Sculpting the maturation, softening and ethylene pathway: the influences of microRNAs on tomato fruits

Abstract

Background: MicroRNAs (miRNAs), a ubiquitous class of short RNAs, play vital roles in physiological and biochemical processes in plants by mediating gene silencing at post-transcriptional (PTGS) level. Tomato is a model system to study molecular basis of fleshy fruit ripening and senescence, ethylene biosynthesis and signal transduction owing to its genetic and molecular tractability. To study the functions of miRNAs in tomato fruit ripening and senescence, and their possible roles in ethylene response, the next generation sequencing method was employed to identify miRNAs in tomato fruit. Bioinformatics and molecular biology approaches were combined to profile the miRNAs expression patterns at three different fruit ripening stages and by exogenous ethylene treatment.

Results: In addition to 7 novel miRNA families, 103 conserved miRNAs belonging to 24 families and 10 non-conserved miRNAs matching 9 families were identified in our libraries. The targets of many these miRNAs were predicted to be transcriptional factors. Other targets are known to play roles in the regulation of metabolic processes. Interestingly, some targets were predicted to be involved in fruit ripening and softening, such as Pectate Lyase, beta-galactosidase, while a few others were predicted to be involved in ethylene biosynthesis and signaling pathway, such as ACS, EIN2 and CTR1. The expression patterns of a number of such miRNAs at three ripening stages were confirmed by stem-loop RT-PCR, which showed a strong negative correlation with that of their targets. The regulation of exogenous ethylene on miRNAs expression profiles were analyzed simultaneously, and 3 down-regulated, 5 up-regulated miRNAs were found in this study.

Conclusions: A combination of high throughput sequencing and molecular biology approaches was used to explore the involvement of miRNAs during fruit ripening. Several miRNAs showed differential expression profiles during fruit ripening, and a number of miRNAs were influenced by ethylene treatment. The results suggest the importance of miRNAs in fruit ripening and ethylene response.

Figure 1

Length comparison of small RNAs at three ripening stages of tomato fruit. The length of the deep sequencing results were mainly between 21-24nt, the number of the 24nt sequences is obviously greater than the other sequence length.

Figure 2

Length distributions of small RNAs at three ripening stages of tomato fruit. The overall sequences (21-24nt) in the breaker stage were more abundant than the other two ripening stages, the number of the 21-23nt sequences in each ripening stages showed no obvious difference, however, there was a sharp increase in the 24nt sequence number.

Figure 3

Conserved miRNAs and their family numbers in tomato fruit. The members of each family were different, the miR156, miR166 and miR171 had more than ten members, in the contrary, miR160, miR319, miR394, miR395, miR399, miR408, miR472, miR482, miR827 had only one member in their corresponding family.

Figure 4

The validation of nine identified non-conserved miRNAs in tomato fruit. The total length of the RTS primer and the miRNA sequence was about 110 bp, the nine non-conserved families were validated, the right two lanes were the miRNAs and the second left lane was the negative control.

Figure 5

The validation of seven novel miRNAs in tomato fruit. The total length of the RTS primer and the miRNA sequence was about 110 bp, the novel seven miRNA families were validated, the right two lanes were the miRNAs and the second right lane was the negative control.

Figure 6

The member distribution of the seven novel miRNA families in tomato fruit. Seven novel miRNA families were found in the libraries, miRZ1, miRZ3 and miRZ5 had only one member, however, and the rest families (miRZ2, miRZ4, miRZ6, miRZ7) had two members.

Figure 7

Distribution of the predicted target genes of known miRNAs in tomato fruit. The targets of the known miRNAs were mainly divided into three types, almost half of them were transcription factors, and nearly one third of them were involved in metabolic process, one fifth of the targets were unknown, in addition, several targets were predicted to participant in disease resistance and their own biosynthetic pathway.

Figure 8

The expression patterns of the known and novel miRNAs in tomato fruit. MiR156 and miR394 were down regulated in the fruit ripening, miR159 showed down regulation in the breaker stage, while, miR396 showed a obvious increase in the breaker stage, miR828 and miR1917 were down regulated in the red ripe stage, miR482 and miRZ7 showed down regulation in the red and softening ripe stage. (Annotation: M-Marker; R-red ripe stage; B-The breaker stage; G- green ripe stage, U6 was used as the reference gene).

Figure 9

The expression patterns of known and novel miRNAs after exogenous ethylene treatment in tomato fruit. Three miRNA families (miR394, miR414 and miR1917) were down regulated, in the contrary, four miRNA families (miR156, miR159, miR396, miR482 and miRZ7) were up-regulated, and however, the miR828 had no obvious change. (Annotation: M1-Marker; M2-1-methylcycloproene treatment; E-exogenous ethylene treatment; C-control, U6 was used as the reference gene).