Study : Next Generation Sequencing Facilitates Quantitative Analysis of bHLH6 OV, spx4, and wild-type Transcriptomes
Next Generation Sequencing Facilitates Quantitative Analysis of bHLH6 OV, spx4, and wild-type Transcriptomes
Purpose: To investigate the expression profiles of genes regulated by bHLH6, we performed RNA-seq analysis using bHLH6 OV, spx4, and wild type treated with high and low P Methods: Roots and shoots mRNA profiles of 7-day-old bHLH6 OV, spx4, and wild-type (WT) rice seedlings treated with 200 μM or 10 μM Pi for 2 weeks three biological replicates were generated by deep sequencing using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA). The mapped reads of each sample were assembled by Hisat2 (v2.0.5). qRT–PCR validation was performed using SYBR Green assays Results: RNA-seq data confirmed that in shoots, 4,694 genes were upregulated and 4,126 were downregulated in bHLH6 OV lines compared with wild type under high-P conditions.Among the differentially expressed genes, 1,805 (38.5%) were upregulated in wild type under low-P conditions, whereas 1,598 (38.7%) of the downregulated genes were downregulated in wild type under low-P conditions, suggesting that bHLH6 overexpression upregulates a subset of PSI genes. In total, 971 genes were upregulated and 853 genes were downregulated in spx4 compared to wild type in high-P conditions. Among these upregulated genes, 371 (38.2%) were also upregulated in shoots of bHLH6 OV plants compared to in wild type; 456 (53.4%) of the downregulated genes were also downregulated in shoots of bHLH6 OV compared with wild type. In roots, 311 (10.5%) out of the 2,969 upregulated genes in bHLH6 OV lines under high-P conditions were also upregulated in wild type under low-P conditions; 501 (19.1%) out of the 2,628 downregulated genes in bHLH6 OV were also downregulated in wild type under low-P conditions. However, 313 (60.2%) out of the 520 genes upregulated in spx4 in roots were also upregulated in bHLH6 OV lines compared to in wild type, whereas 497 (54.6%) out of 910 the downregulated genes were also downregulated in bHLH6 OV compared to in wild type. To validate the RNA-seq results, we selected the upregulated PT and PAP (PURPLE ACID PHOSPHATASE) genes for expression analysis using RT-qPCR. The expression levels of PT2, PT10, PAP10a, and PAP21b were considerably higher in roots of bHLH6 OV plants than in those of wild type and bhlh6 mutants, whereas the expression of SPX2 was much lower in bHLH6 OV plants than in wild type Conclusion: our study is the first to analyze in detail the bHLH6 transcriptome produced by rna-seq technology and perform biological replication. Our results suggest that bHLH6 mediates the regulation of Pi homeostasis by SPX4. Our results advance the current understanding of the Pi-signaling regulatory network and will potentially contribute to the genetic improvement of crop P efficiency. Overall design: Roots and shoots mRNA profiles of 7-day-old bHLH6 OV, spx4, and wild-type (WT) rice seedlings treated with 200 μM or 10 μM Pi for 2 weeks with three biological replicates.