FOLATE METABOLISM & ENGINEERING IN PLANTS

Grant No. MCB-0129944

I. Overview
II. Folate engineering
III. Folate degradation and recycling
IV. References
 

I. Overview

A lack of folates is the world’s commonest nutrient deficiency and has grave consequences for health. There is good evidence that even in developed countries the folate intake is usually suboptimal. Folates – a generic term that groups tetrahydrofolate and its derivatives – belong to the B vitamin family. They are essential cofactors for one carbon transfer reactions in all organisms, but only plants and microorganisms are able to synthesize them de novo. Vertebrates absolutely need folates in their diet. Since plant foods are the single largest source of folate in human diets, enhancing plant folate content by metabolic engineering is an appealing way to improve human nutrition worldwide.

Folates are tripartite molecules that consist of pteridine, p-aminobenzoate (pABA), and one or more glutamate moieties (Fig. 1).
 
 

The plant folate biosynthesis pathway is distributed among three subcellular compartments (Fig. 2). In essence, the cytosol and plastids produce respectively the pteridine and pABA moieties, which are condensed, glutamylated, and reduced in mitochondria to give tetrahydrofolate (Hanson & Gregory, 2002).

By combining genomic, genetic and biochemical approaches, we have identified, cloned and characterized four of the folate biosynthesis enzymes that were until recently unknown in plants –namely, GTP cyclohydrolase I (Basset et al., 2002), ADC synthase (Basset et al., 2004a), ADC lyase (Basset et al., 2004b), and DHN aldolase (Goyer et al., 2004) (Fig. 2).
 
 

II. Folate engineering

Metabolic engineering of folate in plants is an attractive challenge. The chemical instability of folates, their highly compartmented biosynthesis pathway, and the numerous enzymes involved in their metabolism complicate the engineering process. However, the availability of complete plant genomes, the growing number of ESTs, and the advances made in HPLC analysis of folates offer great promise (Hanson & Gregory, 2002).

We are targeting our engineering to tomato fruit for several reasons. The first is that fruits have lower folate contents than leaves, showing that enhancement is in principle possible. The second is that folates are subject to huge losses during cooking, making fruits – which are generally consumed fresh – a more efficient vehicle to deliver folates than grains or vegetables. Finally, tomato is readily transformable and a major world crop.

Our engineering work is designed to explore the extent to which flux in the whole pathway is regulated via the committing steps of its pteridine and pABA branches (i.e., by the activities of GTP cyclohydrolase I and ADC synthase).  As pteridine synthesis capacity is known to drop in ripening tomato fruit (Basset et al., 2002), we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, since this enzyme is predicted to escape feedback control in planta (Díaz de la Garza et al., 2004). This engineering maneuver raised fruit pteridine content by three- to 140-fold, and increased total folate in fruits by up to three-fold (Fig. 3). Most of the folate increase was contributed by 5-methyl- and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in non-engineered tomato fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin, their reduced forms, which are folate biosynthesis intermediates, and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction had the highest folate levels.  pABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with pABA via the fruit stalk increased their folate content by up to ten-fold. These results demonstrate that pteridine engineering can significantly enhance the folate content in food plants, that boosting the pABA supply can produce further gains, and that overkill in pteridine production may depress folate synthesis.  Moreover, our transgenic fruit are a significant first step towards a viable biofortified product, because the highest folate level we achieved so far (~4 nmol g-1 fresh weight) is equivalent to 180 µg per standard 100-g serving. This would provide the entire recommended daily allowance for a young child and almost half of that for an adult.
 

In order to address the pABA depletion in tomato fruit, we produced transgenic plants that over express (under the E8 promoter) the Arabidopsis 4-amino-4-deoxychorismate synthase (AtADCS), which is the first enzyme of pABA synthesis (Basset, et al. 2004).  Fruit at red ripe stage was analyzed to quantify total pABA levels.  On average, transgenic fruit displayed a 30 fold increase in total pABA content compared with the control (Fig. 4).

These plants were crossed with pteridine overproducers to give double transgenics.  Based on our pABA-feeding data (Díaz de la Garza et al., 2004) we can expect an increase in folate of up to 10-fold in the double transgenics.
 
 

III. Folate degradation and recycling

Folates are easily degraded by photochemical or chemical oxidation into pteridine and pABA-polyglutamate (pABA-Glun) moieties (Fig. 5). This breakdown appears to be much faster in plants than in animals. Whereas animals excrete the breakdown products, plants can potentially recycle them to folates. Specifically, in vivo, they can cut the polyglutamyl tail off pABA-Glun and hydrolyze the resulting pABA-Glu to pABA. They may also recycle the pteridine fragment to the folate precursor HMDHP.
 
 

We are combining genomics and biochemistry to find the pABA-Glu hydrolyzing enzyme and the pteridine recycling enzyme(s), and to define their subcellular location. We also plan to use Arabidopsis reverse genetics along with stable isotope labeling and HPLC-MS analysis to evaluate the in-vivo rates and biological impact of the salvage of folate breakdown products.

Folate catabolism is relevant to our engineering goal of enhancing folate levels because it has the potential to wipe out any gains made by engineering synthesis. This makes it essential to find out beforehand the extent of folate turnover in plants. The enzyme (gamma-glutamyl hydrolase) that removes the polyglutamyl tail of pABAGlun also attacks polyglutamylated folates and may in effect target them for destruction since deglutamylated folates are more prone to oxidation in vivo. If so, engineering tomato fruits to suppress gamma-glutamyl hydrolase could limit the folate breakdown that occurs after the fruits are harvested.
 
We first characterized gamma-glutamyl hydrolases (GGH) from Arabidopsis, which has three GGH genes.  We found that individual GGHs exhibit distinct bond cleavage specificities for the polyglutamyl tail, and that this activity was localized in vacuoles (Fig. 6). Interestingly, purified pea vacuoles were found to contain ~20% of the total cellular folate, whereas 60% of beat root folates were vacuolar.  These findings encourage engineering tomato fruit to suppress GGH not only to limit folate breakdown, but perhaps to allow fruit vacuoles to accumulate and store folate polyglutamates.

Figure 6: Localization of GGH in pea leaf vacuoles by subcellular fractionation. Chloroplasts (CP), mitochondria (M), and vacuoles (V) were purified by density gradient centrifugation. A fraction enriched in cytosol and vacuole contents (CS+V) was prepared from pea leaf protoplasts by pelleting intact organelles. The specific activities of GGH (measured using pABAGlu5) and marker enzymes were assayed in each fraction. Markers were   α -mannosidase (vacuole), NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GAPDH, chloroplast), fumarase (mitochondrion), and MTHFR (cytosol). The asterisk indicates that a trace of MTHFR activity was detectable in vacuoles but was too low to quantify; it represented   2% of the total activity of the protoplasts. Data are the means and S.E. of data from 3 to 10 independent preparations of each fraction.

IV. References

Basset, G., Quinlivan, E.P., Ziemak, M. J., Díaz de la Garza, R., Fischer, M.,  Schiffmann, S., Bacher, A., Gregory III, J.F., and Hanson, A.D. (2002) Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proc. Natl. Acad. Sci. USA 99: 12489-12494

Basset, G.J., Quinlivan, E.P., Ravanel, S., Rebeille, F., Nichols, B.P., Shinozaki, K., Seki, M., Adams-Phillips, L.C., Giovannoni, J.J., Gregory, J.F. III, Hanson, A.D. (2004) Folate synthesis in plants: the p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids. Proc. Natl. Acad. Sci. U S A. 101:1496-1501

Basset, G.J., Ravanel, S., Quinlivan, E.P., White, R., Giovannoni, J.J., Rébeillé, F., Nichols, B.P., Shinozaki, K., Seki, M., Gregory, J.F. III and Hanson, A.D. Folate synthesis in plants: The last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase (2004) Plant J. 40(4):453-61.

Goyer, A., Illarionova, V., Roje, S., Fischer, M., Bacher, A., Hanson, A.D. (2004) Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases. Plant Physiol. 135:103-111

Díaz de la Garza R, Quinlivan EP, Klaus SM, Basset GJ, Gregory JF 3rd, Hanson AD. (2004) Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc Natl Acad Sci U S A  101:13720-13725

Hanson, A.D. and Gregory III, J.F. (2002) Synthesis and turnover of folates in plants. Curr. Opin. Plant Biol. 5: 244-249