THE KEY ENZYME GGP REGULATES ASCORBIC ACID CONTENT THROUGH ITS INTERACTION WITH A NEW CLASS OF PHOTORECEPTOR IN TOMATO.

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The D-galacturonate pathway

Although L-galactose pathway is predominant in all the higher plants, there is a possibility that other alternatives pathways may be tissue or specie specific: here is the case of the D-galacturonate pathway.
It had been demonstrated that the D-Galacturonic acid methyl ester could be directly converted to L-ascorbic acid, without involving the reactions of the hexose monophosphate pathway (Isherwood et al., 1953). The alternative D-galacturonate pathway has been shown in strawberry when the gene coding for the involved enzyme, a NADPH-dependent D-galacturonate reductase was identified (Agius et al., 2003). In tomato, the input of D-galacturonate improves the ascorbate content in extract of red ripe fruits (Badejo et al., 2012). The activities of the two enzymes associated with this pathway (GalUR and aldonolactonase) are detectable in the insoluble fractions of fruits suggesting that this path is dependent on the stage of development. However, this does not occur in green fruits. Finally, protists which are capable of photosynthesis, use the D-galacturonate pathway for ascorbate synthesis (Ishikawa and Shigeoka, 2008).

The myo-inositol pathway

The possibility that an animal biosynthetic pathway of AsA is present in plants has never been ruled out. Overexpression in lettuce and tobacco of the gene encoding a rat L-gulono-1,4-lactone oxidase induces an increase in ascorbate content of approximately seven-fold (Jain and Nessler, 2000). More recently, ectopic expression of this gene in Arabidopsis mutant (vtc) leaves restores AsA levels (Radzio et al., 2003). In animals, the substrate used for this enzyme, is L-gulono-1, 4-lactone. It is interesting to note that the mutant vtc1 Arabidopsis produces very little of this intermediate. The myo-inositol pathway uses myo-inositol which is accumulated under stress conditions (Zhu et al., 2014). This osmolite is converted to D-glucuronic acid by myo-inositol oxygenase (Brown et al., 2006). Thus, these data suggest that under some conditions the presence of an alternative pathway can compensate for the L-galactose pathway.

Environmental impact

Environmental factors have a strong influence on the chemical composition of horticultural species (B. P. Klein and Perry, 1982). Biotic or abiotic stresses increase ROS production which can be toxic for the cells resulting in membrane peroxidation, damages to DNA, denaturation of proteins the all leading to cell death. However, there is no evidence of direct modulation of the ascorbate content related to biotic stress (Table 2). The responses of ascorbate to pathogenic infection are presented in the section that deals with its biological roles.

Intake for cultivation

Cultivation conditions also modulate the ascorbate pool. Today, the demand for alternative farming methods limiting the use of fertilizers and synthetic pesticides is growing. There is a notable difference between the antioxidant levels in plants under conventional growing conditions and those grown under more environmental friendly conditions (Lee and Kader, 2000). In the case of tomato, ascorbate levels appear to be higher in organic tomatoes. These observations are controversial in the fruit but remain significant in the leaves (Caris-Veyrat et al., 2004; Woese et al., 1997; Worthington, 2001). In the same way, the use of nitrogen fertilizers has been showed to lead to a decrease in ascorbate content in many potato and cabbage cultivars (Lisiewska and Kmiecik, 1996).
Conversely, the use of potassium fertilizer induces an increase in the ascorbate pool (Nagy, 1980). Several hypotheses have been proposed to explain the impact of nitrogen fertilizers on fruits and vegetables. It seems that beyond its impact on leaf expansion, this type of fertilizer increases the amount of NO3 in the plant (Mozafar, 2008). Other practices such as folding and lowering fruit load influence the nutritional composition and size of the fruit. Similarly, chemicals such as pesticides and growth regulators have important effects. Thus, the application of gibberellins induces an approximately 20% increase in ascorbate content in green tea (Liang et al., 1996).

GGP/VTC2: the key enzyme

GGP/VTC2 enzyme, which is now considered as the limiting enzyme of L-galactose pathway, is also the last to have been identified (Laing et al., 2007; Dowdle et al., 2007; Linster et al., 2007). Despite debates about the nature of the reaction it catalyses, it is now accepted that it is a GDP-L-galactose phosphorylase (Linster et al., 2008). It supports the first committed step in ascorbate biosynthesis in plants, converting GDP-L-galactose to L-galactose-1-phosphate. Also named GGP/VTC2 – according to vtc Arabidopsis mutants – the encoded protein belongs to the HIT (Histidine triad) protein superfamily. The knock-out vtc2 mutants display a drastic decrease in ascorbic acid while keeping a residual amount of it. That makes sense with the activity of VTC5 which is encoded by a homologous gene of GGP/VTC2. The VTC5 gene has a strong sequence homology with GGP/VTC2 but it is 100 to 1000 less expressed (Dowdle et al., 2007; GAO et al., 2011). Furthermore, the seedlings of double mutant vtc2/vtc5 are lethal and do not go beyond the germination stage, only exogenous addition of ascorbate to the medium could rescue the plantlets. Both GGP/VTC2 and VTC5 are strongly involved in the regulation of ascorbate biosynthesis. A lot of studies have shown a positive effect of light on the expression of these genes. GGP/VTC2 gene was constitutively expressed throughout the plant growth and development, with the significant higher expression abundance in green tissues than that in roots.
At cellular level, fluorescent fusion protein have highlighted that GGP/VT2 would be localized in both cytoplasm and nucleus (Müller-Moulé, 2008). The unexpected localization for a metabolic enzyme indicates that GGP might also act as a dual-function protein: a regulatory factor as well as a catalysing enzyme. Using a computer sequence analysis software a putative Nuclear Localization Sequence (NLS) sequence was found in the GGP peptide and this could explain the nuclear localization. However, according to different software used the presence of the NLS sequence could not be confirmed, suggesting the involvement of another partner to be translocated to the nucleus.

Food losses from field to plate: Post-harvest injuries

Food and Alimentation Organization (FAO) indicates that 30 to 50% of global food production would be lost from the field to the consumer plate. At the time of harvest when the products have reached a sufficient maturity to be marketed, fruits and vegetables are very sensitive to biotic and abiotic stresses. Losses can be caused either because of a climatic stress, or by attacks of bio aggressors. These losses are difficult to quantify, because they are very random and variable from one year to another.
Although the greatest risk of damage during the production is in the field, storage and transportation remain critical steps for the conservation of the products. Fresh fruits and vegetables usually are not or not very storable. Perishability depends on the physiology of the products. At 4 ° C, the apple can be stored for up to 8-12 months in a controlled atmosphere, while for raspberry the shelf life is only 2 days. For vegetables, the shelf life ranges from 5 days maximum for leafy vegetables, about 1 week for zucchini and tomatoes, and 2 to 3 weeks for cabbage and carrots.
There are losses at the packing and shipping station for several reasons. Products that do not conform to the specifications (over-maturity, defects in size or appearance etc.) are immediately discarded. Then, during storage of products in the cold room, conservation diseases are likely to develop. For products intended for the fresh market, losses may occasionally be significant at this stage because of a temporary imbalance between supply and demand, or because of overproduction for very perishable products (strawberry, peach, etc …) or many massive imports (tomato, peach, etc …).
Improving product conservation requires coordinated research in genetics and agronomy that implements new tools and systems approaches. Research has also to take into account the agroecology goals. The extension of the shelf life also involves the development of technological innovations such as modelling and simulation (predictive microbiology), new physical or biological processes (fermented products) from food preparation and active packaging or indicators of quality / freshness.

Reverse genetic: Ascorbate-deficient mutants

In the frame of functional characterisation of targeted gene, genetic transformation is useful and commonly used in Arabidopsis such as insertional T-DNA mutagenesis. This kind of technology is easily achievable in tomato but the whole process is time-consuming. Several months are usually necessary to obtain a generation and in the case of MicroTom this period represents about 6 months, which makes it difficult to apply for a large series of genes. Using the very high mutation frequency of the EMS population, TILLING (Targeting Induced Local Lesions IN Genomes) makes it possible to rapidly identify allelic series for a gene of interest.
This approach was used to study the function of genes involved in the modulation of ascorbate in relation with cell wall biosynthesis in tomato. The aim was to isolate in the EMS population from INRA-Bordeaux and Tsukuba ascorbate-deficient mutants, targeting the SlGMP2 gene (Solyc06g051270.2) encoding the GDP-D-mannose pyrophosphorylase, the SlGME1 (Solyc01g097340.2) encoding the GDP-D-mannose epimerase and the SlGGP1 gene (Solyc06g073320.2) encoding the GDP-L-galactose phosphorylase (Baldet et al., 2013, and Baldet et al. unpublished data). Several mutations was identified in MicroTom EMS mutant collection from Tsukuba and Bordeaux. Among the mutations isolated in the exonic region of the selected genes, some display point mutations that lead to the alteration of protein function. Two mutations in SlGMP2, one in SlGME1 and three mutations in SlGGP1, confirmed by sequencing, were selected for further characterization. To determine the physiological effect of the mutations, ascorbate content was assayed in fully expanded leaves. The only ones to display a significant alteration were the two Slggp1 truncation and splice junction lines, with ~95% decrease in ascorbate. This results confirm again the key role of GGP in ascorbate biosynthesis. Moreover, the two GGP homologous genes are also found in tomato (SlGGP1 and SlGGP2). Moreover, this study revealed that the two proteins are not totally and functionally redundant since the KO of SlGGP1 was not fully complemented by SlGGP2, which is about 100 fold less expressed than its counterpart in Tomato wild-type (Massot et al., 2012).

Forward genetic: Ascorbate-enriched mutants

At INRA-Bordeaux, the artificially generated population of MicroTom mutants, with a genetic and phenotypic variability far beyond the natural variation found in domesticated species, has allowed to go further the identification of allelic series of already known genes. Indeed, the screening of this population for a trait of interest, combined with a mapping by sequencing approach makes possible to reveal new genes underlying the phenotypic variations (Garcia et al., 2016).
In the case of the study on ascorbic acid metabolism developed in the INRA group to which I belong, and prior to the start of my PhD, 500 tomato mutant families from the INRA EMS Micro-Tom tomato mutant collection were screened for high ascorbate fruit content. This tremendous phenotyping work on more than 6000 plants, resulted in the finding of five mutant families displaying a significant ascorbate content 2 to 5 times higher than the WT fruits. The morphological analysis of the vegetative and reproductive parts of the plants, showed no obvious phenotype difference in mutant families up to 3 times the WT: P17G9, P20G7, and P21H6 families. For the two other mutant families, P19A6 and P17C5, displaying an increase of ascorbate up to 5 times that of the WT, the fruits exhibited a low seed number and in extreme extend a parthenocarpic phenotype, meaning seedless fruits. The absence of seed production has been a big challenge in the frame of the mapping by sequencing approach. Despite that, two mutant families have been the focus of the attention and finally successfully characterized: the P21H6 and the P17C5 family mutants

The P17C5 mutant family

Among mutant families to have been isolated, P17C5 is the one with the higher ascorbate accumulation (up to 5 times the WT). Moreover, the segregation analysis performed in its progeny seemed to reveal that the responsible mutation is dominant, what is really significant and attractive for breeding programs. However, even being very original, the parthenocarpic phenotype made more complex the way to characterize the mutation. Observations revealed that the flowers was abnormally bigger than the WT, and preliminary analysis of the pollen germination rate also showed an obvious loss of fertility. As mentioned previously, the genetic characterization of the original mutant plant allowed to conclude that this mutation is dominant. Knowing that, it was possible to transfer the P17C5 mutation to another commercial genetic background, at the same time than to attempt to break the link between the AsA + phenotype and parthenocarpy. In that aim the ascorbate enriched P17C5 mutant was crossed with the Solanum lycopersicum cv M82 cultivar, a tomato variety known for both the fresh market and processing. This well-known variety displays morphological and genetic characteristics very different from that of the Micro-Tom cultivar. It has a bushy determinate port, growing up from 1 to 2 meters, and produces large fruits (~8-10cm in diameter compared to MicroTom of 3-4cm). From this first Out-Cross (OC), few seeds have developed after crossing. At the start of my PhD work, among the four OC1F1 plants sown, only one produced fruits displaying a strong AsA+ phenotype, confirming the dominant nature of the mutation in the first mutant P17C5. However those AsA-enriched fruits were all parthenocarpic making impossible to generate the OC1F2 population to perform the mapping by sequencing approach.

Other Related Blue-Light Receptors

The numerous studies of the blue-light receptor families led to the definition of several specific sub-families related to their function. Among them, there is the zeitlupe (ztl) family that, like cryptochromes, contributes to the regulation of the circadian cycle and the control of flowering (Takase et al., 2011). In a similar manner to CRY1/2, the ZTL proteins act by interacting with different partners, controlling the degradation and stability of the compounds for which they are responsible. ZTL proteins localize to the cytosol or the nucleus (Takase et al., 2011) and comprise three members: Zeitlupe (ztl), Flavin-binding kelch repeat F-box 1 (fkf1) and LOV kelch protein 2 (lkp2) (Ito et al., 2012; Suetsugu and Wada, 2013). Each protein contains three domains:(i), a LOV domain which is able to bind oxidized FMN as chromophore in response to blue light (Imaizumi et al., 2003); (ii), an F-box domain, involved in targeting proteins for degradation via the ubiquitin–proteasome system (Ito et al., 2012); (iii), six kelch repeats at the C-terminus utilized to mediate protein–protein interactions and hetero-dimerization.

The SlPLP interacts with SlGGP in the cytosol and the nucleus under the dependence of light signalling

Previous works carried have suggested a potential interaction between AtPLP and AtVTC2, which was governed by the light spectrum (Ogura et al., 2008). Moreover, Müller-Moulé (2008) showed that in Arabidopsis leaf, AtVTC2 (homolog to SlGGP1) was detected in the cytosol and the nucleus. To decipher the function of the SlPLP protein, we first studied its subcellular localization by transiently expressing 35S-GFP-SlPLP either under its wild-type or its truncated form as well as the two 35S-GFP-SlGGP1 and 35S-GFP-SlGGP2 in Nicotiana benthamiana leaves. Confocal microscopy analyses showed that the wild-type form of SlPLP localized like SlGGP1 and SlGGP2 in the nucleus and the cytoplasm (Fig. 4B). Surprisingly, the truncated SlPLP protein displayed the same sub-cellular localization. Next, we tested the physical interactions of SlPLP with SlGGP1 using BiFC experiments in epidermal onion cell. As shown in Figure 4B, this interaction between the two proteins was confirmed, but not at all when the SlPLP mutated protein was used. Interestingly, the interaction occurred both in the cytoplasm, which was expected in light of the GGP activity in the AsA biosynthesis, and into the nucleus. The nuclear localization of these two protein partners raised the question regarding the meaning of such an interaction and the regulatory process involved. With the presence of a LOV domain in the SlPLP, which is (i) defined as a blue light sensor, (ii) is truncated in our mutant PLP protein, and (iii) its impact on AsA metabolism, we hypothesized that such an interaction might be controlled through the regulation by blue light. In that frame, a heterologous system based on animal cell coupled to optogenetic approach was used in a combination of vectors for the SlGGP1, SlGGP2, SlPLP and Slplp sequences (Supplemental Table 4). In our optogenetic system two conditions were experimented, dark and blue light (455nm). It clearly appeared that the WT SlPLP interacts strongly with SlGGP1 in the dark condition whereas under blue light regime a weaker interaction with the SlGGP1 protein was observed, representing only 30% compared of that in darkness (Fig. 5). Interestingly, this experiment revealed also interaction between SlPLP and SlGGP2 which was impaired under blue light.

PLP, a new type of regulator modulating the AsA level by interacting with GGP the key enzyme of the L-galactose pathway

Since now a decade, GDP-L-galactose pyrophosphorylase (GGP1 or VTC2) is presented as a central actor in the regulation of the AsA biosynthesis in plants (Macknight et al., 2017). Several clues led to such a definition. In many plant species, including Tomato, GGP gene is one of the most expressed among the AsA-related genes of the L-galactose pathway, catalyzing the first committed reaction of this pathway (Dowdle et al., 2007). In Arabidopsis, VTC2 has been localized in the nucleus attributing to GGP a putative regulatory role (Müller-Moulé 2008). In that sense, Laing et al. (2015) suggested a negative feedback mechanism involving GGP that could control AsA biosynthesis. According to their model, the regulation was based on the AsA level perceived by cellular system. Hence, the translation of a small regulatory peptide encoded by a uORF located in the 5′-UTR region of the GGP1 gene would be induced in the presence of high AsA level, and reversely when AsA level is low. This peptide could then interact with the polysomal translation machinery and inhibit the production of GGP1 protein. At last, gene expression studies showed that VTC2 is regulated by light and suggested a circadian regulation (Tabata et al., 2002; Dowdle et al., 2007; Müller-Moulé 2008). However, it is quite unlikely that GGP can act alone, or through its interaction with one partner, to control the expression or translation of other actors of the AsA biosynthesis pathway.
Our results synthesize these observations of a regulatory role of GGP and the effect of light, and raise many questions regarding its physiological significance, notably the interaction of SlPLP with SlGGP1 taking place in the cytoplasmic and nuclear compartments. In the photoreceptor family harboring LOV domains, these LOV domains have the property of conformational changes dependent on blue light (Kasahara et al., 2010). Light induces the covalent fixation of flavin which induces the folding of the protein and the recruitment of partner proteins. This reversible process is called the LOV photocycle (Salomon et al., 2001). It is thus tempting to suggest that in this case the partner protein could be GGP1 and GGP2 and that the trigger signal is blue light (Fig. 5). This SlPLP/SlGGP interaction could play a role driving either the degradation of GGP through COP9 signalosome which is known to be involved in light-mediated regulation of AsA synthesis (Mach, 2013), either its inhibition or to act in concert directly on the expression of other genes as a regulatory complex. Moreover, other flavoproteins such as cryptochromes or phototropins, have at least a second functional domain in addition to LOV which recognizes the light signal. In the case of phototropins, whose domains are homologous to those of PLP, a serine/threonine kinase domain allows its autophosphorylation as well as the phosphorylation of its partner proteins to trigger a cascade of response (Chaves et al., 2011; Christie, 2007; Suetsugu and Wada, 2013). These mechanisms would be consistent with those already described involving photoreceptors. In some cases, blue light sensors may modulate the degradation of transcription factors by their interaction with subunits of the signalosome COP9 (see Review Christie, 2015). The second possibility would be the repression of AsA biosynthesis through the inhibition of the GGP activity by PLP. This would be a very simple and rapid way to modulate or stop the biosynthesis of AsA in the cell as a function of the perception of the light signal. In the third option, PLP could alter the partitioning of GGP between the cytoplasm and the nucleus, by sequestrating GGP in the nuclear compartment, and so reducing its capacity to participate to the L-galactose pathway. These assumptions are perfectly relevant given the dual compartmentation of the SlPLP and SlGGP (Fig. 4) and that was already shown for VTC2 by Müller-Moulé (2008).
However, we cannot rule out the possibility that PLP could also participate with GGP in a multimeric complex. Up to now, the PAS domain analysis did not reveal a particular function and no additional PLP function was noted in addition to the ability to interact with GGP1. According to literature, LOV domains are able to dimerize enhancing their affinity with some targeted proteins (Heintz and Schlichting, 2016). In addition to PLP and GGP1, other partners could be recruited to form a bigger regulatory complex. One candidate could be the homolog of AtBLH10 protein that has been identified as a potential interacting with AtPLP by a double-hybrid screening of an Arabidopsis cDNA library (Ogura et al., 2008). BLH10 belongs to the family of TALE homeodomain proteins which are involved in several developmental processes (Hackbusch et al., 2005). Interestingly, in a previous integrative study carried out in Tomato (Garcia et al., 2009), a positive correlation was found between AsA content and the expression level of SlBEL1 gene, homolog of BLH10 (Bournonville C., thesis 2016).

Table of contents :

INTRODUCTION
VITAMIN C – ASCORBIC ACID
HISTORICAL BACKGROUND: LET US GO BACK A BIT IN TIME
CHEMICAL DESCRIPTION OF VITAMIN C
ASCORBIC ACID METABOLISM
i. Biosynthesis
ii. Recycling: redox state
iii. Degradation
REGULATION: VARIABILITY IN PLANTA
i. Genetic variability
ii. Spatiotemporal variability
iii. Environmental impact
iv. GGP/VTC2: the key enzyme
BIOLOGICAL ROLES IN PLANTS
i. Plant development: from seed to fruits
ii. Photosynthesis
iii. Oxidative stress (Post-harvest)
iv. Pathogenic infection
TOMATO – MORE THAN A FRUIT, A MODEL
FROM ORIGINS TO DOMESTICATION
A MODEL SPECIE
i. Agro-economic importance
ii. Food losses from field to plate: Post-harvest injuries
i. Genetic tools
MICROTOM ASCORBATE MUTANTS
i. EMS mutant library
ii. Reverse genetic: Ascorbate-deficient mutants
iii. Forward genetic: Ascorbate-enriched mutants
LIGHT – THE DARK SIDE OF ASCORBATE
DESCRIPTION
i. Some physical aspects of light
ii. Light and plants
i. Light and ascorbate
i. Photo-Sensor Systems
PASLOV PROTEINS
OBJECTIVES OF THE THESIS WORK
RESULTS
PART 1: A BLIND REGULATOR
THE KEY ENZYME GGP REGULATES ASCORBIC ACID CONTENT THROUGH ITS INTERACTION WITH A NEW CLASS OF PHOTORECEPTOR IN TOMATO.
Introduction
Results
Discussion
Materials and Methods
Supplemental
References
PART 2: STERILE DEREGULATION
KNOCKING-OUT SLGGP RETRO-REGULATION LEADS TO HIGH ASCORBATE ACCUMULATION RESULTING IN A DEFECT OF THE REPRODUCTIVE SYSTEM IN TOMATO.
Introduction
Results
Discussion
Materials & Methods
Supplemental
References
PART 3: CHILLING STRESS
ANTIOXIDANT POTENTIAL IN POSTHARVEST FRUIT QUALITY.
Introduction
Results
Discussion
Materials and Methods
CONCLUSION
ANNEXES
Primers and vectors used for GATEWAY® cloning
Agrobacterium-mediated stable transformation of tomato
Biolistic transformation of onion epidermis
Agroinfiltration of Nicotiana Benthamiana leaves
Setting of the light experiment in the growth chamber.
CRISPR Cas9
Optogenetic assay
Ascorbic acid measurement
REFERENCES

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