**6. Role of Ascorbate in Photosynthesis Coordination of the Energy Systems of the Mitochondria and Chloroplast**

Given that ascorbate production is tightly associated with the mitochondrial electron transport chain and considering the well-documented facts on having profound bidirectional relationships with the rates of photosynthesis via a range of mechanisms, we assume that ascorbate takes part in the coordination of the energy systems between the mitochondria and chloroplast.

This hypothesis is raised from validations on ascorbate roles in mitochondrial electron transport rate (mETC) discussed above, photosynthesis, and TCA cycle regulatory networks [39]. Moreover, genes of the Ascorbate-glutathione cycle (ASC–GSH) cycle are expressed in both chloroplast and mitochondria [90], thus both organelles must coordinately take part in ascorbate biosynthesis and recycling. It has been demonstrated in multiple studies that ascorbate can elevate the rate of photosynthesis by a variety of mechanisms, especially in response to acclimation to high light [46,91,92]. These mechanisms include involvement of ascorbate in protecting against photoinhibition in the water–water cycle by scavenging superoxide and hydrogen peroxide, dissipating excess energy, and contribution to thylakoid acidification leading to the control of PSII activity [93–95]. Ascorbate is also considered as an alternative electron donor for PSII, whereby it prevents photo-oxidation [96,97]. The ascorbate-redox state is also known to affect photosynthetic activity through guard cell signaling and stomatal movement and also through changing the expression of the nuclear and chloroplastic encoded genes [40]. Furthermore, feeding Arabidopsis *vtc1* mutants with ascorbate had a great impact on photosynthetic gene expression, leading to an increased and decreased expression of some of the chloroplast- and nuclear-encoded genes, respectively [98].

However, recently observed differences in the photosynthetic responses of *vtc2-1* and *vtc2-4* mutants under high light raised the hypothesis that higher susceptibility of the *vtc2-1* mutants to photoinhibition, previously reported in the literature [46,92], might not have been caused by the lower ascorbate levels in the mutants [99]. Unlike *vtc2-1,* which carries a point mutation, the *vtc2-4* mutant is a T-DNA insertion line with a complete loss of function [99]. Contrary to *vtc2-1*, the *vtc2-4* mutants have unchanged levels of zeaxanthin contents and, despite having similar levels of NPQ under high light (lower than the WT), *vtc2-1* had greater photochemical quenching in the dark (qPd) values than the WT. Therefore, the authors suggested ascorbate as an essential component for growth, but not for photoprotection [99].

Intriguingly, observations on transgenic tomato plants antisensed in mitochondrial malate dehydrogenase (*mdh*), strengthening the hypothesis of ascorbate acting in the coordination of the energy systems of the mitochondria and chloroplast [13,100]. The reduction of the TCA cycle occurring in these transgenic lines via down-regulation of the expression of the mitochondrial MDH did not affect the respiration, but resulted in a fourfold increase in ascorbate following an upregulation of the activity of GLDH. Detailed studies revealed that this was because flux, through the GLDH activity, was upregulated in these lines, with the consequence that electrons were supplied to the mitochondrial electron transport chain [100]. Furthermore, incubating tomato leaf discs with ascorbate under constant illumination increased the amount of carbon assimilation and starch levels [100]. This further strengthens the link between chloroplast and mitochondria in ascorbate levels. Evaluation of tomato plants in which the GLDH was reduced in expression by RNA interference had dramatic consequences on both plant and fruit growth and development [101]. This observation is, however, complicated by a report of Tomaz et al. [102], in which they examined the *mdh* double mutants in Arabidopsis. The Arabidopsis *mdh* double mutants had increased levels of ascorbate, but higher levels of mitochondrial respiration and a lower activity of GLDH, contrary to what had been observed in

tomato plants [102]. More investigations are needed to clarify the cause of this difference observed between the two species.

In conclusion, considering all the described effects of ascorbate on photosynthesis and vice versa, and its intimacy to the respiratory electron transport chain, ascorbate can be considered as an important component in plant central metabolism, modulating the energy systems between the chloroplast and mitochondria [39]. However, the molecular mechanisms and the signals responsible for these network of interactions need further investigations.

## **7. Thiamin Biosynthesis**

In plants, thiamin is synthesized from pyrimidine and a thiazole moiety, both of which are synthesized in the chloroplast. Synthesis of pyrimidine moiety is catalized by thiamin C synthase (THIC) by converting 5-aminoimidazole ribonucleotide (AIR) and S-adenosylmethionine (SAM) as substrates to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate (HMP-P) [103–105]. Thiazole moiety is synthesized by the action of 4-methyl-5-b-hydroxyethylthiazole phosphate (HET-P) synthase (THI1) [106], catalizing the conversion of nicotinamide adenine dinucleotide (NAD+) and glycine as substrates to an adenylated thiazole intermediate (ADT) [107–109]. ADT is then hydrolyzed to HET-P, by an uncharacterized enzyme. Phosphorylation of HMP-P to HMP-PP, and subsequently condensation of HMP-PP and HET-P, is done by the action of a bifunctional enzyme, thiamin monophosphate pyrophosphorylase (TH1), which eventually leads to the formation of thiamin monophosphate (TMP) [30,110]. TMP is dephosphorylated to thiamin by a haloacid dehalogenase (HAD) family phosphatase (TH2) [111], and subsequently pyrophosphorylated to thiamin pyrophosphate (TPP) by TPP kinases (TDPKs), which are located in the cytosol (Figure 3) [110,112].

In the past years, different attempts have been made to biofortify staple crops with thiamin [112–114]. These efforts were possible thanks to a recent increase in the understanding of thiamin metabolism in plants. Several genes involved in thiamin biosynthesis, regulation, transport, and salvage have been identified owing to the availability of complete plant genome sequences [30,115,116]. Although this determined the beginning of thiamin engineering strategies, several aspects of thiamin metabolism are still unclear [114].
