- Pi-starvation induced transcriptional changes in barley revealed by a. - Results: In this study, we describe Pi-starvation-responsive small RNAs and transcriptome changes in barley (Hordeum vulgare L.) using Next-Generation Sequencing (NGS) RNA-Seq data derived from three different types of NGS libraries: (i) small RNAs, (ii) degraded RNAs, and (iii) functional mRNAs. - In roots, a more abundant and diverse set of other sRNAs (DESs, 1796 unique sequences, 0.13% from the average of the unique small RNA expressed under low-Pi) contributes more to the compensation of low-Pi stress than that in shoots (DESs, 199 unique sequences, 0.01. - Additionally, in barley shoots, up-regulation of small RNAs is accompanied by strong induction of two nucleases (S1/P1 endonuclease and 3 ′ -5 ′ exonuclease). - Novel P-responsive genes were selected as putative candidates to overcome low-Pi stress in barley plants.. - The most widely studied molecules in this con- text—miRNA399 molecules—are up-regulated in barley shoots and roots under low-Pi conditions [36]. - In Arabidopsis, the level of mature miR778 was up- regulated in shoots and roots in low-Pi conditions, while its target gene expression SUVH6 (SU(VAR)3–9 HOMO- LOG 6) was accordingly reduced [59]. - However, there is still a gap in understanding how Pi- starvation affects the quantity and quality of sRNAs dis- tributed in barley shoots and roots. - In this paper, we analyzed changes in the expression levels of RNAs in barley growing under Pi-starvation, as compared to control/Pi sufficient conditions. - In addition, we performed tran- scriptome analysis of the protein-coding gene expression in barley shoots upon Pi-starvation. - Subsequent analyses were performed (GO analysis, chromosomal mapping, and Pi-responsive motifs localization) to characterize specific stress responses in barley plants to accomplish Pi homeostasis.. - Barley plants display low-Pi symptoms at the morphological and molecular levels. - Severe low-Pi responses were induced in the barley plant line Rolap grown in the soil containing 8 mg P/. - To examine the in- duction of changes at a molecular level by low-Pi stress in barley plants, we measured the absolute gene expression of the low-Pi-responsive marker gene IPS1.. - Identification of barley differentially expressed miRNAs (DEMs) under low-Pi. - We performed small RNA deep-sequencing to find out which small RNAs are up- or down-regulated by Pi star- vation in barley shoots and roots. - 1 The validation of barley line Rolap plant material under low-Pi stress. - a Pictures of the plants (n = 3) collected on the 23rd day after sowing, grown under low-Pi, 8 mg P/kg soil (left) and control-Pi, addition of 60 mg P/kg soil (right), conditions. - The low-Pi stress-specific subsets of RNAs were generated following (i) deep sequencing of small RNAs from barley shoots and roots, (ii) transcriptomic RNA-Seq for barley shoots, and (iii) degradome profiling for barley shoots and roots. - 0.05) in barley shoots and roots, respectively. - The ShortStack analysis supports two more miRNAs identified in barley shoot:. - The ab- solute expression level of miR827 is significantly up- regulated in both shoots and roots under a low-Pi re- gime (Fig. - Barley plants express an organ-specific set of microRNAs in response to low-Pi conditions. - Interestingly, out of 15 miRNA, only miR- NA166d (ID: 2004) was down-regulated in shoot under low-Pi (log 2 (fold change. - Similarly, only miRNA319b (ID: 51) out of 13 DEMs was down-regulated in low-Pi treated roots (log 2 (fold change. - A specific set of miRNAs was expressed in barley shoot or root under low-Pi (Table 1). - Different classes of small RNAs in barley accumulate in an organ-specific manner under low-Pi regime. - We found that small RNAs, other than miRNAs, differentially expressed sRNAs (DESs) in barley under Pi starvation were represented by 199 unique se- quences identified in shoot (0.01% of the average of unique small RNA found in shoots of barley growing under Pi starvation (Additional file 5) and by respectively) unique sequences identified in roots (Fig. - We analyzed whether different lengths (taking se- quences from 18 to 25 nt in lenght) and classes of small RNAs contributed to either root or shoot response to low-Pi conditions. - The DESs obtained from low-Pi roots were mostly annotated to protein-coding mRNAs (38.54. - While in shoot, we found 199 DESs under the low-Pi regime. - Additional file 8).. - We did not find any DESs annotated to the snRNAs, SRP-RNAs, or tRNAs from barley shoot upon low-Pi. - 3 The induced expression level of miR827 (root ID: 114, shoot ID: 2073) correlates with downregulation of its target SPX-MFS1 in barley. - b Detection of hvu-miR827 expression pattern in barley samples used in this study for NGS analysis. - Furthermore, in roots, the most abundant small RNA was a 25 nt DES ID and 65,590.5 mean of normalized counts in barley root in control and low-Pi conditions, log 2 (fc. - In our results from low-Pi treated shoot samples, the highest fold change was represented by a 24 nt DES ID:. - The most abundant small RNA was a 19 nt DES ID and 49,914.1 normal- ized mean counts in barley shoot in control and low-Pi, respectively, log 2 (fc. - Additionally, 613 rRNA- derived sRNAs are up-regulated, whereas 176 rRNA- derived sRNAs are down-regulated in barley roots (Add- itional file 6). - 4 Differentially expressed other small RNAs (DESs) in barley plants under the low-Pi regime. - The annotation distribution of DESs in barley shoots and roots based on the calculations present in Additional file 8 (right panel). - Interestingly, four genes were induced to a higher extent than the low-Pi stress marker, IPS1 gene. - Table 2 List of 98 DEGs from barley shoots (low-Pi vs. - Molecules which exhibited a single mismatch (or more) may still function as miRNA in barley. - In roots, the most downregulated miR319b (22 nt, ID: 51) has pre- dicted three different target loci in barley. - The plant GAMyb TFs, have been shown to activate gibberellin-responsive gene expression of α-amylase in barley [90, 91].. - Degradome profiling was performed to test whether any of the sequences from the 1796 DESs found in roots or 199 DESs found in shoots contribute to the complexity of gene regulation during low-Pi stress. - RNA-Seq data for potato (Solanum tubero- sum L.) proved that expression of the MYB44 gene is highly downregulated under low-Pi in roots [92], which may be the result of miRNA-guided PTGS.. - a Quantification by ddPCR of the absolute expression levels of the DEGs belonging to two selected pathways in barley shoots. - 7 Degradome profile of SPX-MFS1 mRNA directed for cleavage by miR827 in barley shoot using the PAREsnip2 approach. - Thus, we suspect that such sRNA may function as another miR399 isomiR in barley. - In this study, we used a tripartite approach (sRNA-Seq, mRNA-Seq, and degradome-seq) to describe the set of small RNAs differentially expressed in barley roots and shoots under low-Pi stress. - Integrated deep- sequencing data were used to describe organ-specific ad- aptations to low-Pi through either activation or repres- sion of different classes of 18–25 nt small RNAs.. - In both plant species, authors have shown that the num- ber of various miR399 isomiRs was the most abundant in shoots and roots under low-Pi. - Eight of the 15 DEMs (after Bonferroni p-value adjustment) we found in barley shoots belonged to the miR399 family. - Previously, our abso- lute copy number analysis of mature miR399 demon- strated that its normalized expression level is 4-fold downregulated in barley roots, as compared to in shoots, under a low-Pi regime [99]. - In roots, a more diverse set of miRNAs contrib- uted to the compensation of low-Pi stress, compared to that in shoots. - These DEGs were mostly engaged in Pi mobilization and utilization upon Pi- starvation in barley shoots. - published RNA-Seq data describing the barley transcriptome under low-Pi stress [109]. - The authors compared the transcrip- tomes of two barley genotypes with contrasting low-Pi stress tolerance. - The low-Pi induced feedback loop is located in the middle part, which is involved in the positive regulation of phosphate transporters (i.e., PHT1, PHO1) prompting Pi uptake. - Dotted lines display wide area of molecular networks, connecting most of the plant low-Pi stress responses. - This class of genes is responsible for the in- activation of oxalic acid, which mediates fungal – plant pathogenesis in barley [120].. - Received data showed proper library quality and low-Pi induced changes in small RNA levels. - Experiments were performed in three biological replicates of plants grown under low-Pi and control conditions.. - Additional file 1 Normalized copy numbers of barley IPS1 gene transcript in low-Pi treated root material. - Additional file 2. - Additional file 3 MicroRNAs and small RNAs (other) for which expression is significantly changed during Pi-starvation in barley roots and shoots. - Samples R4 – R6 = low-Pi root. - S4 – S6 = low-Pi shoot. - describing molecules that were exclu- sively expressed in low-Pi or control samples. - Additional file 4. - Identification of differentially expressed miRNAs (DEMs) in barley plants under low-Pi regime. - Additional file 5 List of differentially expressed other small RNAs (ID in barley shoots (low-Pi vs. - Additional file 6 List of differentially expressed other small RNAs (ID in barley roots (low-Pi vs. - Additional file 7. - Length distribution of DESs identified in barley roots and shoots.. - Additional file 8. - Annotation distribution of DESs identified in barley roots and shoots.. - Additional file 9. - Degradome profile (TargetSeek approach) demonstrates potential mRNA targets for differentially expressed miRNAs (DEMs) identified in barley shoots (low-Pi vs. - Degradome profile (TargetSeek approach) demonstrates potential mRNA targets for differentially expressed other sRNAs (DESs) identified in barley shoots (low-Pi vs. - Degradome profile (TargetSeek approach) demonstrates potential mRNA targets for differentially expressed miRNAs (DEMs) identified in barley roots (low-Pi vs. - Degradome profile (TargetSeek approach) demonstrates potential mRNA targets for differentially expressed other sRNAs (DESs) identified in barley roots (low-Pi vs. - Degradome profile (PAREsnip2 approach) demonstrates potential mRNA targets for differentially expressed miRNAs (DEMs) identified in barley shoots (low-Pi vs. - The t-plots generated by PAREsnip2 software show- ing the potential mRNA targets for differentially expressed miRNAs (DEMs) identified in barley shoots (low-Pi vs. - Degradome profile (PAREsnip2 approach) demonstrates potential mRNA targets for differentially expressed other sRNAs (DESs) identified in barley shoots (low-Pi vs. - The t-plots generated by PAREsnip2 software show- ing the potential mRNA targets for differentially expressed other sRNAs (DESs) identified in barley shoots (low-Pi vs. - Degradome profile (PAREsnip2 approach) demonstrates potential mRNA targets for differentially expressed miRNAs (DEMs) identified in barley roots (low-Pi vs. - The t-plots generated by PAREsnip2 software show- ing the potential mRNA targets for differentially expressed miRNAs (DEMs) identified in barley roots (low-Pi vs. - Degradome profile (PAREsnip2 approach) demonstrates potential mRNA targets for differentially expressed other sRNAs (DESs) identified in barley roots (low-Pi vs. - The t-plots generated by PAREsnip2 software show- ing the potential mRNA targets for differentially expressed other sRNAs (DESs) identified in barley roots (low-Pi vs. - Spike-in quality control of RNA-Seq samples from barley shoots (low-Pi vs. - Development and characterization of polymorphic EST based SSR markers in barley (Hordeum vulgare). - Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley. - Identification and characterisation of a previously unknown drought tolerance-associated microRNA in barley. - Heat stress affects Pi-related genes expression and inorganic phosphate deposition/accumulation in barley. - Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves. - Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat. - Gibberellin signaling in barley aleurone cells
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