- responses to temperature stress. - Our data showed that miR535 and miR156 families may derive from a common ancestor during evolution and jointly play a role in fine-tuning SPL gene expression in banana. - We also identified the miRNA-triggered phased secondary siRNAs in banana and found miR393- TIR1 / AFB phasiRNA production displaying cold stress-specific enrichment.. - Conclusions: Our results provide a foundation for understanding the miRNA-dependent temperature stress response in banana. - The characterized correlations between miRNAs and their response to temperature stress could serve as markers in the breeding programs or tools for improving temperature tolerance of banana.. - Accumulation of soluble sugars in the peel induced by a high temperature has been proposed as a major factor suppressing chlorophyll degradation, causing the stay-green ripening phenomenon [3]. - For more than two decades, miRNAs have been widely studied as import- ant regulatory molecules involved in almost all aspects of the plant life cycle. - Despite these efforts, miRNA investigation in banana has a relatively short history. - Although a number of miR- NAs have been reported in banana so far no banana miRNA sequences have been deposited in the miRNA database - miRBase. - Moreover, the role of miRNAs in the temperature stress response of ba- nana fruit remains largely unknown. - We comprehensively identified the temperature-responsive miRNAs and their targets, and characterized the miRNA- triggered phasiRNA production in banana. - The peel firmness of the heat-stressed fruit decreased much faster than the control, and more strik- ingly a sharp drop of pulp firmness was detected in the heat-stressed fruit after the 6 th day. - However, cold stress increased the peel firmness especially on the 2 nd day while it caused a fluctuation in the pulp firmness (Fig. - 70% were perfectly matched to at least one locus in the banana genome. - The 20~24-nt sRNAs con- stituted over 75% of the identified banana sRNAs, and the 21-nt sRNAs was the most abundant class in all samples. - The total 21-nt sRNAs were less abundant in the temperature-stressed fruit than in the control, espe- cially for the heat-stressed fruit. - In addition, the total 24-nt sRNAs were less abundant in the cold-stressed fruit (Fig. - On the contrary, the unique 21-nt sRNAs were more abundant in the cold-stressed fruit than in the control, and the expression of the unique 24-nt sRNAs was higher than the 21-nt class, but only for the control fruit (Fig. - In the temperature-stressed fruit, both total and unique 18~20-nt sRNAs became more abundant. - These dynamic changes in the sRNA population clearly showed that cold and heat stresses have differential effects on the sRNA biosynthesis.. - MiRNA profiling in banana. - After excluding sRNA reads homologous to known miRNAs (≤1 mismatch, miRBase 21) and other non- coding sRNAs (Rfam v12.0), the remaining 21~22-nt sRNAs were subjected to novel miRNA identification based on a series of stringent criteria recognized by the research community [27]. - To validate the sRNA sequencing data, we performed RNA gel blot analysis for selected miRNAs representing known examples in the control and cold/heat-stressed samples (Fig. - We found that the blotting results for most highly expressed known miRNAs were reflective of the relative abundances of sequenced sRNAs. - For ex- ample, miR164 and miR168 were more accumulated in the heat-stressed sample. - 1 Physiological changes in banana fruit under different temperature stresses. - The experiment was conducted twice independently with similar results, and typical photos of the fruit were presented. - The impact of temperature stress on miRNA expression To detect the effect of different temperature stresses on banana miRNAs, the expression of miRNAs in banana fruit with cold stress, heat stress and without stress were examined. - down-regulated than up-regulated in the heat-stressed fruit. - 2 sRNA and miRNA expression profiling in banana. - a Length distribution of total (left panel) and unique (right panel) sRNA sequences in banana stored at control temperature and subjected to cold and heat treatments. - b Heat map and overall read abundance of the known miRNAs in the control, cold- and heat-stressed samples. - and heat stress. - For example, mac-miR164a/b target- ing a NAC gene was down-regulated in the cold-stressed fruit but up-regulated in the heat-stressed fruit. - of the unique reads can be perfectly aligned to the banana transcriptome (https://banana-genome-hub.. - 0.05), all for 37 of the 13 known miRNAs or families (Table 1 and Additional file 1: Table S5). - Although most of the genes (48 of 60) identified were the conserved targets for these miR- NAs across a wide range of plant species, a few of them (12 of 60) had not been reported in other spe- cies. - 3 Expression and temperature responsiveness of miRNAs in banana. - SPL gene family co-targeted by miR156 and miR535 in banana. - Considering the abundantly and differentially expressed miR535 isoforms, our result suggest that miR535 family may play an equally considerable role, as the well-conserved miR156 family does, in fine tuning SPL gene expression in banana.. - Characterization of PHAS loci and their triggers in banana PhasiRNAs are a major class of sRNAs in plants [9].. - 400 bp), compared to coding PHAS loci, suggesting that in banana non-coding PHAS loci mainly generate relatively short transcripts.. - variations of PHAS loci distribution and phasiRNA pro- duction in banana fruit upon temperature stress. - In general, more PHAS loci were found in the cold- stressed fruit while less loci in the heat-stressed fruit, regardless of coding or non-coding PHAS loci (Fig. - As examples, two PHAS loci exhibited distinct phasiR- NAs production in the temperature-stressed samples.. - MiRNA and sRNA expression in banana. - 5 MiR156 and miR535 families co-targeting SPL gene family in banana. - The cleavage site detected in the degradome is indicated in blue and yellow letters. - The cleavage sites detected in the degradome are highlighted in blue letters. - 6 PhasiRNA-generating ( PHAS ) network in banana. - a All PHAS loci were grouped into coding and non-coding genes, and coding PHAS loci were further classified based on their annotation, as shown in the pie chart. - b Venn diagram for coding and non-coding PHAS loci differentially accumulated in the control and temperature-stressed samples. - c Two examples of coding PHAS loci displaying differential variations in phasiRNA production in banana upon cold and heat stress. - In both panels, each track represented small RNA abundance based on mapping results in the control, cold- and heat-stressed samples. - Changes of the phasiRNA accumulation were highlighted in the blue boxes. - Table 2 PHAS loci and trigger miRNAs in banana. - done on characterizing miRNAs in banana [23, 24], espe- cially in banana fruit subjected to different temperature stress. - Deep sequencing of the banana sRNA libraries revealed 113 known miRNAs as well as 26 banana-specific miRNAs (Additional file 1: Tables S2 and S3). - Deep sequencing and whole- genome data mining enable us to discover probably most of the miRNAs in banana under temperature stress.. - And banana apparently has evolved an expanded miR156/miR535 gene family to conform to their target SPL family that comprises a large number of gene members, with the functional importance in banana stress response to be further determined.. - Among the three major protein-coding PHAS gene families in eudicots, only NB-LRR s yielded abundant phasiRNAs in banana. - For other phasiRNA pathways conserved in plants, only miR828- MYB and miR393- TIR1 / AFB were identified in banana. - All these results indicate the dynamic feature of PHAS pathways in banana dur- ing evolution. - In modern plants, auxin plays an import- ant role in the regulation of leaf morphology, lateral root growth, and developmental patterning [37, 38], with ARF and TIR genes being the main components.. - However, the well-conserved miR390- TAS3 - ARF pathway appeared to be minimized in banana and the reason for such functional diversifi- cation of auxin-related pathways deserves further investigation.. - The temperature-responsive miRNAs in banana. - On the other hand, high temperature stress can result in the ‘stay-green’ ripening disorder of ba- nana fruit. - In this study, the expression levels of miR- NAs in banana with and without temperature stress treatments were separately compared. - around 30% of the miRNAs showed significant changes under temperature stress (Additional file 1: Table S4). - On the other hand, strong suppression of miR167b, miR398, miR528 and miR408 expression by cold was widely ob- served in banana. - Likewise, some previously identified cold-induced conserved miRNAs, such as miR319, miR393 and miR402 in Arabidopsis [16], were not found in our data, suggesting that the induction levels of certain miRNAs may be too low to be detected as significant, or they were unaffected by cold in banana. - These data suggest that both kinds of regulation for miR- NAs were involved in the banana cold response.. - Our result indicated that the miR156- SPL module might also mediate the heat stress response in banana. - MiRNA-mediated pathways involved in banana temperature stress response. - Based on miRNA expression profiling and dagradome combined with qRT-PCR validation, we found that sev- eral miRNA-target pairs might have played important roles in the regulation of banana temperature stress re- sponse. - Moreover, an inverse expression pattern of the two tested SPL genes was observed at the later stage of storage, suggesting that SPL transcript accumulation may be balanced via the co-regulation of miR156 and miR535.. - Another well anti-correlated module in banana was miR159 targeting PCF/TCP transcription factor. - Such pattern was opposite in the heat-treated sample (Fig. - In addition, we did not identify any miR319 variants in banana. - We suggest that the downregulation of LAC was mediated by the induction of miR397, which might be involved in the thermotolerance through protection from oxidative dam- age. - However, there has been no direct evidence for the re- lationship between oxidative damage and ripening disorder upon heat stress in banana. - Altogether, our work delivered new insights into the role of miRNAs in the temperature stress response of banana fruit. - Most targets of temperature-responsive miRNAs in banana were transcription factors including a large group of target genes involved in auxin signaling, and other functional genes associated with redox/nutrient homeostasis. - Length variation at both 3′ and 5′ ends and one mismatch inside of the sequence were allowed in the alignment. - (5) Libraries were sequenced using the 5′ adaptor only, resulting in the sequencing of the first 36 nucleotides of the inserts that represented the 5 ′ ends of the original RNAs. - The extracted sequen- cing reads were used in the standard data analysis. - TBtools software was used for the construction of the heat map [56].. - Two-nucleotide offset was added for sRNA matching to the antisense strand due to the 2-nt overhang at the 3′ end of the sRNA duplex. - more than half of the mapped distinct sRNAs were 21-nt in length, and with ≥3 distinct reads in a certain register. - (2) over 30% of the siRNAs from a given PHAS locus were produced in phase. - Summary of known miRNAs in banana. - Summary of species-specific miRNAs in banana. - Summary of differentially expressed miRNAs in respond to temperature stress in banana. - Target genes of known miRNAs in banana. - PHAS loci in banana. - All raw sequencing data were deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE77590.. - All of the authors read and approved the final manuscript.. - Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit. - MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. - The miRNA156/157 recognition element in the 3 ’ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings
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