- Long noncoding RNAs (lncRNAs) have recently been reported to be key regulators in the responses of plants to stress conditions, but the mechanism through which LP stress mediates the biogenesis of lncRNAs in soybean remains unclear.. - GO and KEGG analyses indicated that numerous DE lncRNAs might be involved in diverse. - Moreover, lncRNA-mRNA-miRNA and lncRNA-mRNA networks were constructed, and the results identified several promising lncRNAs that might be highly valuable for further analysis of the mechanism underlying the response of soybean to LP stress.. - Our findings increase the understanding of and provide new insights into the function of lncRNAs in the responses of soybean to P stress.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - As one of the major mineral macronutrients present in all living things, P is essential for plant growth and development due to its key role in the regulation of energy metabolism and the synthesis of nucleic acids and membranes [20, 21]. - RNAs serve as one of the key regulators involved in the P starvation response network. - Previous studies have provided an understanding of the protein-coding genes and miRNAs involved in the response of soybean to phosphate starvation but the role of lncRNAs in the response of soybean to LP stress has rarely been reported.. - Using genome-wide high-throughput RNA sequencing (RNA-seq) technology, we identified and characterized a total of 4,166 lncRNAs that are respon- sive to LP stress in the roots of soybean seedlings, validated 14 lncRNAs by qPCR, and identified 525 dif- ferentially expressed (DE) lncRNAs related to the regula- tion of the tolerance of soybean to LP stress. - The results lay the foundation for obtaining a more in-depth understanding of the mo- lecular mechanisms related to the roles of lncRNAs in response to LP stress. - This study increases our know- ledge of lncRNAs and provides new insights into the function of lncRNAs in LP stress.. - Identification and characterization of lncRNAs across two soybean genotypes under different P levels. - of the 4,166 lncRNAs were lo- cated in intergenic regions, and the remaining . - 1 Identification and characterization of lncRNAs in soybean roots of two genotypes. - b Chromosome-wise distribution of lncRNAs. - c Numbers of predicted exons and introns in the lncRNAs. - of the lncRNAs. - e Sequence length distribution of lncRNAs. - We subsequently analyzed the chromosomal location of all the lncRNAs in the soybean genome. - The majority of lncRNAs have GC percent in the range of 30–45 % (Fig. - of the lncRNAs were shorter than 2,000 nucleotides (Fig. - 3 DE lncRNAs and expression patterns in soybean root plants under LP stress. - b Number of DE lncRNAs in the same genotype between different P levels. - c Number of DE lncRNAs between different genotypes at the same P level. - d Cluster analysis of the expression levels of common DE lncRNAs in the same genotype at different P levels. - e Cluster analysis of the expression levels of common DE lncRNAs in different genotypes at the same P level. - transcript abundance of lncRNAs. - In total, 525 DE lncRNAs were identified among the two different genotypes under HP and LP conditions, and these included 116 DE lncRNAs between different P levels in the same genotype, 456 DE lncRNAs between different genotypes at the same P level, and 47 shared DE lncRNAs (Table S2). - To identify the effect of LP stress on lncRNAs, we compared the DE lncRNAs of dif- ferent genotypes under the same P condition and in the same genotype at different P levels (Fig. - As shown in the volcano plot, the LP treatment of Bogao and NN94156 resulted in more downregulated DE lncRNAs than upregulated DE lncRNAs, and the downregulated DE lncRNAs presented a more substantial change in dif- ferential expression than the upregulated DE lncRNAs (Fig. - The number and fold change in expres- sion of the upregulated and downregulated DE lncRNAs were relatively consistent in the Bogao and NN94156 ge- notypes under the same P level (Fig. - Because the two genotypes showed markedly different responses to LP stress, we performed a Venn diagram analysis to elucidate the DE lncRNAs between the two genotypes under LP conditions. - The number of common and unique DE lncRNAs between the two genotypes is indicated in the Venn diagram (Fig. - NN94156 and Bogao shared 21 common DE lncRNAs in the HP vs. - LP comparisons, and Bogao exhibited more genotype- specific DE lncRNAs (72) than NN Fig. - 3b), which is consistent with the results shown in the vol- cano plot (Fig. - We found that the 21 common DE lncRNAs in Bogao were all downregulated under LP conditions, whereas most of these downregulated. - To determine whether the effect of LP stress on lncRNAs is related to genotype, we compared the changes in DE lncRNAs be- tween Bogao and NN94156 under LP or HP conditions.. - The results identified 133 and 139 unique DE lncRNAs under the LP and HP conditions, respectively (Fig. - The 184 common DE lncRNAs showed the same up- or downregulation trend: 123 were downregulated, and 61 were upregulated (Fig. - Validation and quantification of lncRNAs. - Both the qPCR and RNA- seq assays revealed a positive correlation in the expres- sion fold-change with an R 2 of 0.7878 (Fig. - 4b), which indicated the robustness of our analysis and the reliabil- ity of the lncRNA expression patterns identified in the current study. - Functions and expression patterns of DE lncRNAs and their target genes. - To reveal the potential functions of the differentially expressed lncRNAs under LP stress in two contrasting genotypes, we predicted the candidate targets of cis-, trans- and antisense-acting DE lncRNAs. - In total, 785 targets of 374 DE lncRNAs were identified, and for 960 pairs, one lncRNA might have several targets and/or one mRNA target might be targets of several lncRNAs. - 4 Confirmation of the expression patterns of lncRNAs by qPCR. - The GO analysis of DE lncRNAs in one genotype at different P levels revealed that 403 GO terms (195 in the biological process category, 146 in the mo- lecular function category, and 62 in the cellular component category) were significantly enriched (P <. - The analysis of the DE lncRNAs in Bogao or NN94156 exposed to the same P level showed that 1,086 GO terms (497 in the biological process category, 362 in the molecular function cat- egory, and 227 in the cellular component category) were significantly enriched (P <. - Al- though the numbers of GO terms in the two geno- types were different, their trends were relatively similar. - We subsequently analyzed the enrichment of the pre- dicted target genes of DE lncRNAs in KEGG pathways (Table S5). - The targets of DE lncRNAs in the same genotype between different P levels were enriched in 42 KEGG pathways, including several KEGG pathways re- lated to carbohydrate metabolism, lipid metabolism, and amino acid metabolism (Fig. - 5 Gene ontology (GO) enrichment of DE lncRNAs targets. - a Targets of DE lncRNAs in the same genotype between different P levels. - b Targets of DE lncRNAs between different genotypes at the same P level. - The analysis of targets of DE lncRNAs be- tween Bogao and NN94156 under the same conditions (HP and LP) showed that 74 KEGG terms were enriched, and these included environmental adaptation, carbohydrate metabolism, biosynthesis of other second- ary metabolites, lipid metabolism, and signal transduc- tion. - These findings suggest that DE lncRNAs might regulate genes involved in many biological processes, including molecular metabolism, energy synthesis and signal transduction, in response to LP stress.. - 00068024 (a novel identified mRNA) were targets of lncRNAs and several miRNAs. - TFs regulate a diverse group of genes during stress re- sponses and are important components of gene regula- tory networks, and many TFs belonging to some families have been proven to play an important role in the main- tenance of P homeostasis, such as the phosphate. - P-related genes such as PHO2 and PHR1 play important roles in the P starvation response. - To further study the function of lncRNAs in the responses of soybean roots to LP stress, we constructed a lncRNA-mRNA network of mRNAs of interest (including transcription factors and P-related and plant hormone targets) and corresponding lncRNAs according to the GO, KEGG and functional annotations of the target genes (Table S7). - Twenty-three lncRNAs might be in- volved in the regulation of gene transcription because their target genes have transcription factor activity. - Twenty-six TFs be- long to diverse families, such as MYB, bHLH, NAC, and AP2, and among these, MYB and bHLH reportedly play roles in the maintenance of P homeostasis [40]. - analysis of lncRNAs in two contrasting soybean geno- types subjected to phosphate starvation.. - The number of lncRNAs varies greatly across plant species. - For example, 48,345 lncRNAs have been identi- fied in the maize transcriptome [15], and 1,212 novel lncRNAs have been found in Arabidopsis seedlings grown under P-sufficient and P-deficient conditions [14]. - To identify the lncRNAs that were responsive to P stress, we identified the DE transcripts of the lncRNAs through pairwise comparisons between the two soybean genotypes under HP and LP conditions, and a total of 525 DE lncRNAs were identified among the two differ- ent genotypes under HP and LP conditions. - S1, the number of DE lncRNAs under LP stress identified in Bogao was greater than that found in NN94156, which indicated that Bogao is more sensitive to LP treatment, and this result is consistent with our previous findings that Bogao and NN94156 are P-sensitive and P-tolerant genotypes, respectively [30, 33]. - LP comparisons, and Bogao exhibited more genotype-specific DE lncRNAs (72) than NN Fig. - Most of the common DE lncRNAs were constitutively downregu- lated in the two genotypes, which indicated that the biological mechanisms of lncRNAs involved in basal re- sponsiveness to LP stress were conserved in both soybean genotypes. - To determine whether the ef- fect of LP stress on lncRNAs is related to genotype, we compared the changes in DE lncRNAs between Bogao and NN94156 under LP or HP conditions. - We identified 317 DE lncRNAs (181 downregulated, 136 upregulated) under LP conditions, which suggested that these lncRNAs were constitutively but differentially expressed between the two genotypes under LP conditions. - The 133 DE lncRNAs unique to LP conditions might play a role in LP tolerance. - In contrast, 139 lncRNAs were dif- ferentially expressed in the two genotypes only under HP conditions, which suggested that their differential ex- pression is specific to LP stress.. - To reveal the potential functions of the DE lncRNAs under LP stress in the two contrasting genotypes, we predicted the candidate tar- gets of the DE lncRNAs and then analyzed the GO terms and KEGG pathways of their putative target genes. - The analysis of the DE lncRNAs in one geno- type at different P levels and DE lncRNAs in Bogao or NN94156 exposed to the same P level revealed that 403 and 1,086 GO terms and 42 and 74 KEGG pathways were significantly enriched (P <. - In this study, targets of lncRNAs were enriched in various KEGG pathways, including flavonoid, isoflavonoid and phenyl- propanoid biosynthesis, which indicates that these lncRNAs might be involved in the synthesis of secondary metabolites to regulate P-responsive genes, but this hy- pothesis needs further research.. - Therefore, lncRNAs might be partly involved in ethylene-mediated LP stress tolerance, and both genes are candidate genes that merit further investigation to gain further understanding of the in- volvement of lncRNAs in LP stress tolerance. - The identification of other genes using our preliminary scenario suggested that lncRNAs are involved in the re- sponse to LP stress through the manipulation of genes with a variety of functionalities, and many of these genes might also be cotargets of P-associated miRNAs. - In addition to ethylene, auxin, GA and salicylic acid might be involved in the response to LP stress in soybean. - LP stress can alter the genome-wide expression levels of lncRNAs, particularly in the P- sensitive genotype Bogao. - These findings might provide a first look at the landscape of lncRNAs in soybean in response to LP stress. - promising lncRNAs that might have potential value for further analysis of the mechanism underlying the re- sponse of soybean to LP stress. - Overall, this study en- riches the knowledge concerning lncRNAs and provides some clues for exploring the function of lncRNAs in the response of soybean to LP stress.. - The soybean plants were placed in the hydroponics box using a completely randomized block design. - Identification of lncRNAs. - Quantitative PCR (qPCR) validation of lncRNAs. - Analyses of lncRNAs and/or mRNAs with miRNAs. - Number of up- and downregulated DE lncRNAs under LP and HP conditions in the two soybean genotypes.. - DE lncRNAs in different genotypes and P levels.. - GO enrichment analysis of the targeted mRNAs of significantly DE lncRNAs in two comparisons.. - KEGG pathway annotation of the predicted target mRNAs of DE lncRNAs in two comparisons.. - The datasets generated and/or analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) Database, accession number SRP233239 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP233239).. - Genome-wide analysis of long non-coding RNAs affecting roots development at an early stage in the rice response to cadmium stress.. - Long Non-Coding RNAs Responsive to Salt and Boron Stress in the Hyper-Arid Lluteno Maize from Atacama Desert. - Research on plant abiotic stress responses in the post-genome era: past, present and future. - Novel signals in the regulation of Pi starvation responses in plants: facts and promises. - LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions
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