sulfate limitation and As toxicity (Kumar and Trivedi, 2019). The As accumulation data suggests that miR408OX and exogenous miPEP408 treated seedlings show lower accumulation of As due to a lower GSH pool and the mutated lines have a higher GSH pool and higher accumulation of As (Supplemental Figure 21). Therefore, we came to the conclusion that miR408 and its associated peptide may regulate sulfur metabolism and modulate and reduce glutathione levels to regulate the As detoxification mechanism. There must be a feed-back regulation for the sulfur assimilation pathway mediated by miR408, that may lead to increase of basal sulfate level in miR408OX because it is not utilized in the S assimilation process. Higher level of sulfur inside cell may cause the lower sulfur demand from outside that leads to lower expression of Sultr genes.

On the basis of our results, we propose a model representing miPEP408 regulating the expression of miR408, which negatively regulates the GSTU25 and sulfur reduction pathway genes in Arabidopsis under nutrient and As stress leading to its involvement in the detoxification mechanism against heavy metal and nutrient deficiency (Figure 9).

Materials and methods

Plant materials and growth conditions

Arabidopsis (Arabidopsis thaliana) (Col-0) was used as the WT plant and for overexpression and the editing of miR408 through the CRISPR–Cas9 approach. Seeds were surface sterilized and grown on one-half-strength MS medium (Sigma-Aldrich, USA) according to Shukla et al. (2015). For the control condition, an optimum concentration (1500 µM) of sulfate was used in the medium. For limiting sulfate conditions, MgSO₄ was replaced by MgCl₂ and a 10 µM concentration of sulfate was supplemented. For As(III) treatment, 10 µM Na₂AsO₃ (Stock solution 50 mM Na₂AsO₃, ICN, USA) was added to the one-half-strength MS medium (Sigma-Aldrich, USA). The pH of all the media was adjusted to 5.75-5.85 with 0.1 M KOH or HCl. Seeds were grown in a growth chamber (Conviron, USA) under controlled conditions of 16-h-light/8-h-dark photoperiod cycle, 22°C temperature,150 to 180 µmol m⁻² s⁻¹ light intensity, and 60% relative humidity for 10 days.

Small RNA sequencing and analysis

For the small RNA sequencing, the plant samples were prepared after 10 days of low sulfur and As treatment along with the control condition. The NEB Next Small RNA Sample Preparation protocol is used to prepare a sample sequencing library. The library is prepared as per the kit protocol. The adapters are ligated to each end of the RNA molecule and an RT reaction is used to create single-stranded cDNA. The cDNA is then PCR amplified using a common primer and a primer containing one of 48 index sequences. After libraries are constructed, sequencing was performed on HiSeq 2500 with 1 × 50 bp reads to obtain 40–50 million reads. After the sequencing run, Illumina smallRNA-Seq data was processed to generate FASTQ files.

miRNA target prediction

The computational tool, psRNATarget (http://plantgrn.noble.org/psRNATarget/), was utilized to identify different targets of miRNA408 (Dai and Zhao, 2011). Predicted miRNA targets generated from the 5p and 3p-arm of the hairpin in Arabidopsis.

RLM-RACE analysis for validation of miR408 target and identification of transcription initiation site

To map the cleavage sites of the candidate targets of miR408 in planta, modified RLM-RACE was performed using a First Choice RLM-RACE Kit (Invitrogen) as per manufacturer instruction. Total RNA was extracted from the rosette leaves of the miR408OX line and ligated directly to 5′RNA oligonucleotide adapter and after that cDNA synthesis was performed using oligo (dT) primers using the RevertAid H Minus First- Strand cDNA Synthesis Kit (Applied Biosystems). 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 pT/Z cloning vector (Fermentas) and sequenced to locate the miRNA cleavage sites.

For transcription initiation site identification, full-length mRNAs isolated and treated with calf intestinal phosphatase (CIP) to remove the 5′-phosphate from all molecules which contain free 5′-phosphates (ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA). Full-length mRNAs are unaffected due to capping. 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 is ligated to the RNA population—only molecules containing a 5′-phosphate, the uncapped, full-length mRNAs, will accept the adapter. Random-primed, reverse transcription reactions and nested PCR are then performed to amplify the 5′-end of your specific transcript.

RNA isolation and gene expression analysis

Ten-day-old seedlings were used to isolate total RNA using Spectrum Plant Total RNA Kit (Sigma-Aldrich, USA) as per the manufacturer's instructions. RNA quantity and quality were analysed using NanoDrop spectrophotometer (NanoDrop, Wilmington, DE, USA) and agarose gel electrophoresis. RNase-free-DNase-I (Fermentas, LifeSciences, Canada) was used to remove DNA contamination. For the cDNA preparation, Revert Aid First Strand cDNA synthesis kit (Fermentas, LifeSciences, Canada) was used to reverse transcribe total RNA. Reverse transcription quantitative PCR (RT-qPCR) was carried out in an ABI7500 instrument (ABI Biosystems, USA) using SYBR Green Supermix (ABI Biosystems, USA). As an internal control, the tubulin gene was used to quantitate the relative transcript level of the genes of interest using gene-specific oligonucleotides. Data were analyzed using the comparative Ct (2^−ΔΔct) method (Schmittgen and Livak, 2008). For the expression analysis of mature miR408, 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 for the expression analysis are listed in Supplemental Table 7.

Plasmid construction

For the editing of AtmiR408 using the CRISPR–Cas9 system in A. thaliana, gRNA for AtmiR408 (AT2G47015) was designed using the CRISPR Arizona software (http://www.genome.arizona.edu/crispr). This gRNA was cloned into the binary vector pHSE401 using the BsaI restriction site. This vector contains Cas9 endonuclease-encoding gene under dual CaMV35S promoter as well as genes encoding neomycin phosphotransferase and hygromycin phosphotransferase as selection markers and the construct was sequenced from both the orientations using plasmid-specific forward and vector reverse primers.

For the generation of miR408OX lines, the precursor miR408 (218 bp) was cloned into pBI121 under the control of CaMV35S promoter using XbaI and SacI restriction sites. For the overexpression of miPEP408 (35 aa), the ORF (108 bp) was amplified using genomic DNA of Col-0. The amplicon was cloned into pTZ57R/T and then transferred into plant expression vector pBI121 containing the CaMV35S promoter. For the Histochemical GUS staining, the promoter region (1379 bp) from precursor containing both the ORFs (108 and 129 bp) was PCR amplified using High-Fidelity enzyme mix (Fermentas) from genomic DNA. The amplicon was cloned into pBI121 plant binary vector followed by GUS, using the HindIII and XbaI restriction sites and transformed into Agrobacterium GV3101. These PromiR408::GUS transgenic lines were used to perform GUS assay on different stress condition and validation of functional ORF.

Generation of overexpression and knockout mutant Arabidopsis plants

The pHSE401 vector having gRNA for miR408 and pBI121 (pre-miR408 under the CaMV35S promoter) and pBI121 having miR408 promoter were transformed into Agrobacterium (strain GV3101) using the freeze–thaw method (Clough and Bent, 1998). Arabidopsis thaliana (Col-0) plants were used for transformation by using the floral dip method (Clough and Bent, 1998). The T0 seeds were collected and grown onto half-strength MS plates containing kanamycin (50 mg/ml) for pBI121 and hygromycin (20 mg/ml) for pHSE401. Positive plants were selected and transferred to soilrite for the maturation. Genomic DNA from the leaves was isolated using the GenElute Plant Genomic DNA Miniprep Kit (Sigma). Regions around the target sites were amplified by PCR, and mutations were detected using the Takara Guide-it Mutation Detection Kit (Takara). PCR products from positive plants were cloned in the cloning vector pTZ57R/T and sequenced using M13F and M13R primers. For miR408OX, the cloned sequences were amplified using CaMV35S-F and NosT-R primers and sequenced using gene-specific primers to confirm the construct. For each construct, we generated multiple independent lines.

Synthetic peptide assay

The synthetic peptides (purity > 95%) were synthesized through Link Biotech (http://www.linkbiotech.com). The peptides were dissolved in water (stock concentration, 5 mM). The seedlings were treated with concentrations from 0.25 to 1 μM peptide diluted in the agar medium or in water.

Peptide sequences

• miPEP408-42 aa- MNIRFSQIAVQDFAKQGSTNISGEFWCSTSQKAYRNTIPKSI

• miPEP408-35 aa- MYFGSYHVAAKLFLSTFRFNTHSRKNQNPPANLEG

Biochemical analysis

Lipid peroxidation assay is a biochemical marker for oxidative stress, which is estimated by malondialdehyde (MDA) produced using the thiobarbituric acid (TBA) method (Baryla et al., 2000). Briefly, 0.1 g of sample was ground into powder using liquid nitrogen and was homogenized in 1 ml 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000xg for 10 min. The supernatant was mixed with 4 ml of 20% (w/v) TCA containing 0.5% (w/v) TBA, heated in a boiling bath (950C) for 15 min and then allowed to cool rapidly in an ice bath. The mixture was centrifuged at 10,000xg for 5 min and the resulting supernatant was used for the determination of MDA content. The concentration of MDA was calculated by measuring absorbance at 532 nm (correction was made by subtracting absorbance at 600 nm for turbidity) by using an extinction coefficient of 155 mM^–1 cm.⁻¹

Histochemical GUS staining

The GUS staining was performed using a previously described method (Jefferson, 1989). Seedlings of the PromiR408::GUS transgenic lines were immersed in a solution containing 100 mM sodium phosphate buffer (pH 7.2), 10 mM EDTA, 0.1% (v/v) Triton X-100, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide and 1 mg ml⁻¹ 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide at 37°C for 4 h. The chlorophyll was removed by incubation and multiple washes using 70% (v/v) ethanol. The seedlings were observed under a Leica microscope (LAS version 4.12.0, Leica Microsystems) for the GUS staining. For GUS activity for functional peptide, both the PromiR408::GUS transgenic lines having the ORF regions were grown on half-strength MS for five days and supplemented with miPEP408-35aa and miPEP408-42aa for 2 h and GUS staining was performed to validate the functional miPEP.

Histochemical detection of superoxide and hydrogen peroxide accumulation

NBT staining was used to detect the production of superoxide radicals and was carried out according to the method (Jabs et al., 1996). Ten-day-old seedlings were immersed in 50 mM potassium phosphate buffer (pH 7.8) containing 0.1 mg ml⁻1 nitrobluetetrazolium (NBT) at 25°C in the dark for 2 h. Stained samples were bleached in 80% ethanol and incubated at 70°C for 10–20 min. To analyze the accumulation of hydrogen peroxide (H₂O₂) in the samples, DAB staining was performed according to the method (Daudi and O’Brien, 2012). Ten-day-old seedlings were immersed in 1 mg ml⁻¹ DAB solution. To enhance the uptake of DAB, samples were vacuum infiltrated for 5–10 min. Staining was carried out in dark for 4–5 h with mild shaking. Finally, the samples were de-stained using a 1:1:3 mixture of acetic acid, glycerol and ethanol at 95°C for 15 min. Seedlings were visualized using a Stereoscope zoom binocular microscope (Leica LAS version 4.12.0, Leica Microsystems)

Sulfur estimation

Plant seedlings (200 mg) were homogenized in 4 ml of 0.1 M HCl for 2 h at room temperature. After centrifugation at 12,000xg supernatant was recovered and used for the determination of sulfate content using the turbidimetric method (Tabatabai and Bremner, 1970). The standard curve was plotted using sodium sulfate as a standard and the readings were recorded at 420 nm.

Reduced glutathione (GSH) estimation

A homogenate of ten-day-old seedlings (0.5 g) was prepared in 2.5 ml of 5% (v/v) TCA. Homogenate was centrifuged at 4,000xg for 10 min. The supernatant (0.1 ml) was used for the estimation of GSH. The supernatant (0.1 ml) was made up to 1.0 ml using 0.2 M sodium phosphate buffer (pH 8.0) followed by the addition of 2.0 ml of freshly prepared DTNB (5,5′- dithiobis (2-nitrobenzoic acid) solution. The concentration of GSH was determined spectrophotometrically at 412 nm after 10 min of incubation (Moron et al., 1979). The values are expressed as µmoles g ⁻¹ sample.

Statistical analysis

Data are plotted as means ± SD with error bars as standard deviation. 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 two-tailed Student's t tests using GraphPad Prism version 8.4.3 software (* P < 0.1; ** P < 0.01; *** P < 0.001). All the experiments were repeated at least three times independently, with similar results.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number PRJNA830335.

Supplemental data

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

Supplemental Figure S1. Expression analysis of miRNA(s) in LS, As(III) and LS + As(III) conditions compared to control.

Supplemental Figure S2. Confirmation of GSTU25 as the target of miR408.

Supplemental Figure S3. Representative image of mature WT, miR408 overexpressing and CRISPR-edited plants.

Supplemental Figure S4. Analysis of CRISPR/Cas9-edited miR408 mutants.

Supplemental Figure S5. Quantification of pre-miR408 expression.

Supplemental Figure S6. NBT staining in miR408OX and CRISPR-edited plants in limiting sulfur and As(III) stress.

Supplemental Figure S7. DAB staining in miR408OX and miR408^CR plants in limiting sulfur and As(III) stress.

Supplemental Figure S8. MDA content in miR408OX and miR408^CR seedlings.

Supplemental Figure S9. Relative expression of sulfur reduction pathway genes in miR408OX plants.

Supplemental Figure S10. Relative expression of Sulfur reduction pathway genes in miR408^CR plants.

Supplemental Figure S11. Relative expression of SULTR1; 1 and SULTR4; 1 in miR408OX and edited miR408^CR plants.

Supplemental Figure S12. Total sulfur content in miR408OX and edited miR408^CR plants.

Supplemental Figure S13. Total glutathione (GSH) estimation in miR408OX and mir408^CR plants.

Supplemental Figure S14. Upstream sequence (1000 bp) from pre-miR408 represents putative ORFs.

Supplemental Figure S15. Expression analysis of pre-miR408.

Supplemental Figure S16. Determination of the miR408 transcription start site (TSS) by 5′ RACE.

Supplemental Figure S17. Expression analysis of pre-miR408 and its target in miPEP408OX plants.

Supplemental Figure S18. DAB staining of exogenous miPEP408-supplemented and miPEP408OX 10-day-old seedlings grown on limiting sulfur and As(III) stress.

Supplemental Figure S19. miPEP408 regulates sulfur metabolism and affects plant tolerance toward stress.

Supplemental Figure S20. Overexpression of miPEP408 regulates the sulfur assimilation pathway and plant stress response

Supplemental Figure S21. miR408 affects the arsenic accumulation leading to the sensitivity and tolerance phenotype of Arabidopsis seedlings in heavy metal stress.

Supplemental Table S1. The small RNA sequencing summary.

Supplemental Table S2. Overall summary of raw reads of samples after adapter removal.

Supplemental Table S3. Summary of known miRNAs predicted from samples with the reference genome.

Supplemental Table S4. List of significant differentially expressed miRNAs in limiting sulfur and As(III) conditions compared to control

Supplemental Table S5. List of common miRNAs differentially regulated in limiting sulfur and As(III) conditions compared to control.

Supplemental Table S6. List of predicted targets of miR408.

Supplemental Table S7. Oligonucleotides used for the development of constructs and expression analysis.

Acknowledgments

We thank Q.-J. Chen at China Agriculture University for the pHSE401.

Funding

This research was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, in the form of NCP project no. MLP006. P.K.T. also acknowledges the Department of Biotechnology, New Delhi, for financial support in the form of projects on pathway engineering and genome editing. PKT also acknowledges the Department of Science and Technology for the JC Bose. R.S.K., H.S., and T.D. acknowledge the Council of Scientific and Industrial Research, Department of Biotechnology, New Delhi, for a Senior Research Fellowship. CSIR-CIMAP Publication No.: CIMAP/PUB/2022/140; CSIR-NBRI Publication No.: CSIR-NBRI_MS/2022/12/02.

References

Author notes