Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene

Abstract

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.

Impact of 1-methylcyclopropene and controlled atmosphere storage on polyamine and 4-aminobutyrate levels in “Empire” apple fruit

“…However, we found no evidence for elevated levels of total PAs, Put, Spd, or Spm during the steady-state period (8–46 weeks) in response to 1-MCP treatment; indeed the levels tended to be lower regardless of CO 2 ( Figures 2 – 5 ), suggesting that the availability of SAM for PA biosynthesis in apple fruit was not influenced by the 1-MCP treatment. These findings are consistent with previous studies of ripening apple and tomato fruits, which suggest that the requirement for SAM in PA biosynthesis is not limited by the requirement in ethylene biosynthesis (Van de Poel et al, 2013; Lasanajak et al, 2014), and suggest an unknown biochemical or transcriptional mechanism, possibly altering the PA biosynthetic rates from glutamate (Alcázar et al, 2010; Majumdar et al, 2013), was responsible for the lower total PA levels in 1-MCP-treated fruit under CA storage. However, these findings are at odds with the generally accepted view that PAs, especially Spd and Spm, accumulate in respond to abiotic stress (Groppa and Benavides, 2008; Bassard et al, 2010; Shelp et al, 2012c), which could be due to a variety of reasons: that view is generally based on free PA levels, rather than total PAs; apple fruit experience multiple stresses during CA storage; and, ripening apple fruit, like tomato fruit (Mattoo et al, 2010), are at a terminal developmental stage.…”

“…During the ripening of a bulky fruit such as tomato, there is an inverse relationship between the production of higher PAs and ethylene (Lasanajak et al, 2014), 1-MCP treatment inhibits autocatalytic ethylene production without affecting SAM levels (Van de Poel et al, 2013), and decreased levels of ethylene in RNAi-1-aminocyclpropane-1-carboxylate synthase fruits enhance PA levels and upregulate PA biosynthesis genes (Gupta et al, 2013). Research with non-bulky fruits such as rice grain also suggests interaction between PAs and ethylene in regulating plant growth and in response to environmental stress (Chen et al, 2013).…”

Polyamine catabolism adds fuel to leaf senescence

“…Being a major substrate in 1-C metabolism and involved in methylation processes, SAM diverted to PAs and/or ethylene during leaf senescence needs to be assessed metabolically, as has been described recently in the tomato system (Lasanajak et al 2014). Regulation of SAM levels in mammalian cells was found regulated involving post-translational inhibition of glycine N -methyltransferase by folate (Luka et al, 2009).…”

Genetic Research Progress of Fruit Ripening and Quality Control

“…PAs together with salicylic acid regulated ethylene biosynthesis by affecting the accumulation of ACC synthase transcripts. PAs and ethylene acted on the contrary, by sharing S-Adenosyl methionine(SAM) as precursors to affect plant growth and senescence, plant cells transferred SAM into PA biosynthesis, or ethylene biosynthesis, or both (Lasanajak et al, 2014). Nambeesan et al (2010) confirmed that lycopene content and juice viscosity increased by studying the transgenic over-expression of yeast SAMPC and yeast SPDC of tomato.…”