Can putrescine alleviate effects of K deficiency?
Putrescine addition partly alleviated the effects of low K on glycolysis (regulation of pyruvate kinase content under low K) (Figs. 6, S5c-d), photosynthesis (Fig. 2) and N assimilation (Fig. 3), and typically increased root glutamine and asparagine content (Fig. 5). However, putrescine did not suppress the increase in leaf respiration at low K (Fig. 2) despite the down-regulation of several enzymes involved in respiratory metabolism (Fig. S5d). Diverse mechanisms have been suggested to explain why putrescine is accumulated under low K, including the regulation of mitochondrial ion channels to avoid excessive oxidative damage and mitochondrial permeability transition (Cui et al. , 2020). Here, the addition of putrescine did not have a clear effect on mitochondrial proteins, except for alternative NAD(P)H dehydrogenase in roots (Table 1, Fig. S5).
The fact that putrescine (i ) had rather contrasted effects (different effects in roots and leaves, e.g. Fig. 6), and (ii)caused a decline in pyruvate kinase content at high K, suggests that putrescine is not a molecule that only compensates for cellular K scarcity but rather, is involved (or one of its products) in low-K signaling, perhaps including root-to-shoot signaling. It has been proposed that putrescine regulates metabolism via an increase or a decrease in reactive oxygen species (ROS) depending on its concentration and the potential involvement of polyamine oxidase, which generates H2O2 (Verma & Mishra, 2005; Zepeda-Jazoet al. , 2011; Shelp et al. , 2012; Zhang et al. , 2014). It is worth noting that if putrescine is effectively oxidized, this can interact directly with Ca and K absorption since K efflux and Ca influx in root cells have been found to be regulated by ROS (Demidchik et al. , 2003). Here, we did not find any protein annotated as polyamine oxidase but found two peptides associated with copper amine oxidase (also referred to as diamine oxidase; CAO). Interestingly, putrescine addition caused an increase in CAO in roots but not in leaves (Fig. S6). It is plausible that putrescine addition thus led to an increased production of NH4+ and H2O2 in roots, thereby triggering ROS signaling and improving N nutrition (higher glutamine and asparagine content in roots). In leaves, the most significant metabolites under putrescine addition were serine and homoserine. Putrescine oxidative deamination produces γ-aminobutyrate semialdehyde further oxidized to GABA (Shelp et al. , 2012), which can be in turn incorporated into the TCAP via the GABA shunt and thus feed the synthesis of asparate (precursor of homoserine). That said, the simultaneous increase in serine and homoserine also likely reflects the regulation of sulfur (S) metabolism by putrescine (serine and homoserine are precursors of cysteine and methionine, respectively) (illustrated in Fig. S7).
In effect, low K conditions reconfigured S metabolism. Under low K availability, the sulphur elemental content has been found to change significantly in sunflower (increase in leaves, decline in roots) (Cuiet al. , 2019a) and in Arabidopsis, the tissue content in sulphate, cysteine and O -acetylserine decrease strongly (Forieriet al. , 2017). Here, we observed at low K an increase in cysteine metabolism (increased cysteine synthase, Table 1, lower cystathionine content, Fig 4) at the expense of methionine metabolism (decreased methionine synthase and SAM synthase, Table 1, lower methionine-to-cysteine ratio, Fig. S7). That is, cysteine was consumed to synthesize homocysteine (trans-sulfuration onto homoserine), which was then recycled back to cystathionine by cystathionine β-synthase (CBS), thereby consuming serine and avoiding methionine synthesis. Such an effect of low K on S metabolism is very likely a consequence of the inhibition of SAM synthase catalysis, which has been shown to be K+-dependent (Takusagawa et al. , 1996). Parenthetically, the inhibition of SAM synthase further exaggerates putrescine accumulation since the conversion of putrescine to other polyamines requires SAM. It is also interesting to note that homocysteine production liberates pyruvate (Fig. S7). Therefore, homocysteine production and recycling to cystathionine represents an alternative pathway for pyruvate production (from serine). This is clearly advantageous under low K conditions where pyruvate kinase is inhibited.
Quite critically, the effect of low K on cysteine metabolism appeared to be modulated by Ca and putrescine: low Ca led to an increase in cystathionine β-synthase in leaves, and putrescine addition caused a decline in cysteine synthase in roots (Table 1). This suppression effect by putrescine was probably sufficiently strong to explain why both serine and homoserine increased. Also, putrescine impacted modestly on enzyme content in leaves (the effect of putrescine and K xputrescine on SAM synthase and CBS was associated with a P -value (3.64 10-5 and 4.5 10-5, respectively) just above the Bonferroni threshold (3.54 10-5), Table S2).