Auxin signaling mutant slr1 is susceptible to low temperature
To decipher microRNA mediated auxin signaling during plant cold stress response, we first focused on the identification of the cold-responsive auxin mutants. For the selection of cold-responsive auxin mutants, we used the root growth recovery assay developed in our lab earlier [9]. 12 h cold treatment at 4°C typically results in approximately 40-50% root elongation inhibition during the recovery phase at 23°C [9]. Hence, we selected 12 h incubation at 4°C as an optimal treatment for analyzing the effect of cold stress. Both auxin signaling mutants slr1, tir1, axr1-3, and afb2-1, and auxin transport mutants aux1-7, eir1-1, pin3-3 and pin4-3 were subjected to cold stress screening [56-63]. Percent root elongation recovery, compared to control (grown at 23°C) of each genotype was used as a measure of screening. 5-day-old seedlings grown at 23°C were transferred to new plates and kept at 4°C for 12 h and brought back at 23°C for recovery. The root elongation recovery was analyzed after 6 h and 24 h of recovery (Fig. 1) [9]. Consistent with previous results, we also found that cold stress inhibits root growth recovery by approximately 50% in the wild-type after 6 h (Fig. 1A). Through root growth recovery screening, we could identify slr1, an Aux/IAA14 mutant as a potential candidate as slr1 showed slower root elongation recovery at both 6 h and 24 h time point (Fig. 1, Supplementary Fig. S1). The other mutants did not show any significant difference compared with wild-type for root elongation recovery except auxin transport mutant pin4-3, which shows a slight but statistically significant slower root recovery response at 24 h (Fig. 1). To further confirm whether slr1 response to cold stress persists for a longer time, we measured the root growth recovery till 24 h, and found that slr1 shows slower root growth recovery at all-time points we tested (Fig. 2A and 2B). Because the slr1 response to cold stress was consistent at all-time points, we selected slr1 for miRNA study.
High-throughput sequencing of small RNA libraries
To identify the microRNAs that are responsive to auxin-mediated cold stress response, root tissues from 5 days old seedling grown on modified Hoagland media followed by treatment at 4°C for 12 h, and control grown at 23°C were used to construct small RNA libraries. We made eight small RNA libraries; four from wild-type (two each from 23°C and 4°C) and four from auxin mutant slr1 (two each from 23°C and 4°C). By performing high-throughput sequencing on Illumina platform, a total of 270,303,139 reads (47,318,921 and 39,919,856 reads from Col-0 23°C; 30,230,648 and 23,124,483 reads from Col-0 4°C; 22,247,497 and 43,063,180 reads from slr1-23°C; 25,807,408 and 45,591,146 reads from slr1-4°C respectively) were generated. After trimming the adaptor and low-quality reads, the sequence reads were generated (Table 1). A total of 2,556,265 different tags were found from the trimmed reads, which comprises 933,996 different tags from Col-0 23°C; 441,338 different tags from Col-0 4°C; 626,812 different tags from slr1-23°C and 554,119 different tags from slr1-4°C respectively. The nucleotide length distribution in all the libraries showed that the majority of sequences ranged from 19 to 29 nt in size. We found 21 nt long small RNAs were the most abundant in all four libraries, followed by 24 nt. The total abundance of 2 types of small RNA population, i.e. 21 nt and 24 nt were 46.26% in Col-0 23°C, 56.44% in Col-0 4°C, 42.66% slr1-23°C and 30.43% in slr1-4°C respectively (Table 2). The significant change in small RNA abundance in the cold stressed slr1 compared with wild-type suggests the importance of auxin signaling in miRNA modulated cold stress response. The populations of 26 nt-31 nt small RNAs were drastically affected in slr1 backgrounds by cold stress as an increase in 26 nt-31 nt were observed (Table 2). We also found a significant decrease in the 21 nt small RNA population in slr1 under cold stress, while in Col-0 this was increased. These results further reinforce the idea that auxin directly regulates miRNA expression (Fig. 3, Table 2). We did not observe any significant changes in 24 nt reads for treatments and genotypes (Table 2, Fig. 3). The significant change in the major population of small RNAs in the slr1 mutant under cold stress confirms the involvement of auxin response in regulating miRNA function linked to cold stress response pathway, and also provides a possible explanation for the hypersensitive response of slr1 to cold stress.
Identification of known and novel miRNAs
To identify the number of known miRNAs in small RNA libraries, we used CLC genomics workbench v12.0 and aligned unique reads to known Arabidopsis miRNA sequences in miRBase v22.1, allowing up to 0 mismatches. A total of 180 known miRNAs, representing 70 families, were identified in eight small RNA libraries made from root tissue (Fig. 4, Fig. 5, Supplementary Table S1). In all the eight libraries, miR166 family was the most abundant, followed by miR165 and miR168 families. During low-temperature stress, we found differential expression patterns of miRNAs in wild type and slr1 (Fig. 4, Supplementary Table S1). The comparative analysis between cold treated Col-0 and slr1 revealed that the expression of 13 miRNAs significantly changed in slr1 during cold stress (Table 3). We also observed altered expression of 10 miRNAs in slr1 background between control and cold treatment (Table 3). Interestingly, except for one, all the miRNAs expression was downregulated in slr1 mutant in comparison to Col-0.
For the identification of novel microRNAs, mirDeep-P pipeline was used. A total of 71 sequences were predicted to be potentially novel microRNAs from unannotated small RNAs (Supplementary Table S2 and S3, Supplementary Fig. S2). The abundance of novel miRNAs was lower compared to conserved miRNAs and their length varied from 19 nt to 30 nt. The comparative analysis of novel miRNAs between Col-0 and slr1 revealed that 7 of them significantly changed in slr1 under cold stress (Table 4, Supplementary Fig. S2).
Validation of miRNA expression patterns
The consistency in microRNA expression identified by deep sequencing was validated using quantitative realtime-PCR. We selected 13 differentially expressed miRNAs that were regulated in response to cold in the root. The validation was performed using 10 known miRNAs (miR156, miR164b-3p, miR169a-5p, miR171-5p, miR390-5p, miR5642a, miR408-5p, miR398a-5p, miR472-3p and miR774a-5p) and 3 novel miRNAs (miR_Pred7, miR_Pred27 and miR_Pred37). In general, qRT-PCR validation results of miRNAs expression patterns were in agreement with the deep sequencing data, confirming the accuracy of the sequencing data (Fig. 6). Collectively, the qRT-PCR data confirm that the observed differences in miRNA expression in NGS are consistent and reproducible.
miRNA Target Prediction
The degree of sequence complementarity between miRNA and its binding site within the target determines the mode of action of miRNA. High sequence complementarity results in cleavage of targets [47, 64, 65], while low sequence complementarity results in translational inhibition [66, 67]. Several online resources such as psRNATarget use the same strategy to identify the plant miRNA targets. We carried out target prediction to understand the function of identified miRNAs by using psRNATarget server with preset values. The predicted targets for these miRNAs were from different classes of proteins associated with development, transport, auxin regulation, signaling and stress response (Fig. 7, Supplementary Table S1 and S2). For instance, cold stress response and signaling related proteins were targeted by miRNAs such as miR396b-3p, which targets MYB like transcription factors and miR156, which targets SQUAMOSA-PROMOTER BINDING-LIKE (SPL) transcription factor (Table 5). miRNAs like miR390a/b-5p targets the TASI-ARF, which is involved in auxin signaling. The possible roles of the target proteins in integrating auxin and cold stress responses have been discussed in detail in the discussion section. In general, we predict that the regulation of essential proteins contributing to cold stress tolerance in slr1 is possibly linked to the cold susceptible phenotype of the mutant.
miR169-NF-YA module is altered in slr1 under low-temperature stress
To understand the biological significance of the RNAseq results, we selected one of the potential miRNA candidates, miR169, which is evolutionarily conserved, reported to be present in various plant species including monocots, dicots, ferns and gymnosperms [68-70], and has been shown to be a central regulator of various abiotic stresses, including drought, salt, cold, heat, oxidative and hypoxia [71]. The miR169 family of Arabidopsis has 14 members, that matures into four types of different isoforms, differing only 1 or 2 nucleotides [71]. Phylogenetic analysis of miR169 revealed that apart from miR169a, b, c, and h, there are three obvious clades: clade I (mir169d, e, f, g), clade II (miR169i, k, m) and clade III (miR169j, l, n) [71]. The miR169 family members show distinct temporal and differential expression patterns and thus regulate diverse target genes [71, 72]. One of the major targets of miR169 for abiotic stress response is NUCLEAR FACTOR Y (NF-YA), a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins [73]. The link of the NF-Y family members in regulating plant developmental and stress response pathways has been demonstrated in several studies [73-81]. Earlier, a direct effect of temperature on miR169h and NF-YA was demonstrated [82]. miR169h abundance is directly influenced by temperature; while at high temperature the abundance was high, at low temperature the abundance was considerably low. As expected, miR169 target gene NF-YA expression was reciprocal to the abundance of the miR169 expression [82]. They further demonstrated that NF-Y complex regulates the temperature-dependent flowering and petiole length through directly binding to the promoters of flowering regulator FT and the auxin biosynthesis gene YUC2. These results make an elegant model linking miR169, NF-Y and auxin. We tested whether a similar module works for cold stress response in slr1. For better clarification of the role of the miR169 family, we selected at least one member from each clade, and miR169a, b and h. Beside miR169m, in cold stressed slr, all the tested miR169 members showed either a significant reduction in expression (miR169a, miR169d, miR169e, mir169h) or no changes in expression (miR169b, miR169g) (Fig. 8). The expression data suggest that among miR169 family members, miR169a, miR169d and miR169h function as major regulators to link auxin response and cold.
Next, we investigated whether cold stress-induced change in miR169 affects the NF-YA abundance reciprocally. In Col-0, under cold stress, we observed a decrease in NF-YAs abundance (8, 15 and 22 fold decrease respectively for NF-YA3, NF-YA5 and NF-YA8 compared with 23°C, while in slr1, there was a higher accumulation of NF-YA transcripts compared with Col-0 (3.94, 4.3 and 4.4 fold increase for NF-YA3, NF-YA5 and NF-YA8 respectively, Fig. 8). Based on the above findings, we speculate that the altered miR169-NF-YA module in slr1 is possibly contributing to its susceptible phenotype to cold stress. Further, miR169-NF-YA module might be playing a pivotal role during cold stress recovery, and miR169 regulates the expression of this module in an SLR dependent manner.