High-throughput sequencing of small RNAs revealed the diversified cold-responsive pathways during cold stress in the wild banana (Musa itinerans)

Abstract

Background: Cold stress is one of the most severe abiotic stresses affecting the banana production. Although some miRNAs have been identified, little is known about the role of miRNAs in response to cold stress in banana, and up to date, there is no report about the role of miRNAs in the response to cold stress in the plants of the cultivated or wild bananas.

Result: Here, a cold-resistant line wild banana (Musa itinerans) from China was used to profile the cold-responsive miRNAs by RNA-seq during cold stress. Totally, 265 known mature miRNAs and 41 novel miRNAs were obtained. Cluster analysis of differentially expressed (DE) miRNAs indicated that some miRNAs were specific for chilling or 0 °C treated responses, and most of them were reported to be cold-responsive; however, some were seldom reported to be cold-responsive in response to cold stress, e.g., miR395, miR408, miR172, suggesting that they maybe play key roles in response to cold stress. The GO and KEGG pathway enrichment analysis of DE miRNAs targets indicated that there existed diversified cold-responsive pathways, and miR172 was found likely to play a central coordinating role in response to cold stress, especially in the regulation of CK2 and the circadian rhythm. Finally, qPCR assays indicated the related targets were negatively regulated by the tested DE miRNAs during cold stress in the wild banana.

Conclusions: In this study, the profiling of miRNAs by RNA-seq in response to cold stress in the plants of the wild banana (Musa itinerans) was reported for the first time. The results showed that there existed diversified cold-responsive pathways, which provided insight into the roles of miRNAs during cold stress, and would be helpful for alleviating cold stress and cold-resistant breeding in bananas.

Keywords: Cold stress; Musa itinerans; RNA-seq-based profiling; miR172; miRNA.

Fig. 1

Morphological changes of leaves from field plants and in vitro plants treated during cold stress

Fig. 2

The length distribution of sequencing reads from 4 small RNA libraries in the wild banana. L0, L4, L13, L28: treated at 0 °C, 4 °C, 13 °C, 28 °C, respectively (the same below)

Fig. 3

Heatmap of DE miRNAs during cold stress in the wild banana. The color represents miRNA expression values from 1.5 (the red corresponds to sRNAs with high expression) to − 1.5 (the blue corresponds to sRNAs with low expression). L0, L4, L13 and L28 correspond to the libraries obtained in the temperature 0 °C, 4 °C, 13 °C and 28 °C respectively. a up-regulation at 28 °C but down-regulation at other 3 temperatures; b: down-regulation at 28 °C but up-regulation at other 3 temperatures; c: down-regulation at 13 °C but up-regulation at other 3 temperatures; d: up-regulation at 4 °C but down-regulation at other 3 temperatures; e: down-regulation at 0 °C but up-regulation at other 3 temperatures; f: up-regulation at 0 °C but down-regulation at other 3 temperatures

Fig. 4

Venn diagrams of DE miRNAs among 3 groups during cold stress in the wild banana

Fig. 5

The plant circadian rhythm of the top 20 KEGG pathways in L_0 vs L28. The map was provided permission from Kanehisa laboratory [–180]. The nodes are marked in red background color indicating the DEGs is up-regulated expression, and the nodes are marked in green background color indicating the DEGs is down-regulated expression. On the other side, the white nodes indicated the genes are DEGs, but there is no distinguishment of the DEGs about the up- / down-regulated expression

Fig. 6

The qPCR validation of partial miRNAs and the target genes during cold stress in the wild banana. a-f. The expression patterns of the 6 miRNAs were opposite to those of the related targets. g-n. The expressions of miR172 and the related targets by qPCR (g-i: different sin3 gene members; j-n: ada1s). Duncan’s multiple range test: * P < 0.05; ** P < 0.01; the number of biological replicates = 3

Fig. 7

The diversified cold-responsive pathways of DE miRNAs targets during cold stress in the wild banana