**8. If AsA is so much important, why did we lose AsA biosynthetic capability?**

We are all aware that vitamin C is not only important for our health but even essential for our own survival. It cannot be fully substituted by any other antioxidant, and it has a specific role in the reactions catalyzed by several dioxygenases. With all this in mind, the loss of the AsA biosynthetic capability that occurred independently in some animal lineages is a mind-boggling paradox. Is there any possible advantage in the loss-of-function of the gene coding for the last step in AsA biosynthesis? The mutations occurring in the *GULO* gene [10] have been conserved in primates, but reversed in some bats and birds, suggesting that the capability to synthesize AsA is a neutral trait [96]. According to a different interpretation, the advantage of losing GULO activity is that the mechanism of the enzyme yields hydrogen peroxide, limiting the beneficial effect of AsA production [73]. An additional hypothesis is advanced, based on the informational content that AsA can bring to the mechanism of 2-ODDs, and the trade-off between stress response and lifespan [97]. As mentioned above, 2-ODDs require 2-oxoglutarate, oxygen, iron, and AsA. While the involvement of the former three co-substrates is easily associated with energy metabolism (Section 4, above), the contribution of AsA in terms of molecular information is not as clear. However,

as discussed in Section 7, the peculiar reactivity of AsA, and more specifically its interaction with different oxidants, makes it the perfect proxy for evaluating general stress conditions occurring in a given cell or cell compartment. The example of the HIF1α transcription factor (**Figure 2**) is especially relevant and useful to understand this point. The HIF1α transcription factor is constitutively expressed, but it becomes active exclusively when the hydroxylation of its proline residues (depending of co-substrate availability) does NOT take place. Like this, the transcriptional response is very fast: HIF is already there and immediately operative. The response is needed whenever there is no oxygen, not enough energy, and the cell is under heavy stress conditions, resulting in AsA depletion. A combination of these conditions variably induces the response. A similar mechanism, in which ligand depletion activates the transcriptional response, has been recently reported in plants [98].

A mechanism in which a multiple anti-stress response is activated by the absence of selected co-substrates usually associated with the effector (transcription factor) has several advantages. The proxies corresponding to different (variable) clues integrate directly at the effector site, producing a graded response. In this model, the co-substrates set the "meters" of the response (**Figure 4**). Many possible intermediate conditions would lie between the two extremes reported in the scheme. However, the quick response to unfavorable conditions also brings a burden to the organism if growth is reduced, and all available resources are diverted to defense. In the case of chronic stress, and the consequent continuous activation of the defenses, the lifespan is shortened, and early senescence occurs. This has been observed, for example, in the case of plants with lower AsA content [77]. The model also accommodates the sometimes conflicting observations on the effects of antioxidants on aging and

### **Figure 4.**

*A tentative model explaining the role of ascorbate (AsA) in putative signaling modules activating stress-responsive genes, in analogy with the HIF1α mechanism.*
