Polyamines (PAs) regulate growth in plants and modulate the whole plant life cycle. They have been associated with different abiotic and biotic stresses, but little is known about the molecular regulation involved. We quantified gene expression of PA anabolic and catabolic pathway enzymes in tomato (Solanum lycopersicum cv. Ailsa Craig) leaves under heat versus cold stress. These include arginase1 and 2, arginine decarboxylase 1 and 2, agmatine iminohydrolase/deiminase 1, N-carbamoyl putrescine amidase, two ornithine decarboxylases, three S-adenosylmethionine decarboxylases, two spermidine synthases; spermine synthase; flavin-dependent polyamine oxidases (SlPAO4-like and SlPAO2) and copper dependent amine oxidases (SlCuAO and SlCuAO-like). The spatiotemporal transcript abundances using qRT-PCR revealed presence of their transcripts in all tissues examined, with higher transcript levels observed for SAMDC1, SAMDC2 and ADC2 in most tissues. Cellular levels of free and conjugated forms of putrescine and spermidine were found to decline during heat stress while they increased in response to cold stress, revealing their differential responses. Transcript levels of ARG2, SPDS2, and PAO4-like increased in response to both heat and cold stresses. However, transcript levels of ARG1/2, AIH1, CPA, SPDS1 and CuAO4 increased in response to heat while those of ARG2, ADC1,2, ODC1, SAMDC1,2,3, PAO2 and CuPAO4-like increased in response to cold stress, respectively. Transcripts of ADC1,2, ODC1,2, and SPMS declined in response to heat stress while ODC2 transcripts declined under cold stress. These results show differential expression of PA metabolism genes under heat and cold stresses with more impairment clearly seen under heat stress. We interpret these results to indicate a more pronounced role of PAs in cold stress acclimation compared to that under heat stress in tomato leaves.

Polyamines have been implicated in ameliorating the detrimental effects of drought and saline conditions on plant growth and development. The independent impact of these two abiotic stresses on polyamine (PA) biosynthesis, catabolism, and homeostasis, as well as on their transcript abundance in tomato leaves, is presented here. We show that the total levels of putrescine (PUT), spermidine (SPD), and spermine (SPM) increase up to 72 h during drought and up to 48 h during salinity stress before their precipitable drop thereafter. Thus, tomato plants maintain survivability to drought as well as salinity stress for up to 3 and 2 days, respectively. Independent multivariant analyses of drought and salinity stress kinetic data separately showed a closer association with levels of free, conjugated, and bound forms of SPD and SPM, but not with free or bound PUT. However, combined multivariant analyses showed a closer association of free SPD, conjugated SPD, and bound SPD with both stresses; SPD-bound and SPM conjugated with drought; and free SPM and conjugated PUT with salinity stress, respectively. PA biosynthesis genes, ARG1, SPDS1, and SAMDc3, segregated with drought and SPDS2 with salinity stress. PA catabolic genes CuAO4-like and PAO4 were associated with drought and salinity stresses, respectively, suggesting differential involvement of PA biosynthesis and catabolic genes in drought and salinity stresses. Pearson correlation indicated mostly positive correlations between the levels of free, conjugated, and bound forms of PUT, SPD, and SPM under drought and salinity stress. However, negative correlations were mostly seen between the levels of various forms of the PAs and their biosynthesis/catabolic genes. Levels of different PA forms had a twofold higher negative correlation during drought as compared to salinity stress (66 vs. 32) and with transcript levels of PA biosynthesis and catabolic genes. Transcripts of light-harvesting chlorophyll a/b-binding genes were generally positively associated with different forms of PAs but negatively to carbon flow genes. Most of the PA biosynthesis genes were coordinately regulated under both stresses. Collectively, these results indicate that PAs are distinctly regulated under drought and salinity stress with different but specific homologs of PA biosynthesis and catabolic genes contributing to the accumulation of free, conjugated, and bound forms of PAs.

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.

Polyamines (PAs) constituting putrescine (Put), spermidine (Spd), and spermine (Spm) are ubiquitous in all organisms and play essential roles in the growth and developmental processes in living organisms, including plants. Evidences obtained through genetic, biochemical, and transgenic approaches suggest a tight homeostasis for cellular PA levels. Altered cellular PA homeostasis is associated with abnormal phenotypes. However, the mechanisms involved for these abnormalities are not yet fully understood, nor is it known whether cellular ratios of different polyamines play any role(s) in specific plant processes. We expressed a yeast spermidine synthase gene (ySpdSyn) under a constitutive promoter CaMV35S in tomato and studied the different phenotypes that developed. The constitutive expression of ySpdSyn resulted in variable flower phenotypes in independent transgenic lines, some of which lacked fruit and seed set. Quantification of PA levels in the developing flowers showed that the transgenic plants without fruit and seed set had significantly reduced Spd levels as well as low Spd/Put ratio compared to the transgenic lines with normal fruit and seed set. Transcript levels of SlDELLA, GA-20oxidase-1, and GA-3oxidase-2, which impact gibberellin (GA) metabolism and signaling, were significantly reduced in bud tissue of transgenic lines that lacked fruit and seed set. These findings indicate that PAs, particularly Spd, impact floral organ identity and fruit set in tomato involving GA metabolism and signaling. Furthermore, we suggest that a nexus exists between PA ratios and developmental programs in plants.

Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines - putrescine (Put), spermidine (Spd), and spermine (Spm) - are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent of ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon.

Shape and size are important features of fruits. Studies using tomatoes expressing yeast Spermidine Synthase under either a constitutive or a fruit-ripening promoter showed obovoid fruit phenotype compared to spherical fruit in controls, suggesting that polyamines (PAs) have a role in fruit shape. The obovoid fruit pericarp exhibited decreased cell layers and pericarp thickness compared to wild-type fruit. Transgenic floral buds and ovaries accumulated higher levels of free PAs, with the bound form of PAs being predominant. Transcripts of the fruit shape genes, SUN1 and OVATE, and those of CDKB2, CYCB2, KRP1 and WEE1 genes increased significantly in the transgenic ovaries 2 and 5 days after pollination (DAP). The levels of cell expansion genes CCS52A/B increased at 10 and 20 DAP in the transgenic fruits and exhibited negative correlation with free or bound forms of PAs. In addition, the cell layers and pericarp thickness of the transgenic fruits were inversely associated with free or bound PAs in 10 and 20 DAP transgenic ovaries. Collectively, these results provide evidence for a linkage between PA homeostasis and expression patterns of fruit shape, cell division, and cell expansion genes during early fruit development, and suggest role(s) of PAs in tomato fruit architecture.