On 2017 Dec 13, Anthony Michael commented:

This paper reports that the single-celled green alga Chlamydomonas reinhardtii accumulates the polyamine putrescine and to a lesser extent norspermidine, with low amounts of 1,3-diaminopropane, spermidine and spermine. It also reports that arginine decarboxylase (ADC) activity is detected at a level 20% that of ornithine decarboxylase (ODC), two enzymes involved in the alternative initial step of putrescine biosynthesis. The authors also tentatively suggest that norspermidine could be synthesized by the aspartate beta-semialdehyde pathway, a bacterial polyamine biosynthetic pathway.

The detection of ADC activity in C. reinhardtii is intriguing because its genome does not encode a homologue of the plant ADC. Rather, C. reinhardtii encodes two paralogues of ODC (XP001697502 and XP00698872) that according to ChloroP analysis, each contain a chloroplast targeting sequence. The authors applied the irreversible, active site suicide inhibitor of ADC, difluoromethylarginine (DFMA) to growing cells and then tested the effect of this inhibitor on activities of ADC and ODC. Against expectation of the authors, DFMA inhibited ODC activity, and the authors reasoned that this was due to DFMA being converted by arginase to difluoromethylornithine (DFMO), a specific inhibitor of ODC. Indeed, the authors found that cell extract supernatant and the pelleted fraction contained arginase activity. Surprisingly, the authors did not question whether the same phenomenon was happening when they assayed ADC activity. ADC activity was determined by measuring the release of [14C] CO2 from [14C] arginine. However, if arginase was converting [14C] arginine to [14C] ornithine, then any ODC activity would release [14C] CO2 from [14C] ornithine that had been formed from [14C] arginine. This would give the impression that there is ADC activity when in fact the [14C] CO2 was being release by ODC activity, in essence, the assay was detecting a phantom ADC activity, particularly relevant in the absence of an ADC homologue in the C. reinhardtii genome. This is a well-known pitfall with detecting plant ADC activity, and the best way to unambiguously detect ADC activity is to measure the product agmatine (decarboxylated arginine) that is produced by ADC activity on arginine.

The authors also reported that C. reinhardtii accumulated norspermidine and spermine, and proposed that norspermidine is produced by an equivalent of the bacterial aspartate beta-semialdehyde-dependent pathway. Both spermidine and spermine in flowering plants are synthesized by dedicated spermidine and spermine synthases. An isomer of spermine, thermospermine, is synthesized by a dedicated thermospermine synthase. In Arabidopsis, spermine synthase has very likely evolved from gene duplication of spermidine synthase, which happened at the origin of flowering plants, whereas thermospermine synthase was likely acquired endosymbiotically from the cyanobacterial progenitor of the chloroplast. C. reinhardtii does not encode more that one spermidine synthase homologue, i.e., there is no spermine synthase present. However, a homologue of the Arabidopsis Acl5 thermospermine synthase is present (XP_001696651). As spermine and thermospermine have the same molecular mass and are difficult to separate by HPLC, the absence of a spermine synthase homologue, and the presence of a thermospermine synthase homologue, strongly suggests that the authors detected thermospermine rather than spermine in C. reinhardtii. The presence of thermospermine, and the application of Occam’s razor, then eliminates the requirement for a specific norspermidine biosynthetic pathway that is based on the bacterial asparate beta-semialdehyde-dependent pathway. This is because a known member of the plant polyamine oxidase family produces norspermidine from the oxidation of thermospermine (Sagor et al., (2015) The polyamine oxidase from lycophyte Selaginella lepidophylla (SelPAO5), unlike angiosperms, back converts thermospermine to norspermidine. FEBS Letts, 589, 3071-3078).

In summary, I respectfully suggest that the authors’ own data indicate that the ADC activity detected by the CO2 release assay may be an artifact of: arginase activity on the [14C] arginine substrate to produce [14C] ornithine, and subsequent ODC activity on the resultant [14C] ornithine. The spermine they detect is likely to be thermospermine, which would explain the presence of the thermospermine catabolite, norspermidine. The effect that the authors found, of the spermidine synthase inhibitor cyclohexylamine, in increasing C. reinhardtii putrescine levels and reducing both spermidine and norspermidine levels suggests that inhibition of spermidine synthesis diminishes thermospermine levels. Thermospermine is made by aminopropylation of spermidine, and therefore the amount of the thermospermine catabolite norspermidine is also reduced.

On 2017 Dec 13, Anthony Michael commented:

This paper reports that the single-celled green alga Chlamydomonas reinhardtii accumulates the polyamine putrescine and to a lesser extent norspermidine, with low amounts of 1,3-diaminopropane, spermidine and spermine. It also reports that arginine decarboxylase (ADC) activity is detected at a level 20% that of ornithine decarboxylase (ODC), two enzymes involved in the alternative initial step of putrescine biosynthesis. The authors also tentatively suggest that norspermidine could be synthesized by the aspartate beta-semialdehyde pathway, a bacterial polyamine biosynthetic pathway.

The detection of ADC activity in C. reinhardtii is intriguing because its genome does not encode a homologue of the plant ADC. Rather, C. reinhardtii encodes two paralogues of ODC (XP001697502 and XP00698872) that according to ChloroP analysis, each contain a chloroplast targeting sequence. The authors applied the irreversible, active site suicide inhibitor of ADC, difluoromethylarginine (DFMA) to growing cells and then tested the effect of this inhibitor on activities of ADC and ODC. Against expectation of the authors, DFMA inhibited ODC activity, and the authors reasoned that this was due to DFMA being converted by arginase to difluoromethylornithine (DFMO), a specific inhibitor of ODC. Indeed, the authors found that cell extract supernatant and the pelleted fraction contained arginase activity. Surprisingly, the authors did not question whether the same phenomenon was happening when they assayed ADC activity. ADC activity was determined by measuring the release of [14C] CO2 from [14C] arginine. However, if arginase was converting [14C] arginine to [14C] ornithine, then any ODC activity would release [14C] CO2 from [14C] ornithine that had been formed from [14C] arginine. This would give the impression that there is ADC activity when in fact the [14C] CO2 was being release by ODC activity, in essence, the assay was detecting a phantom ADC activity, particularly relevant in the absence of an ADC homologue in the C. reinhardtii genome. This is a well-known pitfall with detecting plant ADC activity, and the best way to unambiguously detect ADC activity is to measure the product agmatine (decarboxylated arginine) that is produced by ADC activity on arginine.

The authors also reported that C. reinhardtii accumulated norspermidine and spermine, and proposed that norspermidine is produced by an equivalent of the bacterial aspartate beta-semialdehyde-dependent pathway. Both spermidine and spermine in flowering plants are synthesized by dedicated spermidine and spermine synthases. An isomer of spermine, thermospermine, is synthesized by a dedicated thermospermine synthase. In Arabidopsis, spermine synthase has very likely evolved from gene duplication of spermidine synthase, which happened at the origin of flowering plants, whereas thermospermine synthase was likely acquired endosymbiotically from the cyanobacterial progenitor of the chloroplast. C. reinhardtii does not encode more that one spermidine synthase homologue, i.e., there is no spermine synthase present. However, a homologue of the Arabidopsis Acl5 thermospermine synthase is present (XP_001696651). As spermine and thermospermine have the same molecular mass and are difficult to separate by HPLC, the absence of a spermine synthase homologue, and the presence of a thermospermine synthase homologue, strongly suggests that the authors detected thermospermine rather than spermine in C. reinhardtii. The presence of thermospermine, and the application of Occam’s razor, then eliminates the requirement for a specific norspermidine biosynthetic pathway that is based on the bacterial asparate beta-semialdehyde-dependent pathway. This is because a known member of the plant polyamine oxidase family produces norspermidine from the oxidation of thermospermine (Sagor et al., (2015) The polyamine oxidase from lycophyte Selaginella lepidophylla (SelPAO5), unlike angiosperms, back converts thermospermine to norspermidine. FEBS Letts, 589, 3071-3078).

In summary, I respectfully suggest that the authors’ own data indicate that the ADC activity detected by the CO2 release assay may be an artifact of: arginase activity on the [14C] arginine substrate to produce [14C] ornithine, and subsequent ODC activity on the resultant [14C] ornithine. The spermine they detect is likely to be thermospermine, which would explain the presence of the thermospermine catabolite, norspermidine. The effect that the authors found, of the spermidine synthase inhibitor cyclohexylamine, in increasing C. reinhardtii putrescine levels and reducing both spermidine and norspermidine levels suggests that inhibition of spermidine synthesis diminishes thermospermine levels. Thermospermine is made by aminopropylation of spermidine, and therefore the amount of the thermospermine catabolite norspermidine is also reduced.