Masson pine (Pinus massoniana Lamb.) is a major fast-growing woody tree species and pioneer species for afforestation in barren sites in southern China. However, the regulatory mechanism of gene expression in P. massoniana under drought remains unclear. To uncover candidate microRNAs, their expression profiles, and microRNA-mRNA interactions, small RNA-seq was used to investigate the transcriptome from seedling roots under drought and rewatering in P. massoniana. A total of 421 plant microRNAs were identified. Pairwise differential expression analysis between treatment and control groups unveiled 134, 156, and 96 differential expressed microRNAs at three stages. These constitute 248 unique microRNAs, which were subsequently categorized into six clusters based on their expression profiles. Degradome sequencing revealed that these 248 differentially expressed microRNAs targeted 2069 genes. Gene Ontology enrichment analysis suggested that these target genes were related to translational and posttranslational regulation, cell wall modification, and reactive oxygen species scavenging. miRNAs such as miR482, miR398, miR11571, miR396, miR166, miRN88, and miRN74, along with their target genes annotated as F-box/kelch-repeat protein, 60S ribosomal protein, copper-zinc superoxide dismutase, luminal-binding protein, S-adenosylmethionine synthase, and Early Responsive to Dehydration Stress may play critical roles in drought response. This study provides insights into microRNA responsive to drought and rewatering in Masson pine and advances the understanding of drought tolerance mechanisms in Pinus.

The mechanisms underlying plant response to drought involve the expression of numerous functional and regulatory genes. Transcriptome sequencing based on the second- and/or third-generation high-throughput sequencing platforms has proven to be powerful for investigating transcriptional landscape under drought stress. However, the full-length transcriptomes related to drought responses in the important conifer genus Pinus L. remained to be delineated using the third-generation sequencing technology. With objectives to identify the candidate genes responsible for drought and/or rehydration and clarify the expression profile of key genes involved in drought regulation, we combined the third- and second-generation sequencing techniques to make transcriptome analysis on seedling roots under drought stress and rewatering in the drought-tolerant conifer Pinus massoniana Lamb.. A sum of 294,114 unique full-length transcripts were produced with a mean length of 3,217 bp and N50 estimate of 5,075 bp, including 279,560 and 124,438 unique full-length transcripts being functionally annotated and gene ontology enriched, respectively. Totally 4,076, 6,295 and 18,093 differentially expressed genes (DEGs) were identified in three pair-wise comparisons of drought-treatment versus control transcriptomes, including 2,703, 3,576 and 8,273 upregulated and 1,373, 2,719 and 9,820 downregulated DEGs, respectively. Moreover, 157, 196 and 691 DEGs were identified as transcription factors in the three transcriptome comparisons and grouped into 26, 34 and 44 transcription factor families, respectively. Gene ontology enrichment analysis revealed that a remarkable number of DEGs were enriched in soluble sugar-related and cell wall-related processes. A subset of 75, 68 and 97 DEGs were annotated to be associated with starch, sucrose and raffinose metabolism, respectively, while 32 and 70 DEGs were associated with suberin and lignin biosynthesis, respectively. Weighted gene co-expression network analysis revealed modules and hub genes closely related with drought and rehydration. This study provides novel insights into root transcriptomic changes in response to drought dynamics in Masson pine and serves as a fundamental work for further molecular investigation on drought tolerance in conifers.