“Hypotheses on the role of soil and soil biology in wine aroma synthesis”.
Introduction :
Fifty years of often excessive application of mineral fertilizers have made winegrowers forget the complex and fundamental relationships between soil, soil microbes and vines. The purpose of this article is to recall the fundamental basics of soil microbiology.
Cation supply to vines :
Cations play two roles in vine nutrition:
– Membrane electrical charge. This is the case for monoatomic or alkaline cations: H+, Li+, Na+, K+, Rb+ and Co+ (Mitchell, 1961). These cations, apart from hydrogen, are not constituents of vine tissue. In fact, there are no organic molecules containing lithium, sodium, potassium, rubidium or caesium. These cations only serve to positively charge cell membranes, to attract and absorb anions synthesized by microbes, or as cofactors for certain enzymes.
– A role in vine tissue composition. These are 2 alkaline earth elements: Magnesium and Calcium, which can be absorbed as such by root cells, provided their concentration is high in the soil.
In the first case, that of monoatomic cations, there is no intervention by microbes to make them assimilable by the vine. There is no biogeochemical cycle for these cations. This is logical, since they do not play a part in the constitution of plants. Their absorption is therefore subject to the physical law of concentration. These cations are stored within the clay-humus complex. This is known as C.E.C. or cation exchange capacity. Clays are layered crystals with negative surfaces, attracting cations that lodge between the layers. Humus is also negatively charged and retains cations. Depending on the quality of the clay-humus complex (type of clay and humus), it may therefore contain varying quantities of cations. These will be released into the soil water according to the Law of Concentration.
To positively charge its cells, the vine, like all plants, sends sugar to its roots in the downstream sap.
Sugar is used to synthesize the energy molecule of the living world: A.T.P. With this A.T.P., root cells attract cations (mainly H+, Na+ and K+) to the membrane, charging it positively (Higinbotham et al ,1970).
As these groups withdraw cations from the soil water, the clay-humus complex will release cations under the influence of the Law of Concentration. This mechanism must be seen as dynamic.
In other words, the law of concentration tends to draw membrane cations towards the soil water, but the A.T.P. pumps, which are constantly supplied by the elaborated sap, attract these cations back to the root membrane, ensuring the overall charge necessary for nutrient uptake.
Monoatomic cations are not involved in the synthesis of wine aromas, since they are not constituents. They do, however, influence wine salinity. The work of Vignon et al. has shown that cations such as sodium and potassium at levels of over 10 mg/l give a salty sensation when combined with an anion such as chlorine. These cations can be abundant in wine if the soil is naturally rich in them (salty soils in Australia, or excess potassium chloride added at planting). What’s more, these cations, at doses in excess of 10mg/liter, change some of the perceptions of other primary flavors, such as bitterness and astringency. Sodium, for example, reduces the perception of bitterness. Sodium can be abundant in the dolomitic soils of Italy. It’s interesting to note that Italians appreciate bitterness, whereas the French don’t, and France has very few magnesium-rich soils.
Nutrient supply for vines:
Once its membranes are positively charged thanks to the electrical force created by the elaborated sap and A.T.P. pumps, the vine is able to absorb anions, i.e. negative charges.
In fact, in soil water, all ionic exchanges are influenced by the Law of Concentration, which imposes an equality of ions outside and inside root cells. However, nitrate (NO3), phosphate (PO4), sulfate (SO4), etc. are very rare in soil water; root cells should therefore be poor in these elements, and vines should starve.
To feed itself, the vine creates an electrical force (membrane charges) that opposes the Law of Concentration, allowing elements such as nitrate to accumulate in the root cell.
This explains why vines can only absorb negative elements (anions) that can be electrically attracted by the positive membrane of root cells. However, it is the soil microbes – bacteria, actinomycetes and fungi – that synthesize the negative forms of nutrients that can be absorbed by the vine.
To make an element ionic, i.e. soluble in water, and anionic, i.e. negative, so that it can be attracted to the positive membrane of root cells, microbes use two biochemical pathways: oxidation and chelation.
– Microbial oxidation :
Three elements are soluble in water as oxides: nitrogen as nitrate (NO3), sulfur as sulfate (SO4) and phosphorus as phosphate (PO4). Soil nitrogen is stored in organic matter. It is first mineralized by numerous microbes to ammonium (NH4+), then oxidized to nitrite (NO2) by the Nitrosomonas genus, and finally oxidized to nitrate by the Nitrobacter genus. This nitrate (NO3) is attracted to the positive membrane, and specific transporters move it across the membrane.
Sulfur is oxidized by sulfobacteria to sulfate (SO4), either from the mineral forms of sulfur: H2O and S, or from the organic forms contained in humus.
Phosphorus is released in the form of phosphate (PO4) by Penicillium fungi or bacteria (Pseudomonas, Bacillus) from mineral forms (calcium, iron or aluminum phosphate) (Stewart et al, 1982), or recovered as such from the clay-humus complex by mycorrhizae (Bardgett, 2005).
– Microbial chelation :
All other soil nutrients are precipitated in the oxide state. This is the case, for example, with iron, precipitated as FeO3 or Fe(OH)2, which gives soils their red or yellow color. For these elements, microbes use the chelation pathway. They do this by chelating the nutrient with an organic acid they synthesize, or by methylation (Dolfing et al, 1995). For example, they chelate iron to iron succinate, iron tartrate or iron citrate (Weinberg, 1977). Pseudomonas use siderophores (hydroxamates) to solubilize iron for grapevines. These chelation reactions use organic acids with negative R-C-O-O functions, which are attracted to the positive root membrane. It is at this level of oxidation or microbial chelation that the role of the soil and its biology in the synthesis of wine aromas comes into play.
In criticisms of the role of soil in wine expressions, we commonly hear the following arguments:
– Soil cannot play a role in wine aromas, since these are carbon compounds and carbon comes from photosynthesis, not soil. This type of argument is scientifically invalid, as it ignores one of the fundamental bases of physiology: in living organisms, syntheses are carried out by enzymes. Enzymes are proteins with metal cofactors, and all metals and nitrogen come from the soil (Summers et al., 1978).
– Deep rooting only serves to absorb water. This argument flies in the face of everything known about root physiology. In fact, there is no difference in the metabolism of absorption from the base to the tip of a root. When the vine sends its sugars down the sap, it does not localize them at the base of the roots; there are no valves or closure systems for the sap elaborated in the phloem (Guinochet, 1965). This is confirmed by 2 facts:
- The principle behind systemic herbicides is that they follow the phloem and destroy the entire root system.
- Experiments by Ghorbal et al, 1978, show that results for potassium concentration in roots obtained from root compartments or complete root shreds were in perfect agreement.
What’s more, raw sap circulates rapidly from bottom to top, at speeds ranging from a few meters/hour to 100 m/hour during intense transpiration (Lafon et al, 1988), and it’s hard to see how, at these speeds, it could bring up only water at depth and minerals at the surface.
It can also be argued that biological activity is most intense in the first 10 cm of soil, and weakest at depth. This is due to the fact that at depth, biological activity is localized along the galleries of the soil fauna and along the roots in the so-called rhizospheric zone (Sorensen, 1997). In these 2 zones, biological activity is just as intense as at the surface.
Enzymes, agents of biochemical synthesis :
“Enzymes are the largest and most specialized class of proteins. They represent the direct primary instrument for the expression of gene action, catalyzing thousands of chemical reactions that constitute the intermediate metabolism of cells”, Lehninger, 1972.
An important aspect of enzymes is their ability to carry out complex metabolic or synthetic reactions at ordinary temperatures. For example, Rhizobium nitrogenase is able to open the triple bond of the nitrogen molecule and make 2 ammoniums, whereas to produce this reaction man puts nitrogen under high pressure and raises it to high temperature. To carry out these low-temperature reactions, enzymes are combined either with metal cofactors or with coenzymes containing sulfur, phosphorus, cobalt, etc. The combination of enzyme plus cofactor is called a “coenzyme”. The combination of enzyme plus cofactor or coenzyme is called a holoenzyme.
Cofactors and coenzymes :
Biochemists have isolated thousands of enzymes and studied cofactors and coenzymes. The main metals used as cofactors to facilitate biochemical reactions are summarized in the following table:
| Cofactors | Enzymes | |
| Names | Symbols | |
| Potassium | K+ | Pyruvate phosphokinase |
| Sodium | Na+ and | Membrane ATPase |
| Zinc | Zn++ | Carbonic anhydrase Alcohol dehydrogenase Carboxypeptidase DNA synthesis enzymes |
| Magnesium | Mg ++ | Phosphohydrolases Phosphotransferases Chlorophyll |
| Manganese | Mn++ | Arginase Photosynthesis enzymes |
| Iron | Fe++ or Fe+++ or Fe+++ or Fe+++ or Fe+++ or Fe+++ or Fe+++ or Fe+++ | Cytochromes Peroxidases Catalases Ferredoxin |
| Copper | Copper | Photosynthesis enzymes Tyrosinase Cytochrome oxidases |
| Molybdenum | Mo | Nitrogenase Nitrate reductase Sulfite oxidase DMSO- TMAO |
| Nikel | Ni | Hydrogenase Ureases |
| Selenium | Visit | Dehydrogenase |
Table 1: Examples of metals present as cofactors in certain coenzymes, according to Lehninger, 1972.
Tungsten cofactors (W) and Vanadium (V) cofactors have been isolated from bacterial enzymes, but not yet from plants, where detection is more difficult.
Coenzymes like N.A.D. are organic complexes combining amine, sugar and phosphate groups. Many coenzymes are vitamins, such as vitamin B12, which contains cobalt, a soil-derived element.
Lack of knowledge of enzyme chains:
Although biochemists have isolated thousands of enzymes, we still don’t know all the enzymes involved in all biochemical cycles. Biochemists were initially interested in the enzymes involved in major biochemical cycles, such as the respiratory chain or Lynen’s helix.
We’re still a long way from knowing all the enzymes involved in aroma synthesis, but we do know that without these enzymes, no aroma can be synthesized by the vine. In fact, during the grape ripening process, we know that the fruit first contains non-volatile acids. These acids have been synthesized by enzymes. Then the grapes will contain some aromatic substances derived from the acid-alcohol combinations made by enzymes; these are called esters. Unlike other highly aromatic fruits (apple, melon, apricot), grapes contain little acid and alcohol, but are very rich in sugars. These are transformed into alcohol by yeast enzymes, which combine with the grape acids to create a bouquet that did not exist in the original grape. But all these substances have been synthesized by grape enzymes, then yeast enzymes.
And all these enzymes contain metal cofactors that come from the soil. Given the current state of knowledge, the hypothesis put forward by the LAMS team over the last 20 years remains valid. This hypothesis states that all the metals, nitrogen and phosphorus that go into the enzymes come from the soil, and that consequently all the aromas synthesized by the vine depend on the soil’s content of these metals, nitrogen or phosphorus. Until we have proof to the contrary, we can therefore hypothesize that the role of soil in the taste of wines stems from the fact that each soil has its own specific metal content.
Once we know, for example, which manganese enzymes are involved in the synthesis of which aromas, we’ll be able to explain why Morgon wine, which comes from vines growing on manganese-rich granite, “morgonne” as the winemakers put it. Of course, this hypothesis does not exclude the role of other terroir factors such as climate and topography.
Indeed, photosynthesis, which produces the sugars that give rise to all the molecules present in wines (aromas, acids, tannins, etc.), depends on these factors. The size and shape of the canopy are also fundamental. All these factors combine with soil and geology to give wines their Terroir flavour.
Graphics illustrating the article
Figure 1: Comparison of main element contents (mg/kg) between three vineyard plots from the same wine-growing region, cultivated and vinified by the same winemaker, with identical grape varieties, located in three different appellations and producing three very distinct wines.
Figure 2: Comparison of trace element levels (mg/kg) between three vineyard plots from the same wine-growing region, cultivated and vinified by the same winemaker, with identical grape varieties and located in three different appellations, producing three very distinct wines.
Conclusion:
The vine actively feeds on soil water, enabling it to absorb mono-atomic cations. These serve both to charge the root membrane positively and to release saline flavors when soils are rich in sodium or potassium. These ions enhance the mineral sensations of magnesium and calcium. This absorption of cations enables the vine to absorb the anions (oxides or chelates) synthesized by the microbes. All these anions: nitrate, sulfate, phosphate and metals serve as cofactors for the enzymes that synthesize the aromas.
Vines are even capable of conversing with soil microbes through root exudates. Root exudates stimulate the multiplication and activity of microbes that oxidize or chelate nutrients. When we create deficiencies in plants, they very quickly increase their exudates (Calkmak et al ,1988), and if we take these exudates and put them in a soil, we see the multiplication of microbes that chelate the element the plant is deficient in (Timonin, 1946).
These subtle mechanisms also help to explain the homogenization of wine tastes over the last 30 years. Excessive use of fertilizers and pesticides, as well as the use of overly heavy equipment and deep ripping, have led to two phenomena:
- A drop in the biological activity of soils, leading to lower levels of assimilable elements and trace elements in the soil.
- Root morphology is predominantly horizontal, with deep root mortality. This morphology leads to high vine vigor and the formation of low-quality grapasses, nourished by fertilizers rather than by the subsoil, which contains trace elements specific to Terroirs.
These two phenomena lead to a loss of complexity in the wines, and a loss of identity with their terroir, requiring more and more intervention in the cellar to maintain wine quality.
This is why the LAMS team has been insisting for 20 years on the importance of having a soil in good biological condition, from the surface to the depths, in order to have wines whose taste is conferred by their Terroir.