miRNA expression profiling and zeatin dynamic changes in a new model system of in vivo indirect regeneration of tomato

Abstract

Callus formation and adventitious shoot differentiation could be observed on the cut surface of completely decapitated tomato plants. We propose that this process can be used as a model system to investigate the mechanisms that regulate indirect regeneration of higher plants without the addition of exogenous hormones. This study analyzed the patterns of trans-zeatin and miRNA expression during in vivo regeneration of tomato. Analysis of trans-zeatin revealed that the hormone cytokinin played an important role in in vivo regeneration of tomato. Among 183 miRNAs and 1168 predicted target genes sequences identified, 93 miRNAs and 505 potential targets were selected based on differential expression levels for further characterization. Expression patterns of six miRNAs, including sly-miR166, sly-miR167, sly-miR396, sly-miR397, novel 156, and novel 128, were further validated by qRT-PCR. We speculate that sly-miR156, sly-miR160, sly-miR166, and sly-miR397 play major roles in callus formation of tomato during in vivo regeneration by regulating cytokinin, IAA, and laccase levels. Overall, our microRNA sequence and target analyses of callus formation during in vivo regeneration of tomato provide novel insights into the regulation of regeneration in higher plants.

Fig 1. External appearance of the different stages of in vivo regeneration in tomato.

(A) The decapitated primary shoot; (B),(C) The callus formed on the cutting surface at 15 and 25d after decapitation; (D) The adventitious shoots differentiated from callus.

Fig 2. HPLC analysis of trans-zeatin levels during in vivo regeneration of tomato micro-TOM.

Fig 3. Regenerated adventitious shoots levels in lovastatin and control treated tomato plants.

Fig 4. Length distributions of unique sRNAs in stem and callus.

Fig 5. Reads of known miRNA families at stem and callus.

Fig 6. Secondary structure of identified miRNA precursors.

The red protrusions are the mature sequences. (A) Known miRNA: sly-miR162; (B) Novel miRNA: novel 101.

Fig 7. Cluster analyses of differentially expressed miRNAs.

Red denotes highly expressed miRNAs, while blue denotes weakly expressed miRNAs. The color is from red to blue, indicating that log₁₀ (TPM + 1) is from large to small.

Fig 8. The number of known and novel up- and down- regulated miRNAs in callus vs. stem tissue.

Fig 9. Venn diagram of the number of specifically expressed miRNAs at stem and callus.

(A) Known miRNAs; (B) Novel miRNAs.

Fig 10. The relative expression levels of 6 (four known and two novel) miRNAs by qRT-PCR.

Fig 11. Genetic networks of callus formation during in vivo regeneration of tomato regulated by miRNA-target modules together with their downstream targets.