Comparative Analysis and Identification of miRNAs and Their Target Genes Responsive to Salt Stress in Diploid and Tetraploid Paulownia fortunei Seedlings

Abstract

Salt stress is a global environmental problem that affects plant growth and development. Paulownia fortunei is an adaptable and fast-growing deciduous tree native to China that is environmentally and economically important. MicroRNAs (miRNAs) play important regulatory roles in growth, development, and stress responses in plants. MiRNAs that respond to biotic stresses have been identified; however, how miRNAs in P. fortunei respond to salt stress has not yet been reported. To identify salt-stress-responsive miRNAs and predict their target genes, four small RNA and four degradome libraries were constructed from NaCl-treated and NaCl-free leaves of P. fortunei seedlings. The results indicated that salt stress had different physiological effects on diploid and tetraploid P. fortunei. We detected 53 conserved miRNAs belonging to 17 miRNA families and 134 novel miRNAs in P. fortunei. Comparing their expression levels in diploid and tetraploid P. fortunei, we found 10 conserved and 10 novel miRNAs that were significantly differentially expressed under salt treatment, among them eight were identified as miRNAs probably associated with higher salt tolerance in tetraploid P. fortunei than in diploid P. fortunei. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to predict the functions of the target genes of the conserved and novel miRNAs. The expressions of 10 differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This is the first report on P. fortunei miRNAs and their target genes under salt stress. The results provided information at the physiological and molecular levels for further research into the response mechanisms of P. fortunei to salt stress.

Fig 1. Determination of physiological indices.

PF2:diploid P. fortunei; PF4: tetraploid P. fortunei. Standard error of the mean for three technical replicates is represented by the error bars.

Fig 2. Length (nt) distribution of sRNAs.

PF2U: diploid P. fortunei with0% NaCl; PF2S: diploid P. fortunei with 0.4% NaCl; PF4U: tetraploid P. fortunei with0% NaCl; PF4S: tetraploid P. fortunei with 0.4% NaCl.

Fig 3. Gene Ontology analysis of miRNA targets in P. fortunei.

Fig 4. Relative expression levels of the miRNA in P. fortunei by qRT-PCR.

PF2U: diploid P. fortunei with0% NaCl; PF2S: diploid P. fortunei with 0.4% NaCl; PF4U: tetraploid P. fortunei with0% NaCl; PF4S: tetraploid P. fortunei with 0.4% NaCl. The expression levels of miRNAs were normalized to U6. The normalized miRNA levels in the PF2U were arbitrarily set to 1.Standard error of the mean for three technical replicates is represented by the error bars.

Fig 5. Relative expression levels of the target genes in P. fortunei by qRT-PCR.

PF2U: diploid P. fortunei with0% NaCl; PF2S: diploid P. fortunei with 0.4% NaCl; PF4U: tetraploid P. fortunei with0% NaCl; PF4S: tetraploid P. fortunei with 0.4% NaCl. CL5964.Contig1_All targeted by pfo-miR159b. CL186.Contig2_All targeted by pfo-miR396a. CL13370.Contig1_All targeted by pfo-miR5021a. CL3220.Contig4_All targeted by pfo-miR5239a. CL14209.Contig1_All targeted by pfo-mir28a. CL15613.Contig1_All targeted by pfo-mir11a. CL637.Contig2_All targeted by pfo-mir47. CL14940.Contig1_All targeted by pfo-mir89a. CL1879.Contig1_All targeted by pfo-miR167a. Three independent biological replicates were performed. The expression levels of targets were normalized to 18SrRNA. The normalized miRNA levels in the PF2U were arbitrarily set to 1.Standard error of the mean for three technical replicates is represented by the error bars.