Oxalate Synthesis in Plants:
The involvement of ascorbic acid in response to UV-B

Introduction

Oxalates are salts or esters of oxalic acid. They occur naturally in a large number of plant species, functioning in high capacity calcium regulation and protection against herbivory (Franceschi and Nakata, 2005). Common edible plants that are rich in oxalates include rhubarb, yams, sweet potato, and leafy green vegetables such purslane, spinach and beets. They are also found in beverages such as tea, cocoa and colas.
In mammalian metabolism, oxalates can be seen as the resultant compounds of ascorbate, glyoxylate and glycine pathways. Despite these metabolic occurrences, oxalates can have adverse effects when consumed in excess, and are therefore considered anutritive factors in food.
Oxalate can exist in two forms – soluble and insoluble. Soluble or free oxalate is water soluble and absorbed by passive diffusion in the small intestine and colon of humans. The insoluble form is manufactured when oxalate binds to minerals such as calcium, iron or magnesium. It cannot be absorbed into the blood and is excreted in the faeces, therefore having no system toxicity. Insoluble oxalate can only become soluble in acidic conditions.
Of these two forms, free oxalate is the least desirable. Free oxalates bind to minerals in the body, particularly calcium and iron, reducing bioavailability. They can also precipitate with calcium in the urine forming calcium oxalate crystals, the major component of kidney stones (Noonan and Savage, 1999).
An understanding of factors involved in increased oxalate biosynthesis in plants is necessary for improving the nutritional value of certain foods. In plants, oxalates occur as metabolic end products of the oxaloacetate, glycolate and glyoxalate pathways (Franceschi and Loewus, 1995). A well known plant antioxidant, ascorbic acid, has also been shown to be an oxalic acid precursor capable of large scale oxalic acid synthesis. Increased ascorbic acid turnover in plants, due to factors such as the environment, may very well lead to raised oxalate content. It has already been shown that factors such plant nutrition (Palaniswamy et al., 2004), and different season cultivars (Morrison and Savage, 2003) can influence oxalate content, however environmental stresses, such as the effects of UV-B, have not yet been explored.


Oxalate in plants

Oxalates are common constituents of plants and are found in the majority of plant families. They occur in the forms of oxalic acid, soluble salts of potassium, sodium and magnesium, and as the insoluble salt of calcium (Franceschi and Loewus, 1995). The amount of oxalate in plants ranges from a few percent up to 80% of the total weight of the plant.
These compounds are usually accumulated within the vacuoles of plant cells, although crystalline calcium oxalate forms within the cells walls of some plants. Since plant cells generally have a large vacuolar compartment, often from 75-90% of the cell volume, there is the massive oxalate accumulation potential (Franceschi and Loewus, 1995).


Function of oxalates in plant systems

Oxalates serve as important counter ions for regulation of charge balance, metal detoxification and calcium activity regulators in plant cells. Oxalates regulates calcium through the production of the insoluble salt, calcium oxalate.
Soluble oxalate and calcium oxalate also have protective functions in some plants. High soluble oxalate content can inhibit the activity of sucking insects, such as leafhoppers and aphids, while sharp calcium oxalate crystals deter grazing animals. These crystals also contribute to the specialised stinging hairs in types of stinging nettle. The hairs have large conical cells, the lumen of which contains sharply pointed calcium oxalate crystals. Upon contact with an animal, the thin walled tip of the cell ruptures, and the skin is penetrated by the tips of these crystals and toxins within the cell.
It is well established that oxalate rich plants ingested by livestock can lead to acute and toxic effects often due to renal failure.
While the examples above demonstrate plant resistance to herbivory via oxalate, it is unlikely that plants accumulate oxalate primarily as a protective compound. These defence mechanisms are thought to be only a secondary effect of oxalates in plants, with the main function being regulation of ions and calcium.
The amount and distribution of oxalate within plants is important in plant defence mechanisms, and animal and human nutrition.


Biosynthesis in plants
Oxalate ion
Oxalate ion

The biosynthetic origin of oxalate was uncovered with the use of isotope labeled substrates. Attention was focused on three intermediary compounds of plants metabolism – oxaloacetate, glycolate and glyoxalate (Franceschi and Loewus, 1995). An oxaloacetate hydrolase which cleaves oxaloacetate into acetate and oxalate, is found in red beets and spinach. Glycolate oxidase catalyses an oxygen-dependent oxidation of glycolate to glyoxalate and hydrogen peroxide in plants. Further oxidation of glyoxalate, either non-enzymatic or in the presence of catalase, leads to oxalate.
An alternative biochemical pathway independent of pre-formed glycolate, emerged when it was discovered that oxalic acid is a major product of ascorbate metabolism. A biosynthetic role for L-ascorbic acid in the production of oxalic acid was first discovered when it was found that apices of Pelargonium crispum labeled with 14C-L-ascorbic acid accumulated 14C-oxalic acid (Wagner and Loewus, 1973).
L-ascorbic acid is a constituent of all plants cells, present predominantly in the reduced form. Studies using 14C-radiolabeled isotopes clearly established the pathway in which the carbon chain of glucose is conserved and its numerical sequence preserved (tracing showed C1 through to C6 of D-glucose becomes C1-C6 of L-ascorbic acid). Ascorbic acid undergoes rapid catabolism in young or developing tissues in plants, and C2/C3 cleavage of the carbon chain of L-ascorbic acid is one aspect in this breakdown. Chemical cleavage at the C2/C3 bond was first used to establish the constituents that make up L-ascorbic acid, and the products were found to be L-threonic acid and oxalic acid.

(McKersie, 1996)
(McKersie, 1996)


See oxalate pathways in glyoxylate, dicarboxylate and ascorbate metabolism in detail here



UV-B and ascorbic acid production in plants

Sunlight contains non-photosynthetic wavelengths including shorter wavelengths such as UV-B (280-320nm) radiation. UV-B is damaging to living organisms since cellular components such as proteins and nucleic acids absorb this energy-rich radiation. Due to increasing atmospheric pollution and the subsequent depletion of stratospheric ozone levels, the level of UV-B reaching the Earth’s surface has increased. Many studies have shown a deleterious UV-B effects on plants such as reduced photosynthesis, biomass reduction, decreased protein synthesis, impaired chloroplast function, damage to DNA and so on (reviewed by Jordan, 1996). Much of this damage is often a consequence of reactive oxygen species production (ROS) (Strid et al., 1994). The photosynthetic electron system in plants is the main source of ROS, and even under regular metabolism plant cells are prone to hydrogen peroxide and oxygen production. Under stressful conditions however, i.e. elevated exposure to UV-B, this is increased and induces deleterious photo-inhibitory events in the photosynthetic apparatus. To cope with ROS toxicity, plant cells display numerous, highly efficient antioxidant defence systems that are both enzymatic and non-enzymatic. The non-enzyme antioxidants are generally small molecules such as glutathione and ascorbate, acting in the aqueous phase, whereas the lipophilic antioxidants, tocopherols and carotenoids are active in the membrane environment.
Ascorbate chemical structure
Ascorbate chemical structure

Ascorbate is a major primary antioxidant, reacting directly with hydroxyl radical, superoxide and singlet oxygen. It is also a powerful secondary antioxidant, reducing the oxidised form of α-tocopherol. Ascorbate and glutathione are closely related, since they are both constituents of the anti-oxidative ascorbate-glutathione cycle which detoxifies hydrogen peroxide through a series of enzymatic reactions. Glutathione turnover seems to be enhanced by UV-B radiation with no net change in its concentration, a hypothesis that was confirmed by Masi et al. (2002) in maize plants. While this is interesting, this experiment used artificially enhanced UV-B irradiation, an environment which is very different to plants grown in the field. Field experiments using selective filters to remove UV-B, therefore not disturbing the microenvironment of plants, are of more interest when looking at plants grown for food purposes. In soybeans, an field study was conducted where selective filters were used to remove the UV-B fraction of sunlight (Xu et al., 2006). Results showed that in the control plants where UV-B was not excluded were oxidatively stressed with ascorbate peroxidase, catalase and glutathione reductase activity increased, and ascorbic acid content decreased. This suggests UV-B triggers an increased antioxidant turnover response in plants.

Oxidative Stress in Plants
(McKersie, 1996)

Ascorbic acid turnover and oxalate synthesis

Under high stress environmental conditions, such as increased exposure to UV-B, it seems quite likely that ascorbic acid turnover is enhanced. As a precursor of oxalic acid, this seems an important detail to remember, particularly when looking to improve the nutritional content of plant foods. Current research conducted by Moreau and Savage (2007) confirms that purslane grown in a greenhouse exhibited oxalate levels 10% lower than those grown in the field. It is quite possible that the field grown plants were under oxidative stress compared to the greenhouse specimen, as the damaging UV-B fraction of light was not filtered out as it was by the glass of the greenhouse grown plants. It is quite possible that the field grown plants had increased antioxidative activity and ascorbic acid turnover, which may very have been responsible for the increased oxalate levels seen in this experiment.



Conclusion

As well as cultivar variation, environmental factors may influence the oxalate content in plants. While the effect of plant nutrition on oxalate content has been documented, there has been little investigation into other environmental factors, such as temperature and UV-B exposure, on oxalate production. Almost no literature exists on field trials and oxalate content. For this reason, research is being conducted into this area at Lincoln University. It is hypothesised that as well as analytical differences, environmental effects on plant growth may account for much of the variation seen in reported oxalate content in plant foods.



Tracey Feary, M.Sc. (Biochemistry) student
May 2008


References