Engineered Ripening-Specific Accumulation of Polyamines Spermidine and Spermine in Tomato Fruit Upregulates Clustered...

Ripening of tomato fruit leads, in general, to a sequential decrease in the endogenous levels of polyamines spermidine (SPD) and spermine (SPM), while the trend for the diamine putrescine (PUT) levels is generally an initial decrease, followed by a substantial increase, and thereafter reaching high levels at the red ripe fruit stage. However, genetic engineering fruit-specific expression of heterologous yeast S-adenosylmethionine (SAM) decarboxylase in tomato has been found to result in a high accumulation of SPD and SPM at the cost of PUT. This system enabled a genetic approach to determine the impact of increased endogenous levels of biogenic amines SPD and SPM in tomato (579HO transgenic line) and on the biogenesis, transcription, processing, and stability of ribosomal RNA (rRNA) genes in tomato fruit as compared with the non-transgenic 556AZ line. One major biogenetic process regulating transcription and processing of pre-mRNA complexes in the nucleus involves small nucleolar RNAs (snoRNAs). To determine the effect of high levels of SPD and SPM on these latter processes, we cloned, sequenced, and identified a box C/D snoRNA cluster in tomato, namely, SlSnoR12, SlU24a, Slz44a, and Slz132b. Similar to this snoRNA cluster housed on chromosome (Chr.) 6, two other noncoding C/D box genes, SlsnoR12.2 and SlU24b, with a 94% identity to those on Chr. 6 were found located on Chr. 3. We also found that other snoRNAs divisible into snoRNA subclusters A and B, separated by a uridine rich spacer, were decorated with other C/D box snoRNAs, namely, J10.3, Z131a/b, J10.1, and Z44a, followed by z132a, J11.3, z132b, U24, Z20, U24a, and J11. Several of these, for example, SlZ44a, Slz132b, and SlU24a share conserved sequences similar to those in Arabidopsis and rice. RNAseq analysis of high SPD/SPM transgenic tomatoes (579HO line) showed significant enrichment of RNA polymerases, ribosomal, and translational protein genes at the breaker+8 ripening stage as compared with the 556AZ control. Thus, these results indicate that SPD/SPM regulates snoRNA and rRNA expression directly or indirectly, in turn, affecting protein synthesis, metabolism, and other cellular activities in a positive manner.