**4. Flavonol, anthocyanin, and catechin functions in plants**

Plants are sessile organisms, due to this fact they have developed different methods for pro‐ tection against the stressful conditions of the surrounding, including abiotic and biotic stim‐ uli. The most important mechanism is the production of secondary metabolites, like flavonols, anthocyanins, and catechins [36, 37].

There is an increase in the production of these compounds under adverse or stressing condi‐ tions, such as intense UV radiation, heat, drought, and salt stress, presence of heavy metals, herbivores, insects, nematodes, etc., because reactive oxygen species (ROS), the natural prod‐ ucts obtained from metabolic reactions, play a relevant role in cell signaling and homeostasis. In certain situations, as previously described, ROS levels can undergo a mild increase, trig‐ gering defensive responses as SAR or ISR [38], or suffer a dramatic increase that results in cell damage (lipids, DNA, and protein structures) if not controlled.

Living beings have different methods to get rid of these ROS, enzymatic and nonenzymatic; but if the amount is too high the organism cannot transform all of them, causing the damages described before. Phenylpropanoids and flavonoids, in which flavonols, anthocyanins, and catechins are enclosed, are the nonenzymatic antioxidants known to have high antioxidant activities, because of their capacity to directly quench ROS, thanks to the hydroxyl group present in rings A and B; they also interfere over the enzymatic systems composed of cyclo‐ oxygenase (in animals only), lipoxygenase, glutathione S-transferase, and xanthine oxidase, which is the other system in charge of ROS removal, together with the SOD-APX and the ascorbate-glutathione cycle enzymes that contribute to ROS control [39]. Hence, these com‐ pounds are involved in fine tuning of defensive and adaptive metabolisms, integrating all the external information, to optimize plant energetic resources for survival.

Flavonoids are known to be nonessential regulators auxin transport, modulating different transporters such as PIN proteins, and the transporter superfamily (ABCB) [40–42] proteins involved in their transport along the plant. It has been demonstrated that changes in flavonols accumulation lead to changes in auxin transport, therefore changes in auxin distribution [43], and the corresponding changes in plant physiology.

#### **4.1. Abiotic stress**

Abiotic stress is defined as the negative impact caused by the nonliving factors in the plant. Under adverse conditions, like intense UV radiation, heat, drought, and salt stress, presence of heavy metals, etc., there is a high increase of the reactive oxygen species (ROS) that lead to signal transduction to activate plant defense or to oxidative damage, as described above.

The UV radiation causes a stressful situation for plants [44], which is handled in two ways. First of all flavonoids and other pigments present mainly in the outer parts of the plant (epi‐ dermis and mesophyll tissues) absorb and considerably reduce the amount of radiation; the second one would consist in decreasing the effect of ROS caused by the radiation by scaveng‐ ing of ROS [7]. Among flavonols, the main compound related to light absorption is kaemp‐ ferol 3-O-glucoside because of its monohydroxy B-ring, and the flavonol with the greatest antioxidant properties is quercetin 3-O-glucoside, because of its dihydroxy B-ring. It has been shown that upon different UV exposure, synthesis of phenolic compounds is increased [45]. This may be the primary mechanism of response, which can be followed by others such as accumulation of pigments or lignification processes. Hence, flavonoids and anthocyanins are involved in protection against oxidative stress due to high UV radiation.

The impact of drought and salt stress on flavonoid biosynthesis has been studied in *A. thaliana* [46, 47]. An increase of glycosides of quercetin, cyanidin, and kaempferol during drought stress has been reported, being kaempferol glycosides the most significantly increased [47]. Although the behavior of flavonoids during these types of stresses is still not well docu‐ mented, this evidence their role against salt and drought stress.

These studies carried out in *A. thaliana* are very convenient to elucidate the mechanism of action of these flavonoids and to see the flavonoid profile. However they cannot be directly extrapolated to evaluate behavior in fruit production, fruit quality, or fruit endurance, as *A. thaliana* does not have edible fruits. For these purposes, other model plants are used, such as tomato or strawberry among the berries.

Concerning fruit quality, there is a great concern in the endurance of the fruit after harvest; it is one of the most important traits for commercial value and economic profit. The relationship between the overripening and the antioxidant properties has been evaluated [48]. A study on tomato overexpressing AtMYB12, the transcription factor activating the flavonol anthocy‐ anin pathway, showed a notable increase in flavonoid biosynthesis, as well as its antioxidant capacity. The high anthocyanin and high flavonol profiles resulted in a longer, more durable shelf life, comparing with control plants, indicating that the endurance is directly correlated with this profile. Based on this data, it seems that the overripening time is determined by the oxidative damage of the fruit under changing conditions [49]. Therefore, an increase in flavonoids and anthocyanins is related to better fruit quality during the postharvest period.

Another stress factor is the levels of heavy metals. As a consequence of industrial develop‐ ment, pollution with heavy metals has dramatically increased. Heavy metals toxicity can result from different mechanisms, the first one is the generation of ROS by Fenton reaction and autoxidation [7], blocking of essential functional groups in biomolecules, and displace‐ ment of essential metal ions from biomolecules. Cadmium and other metals provoke a deple‐ tion of GSH and inhibit mainly the glutathione reductase (among other enzymes implicated in the ROS cycle) [45]; in consequence, the plant has to increase dramatically other antioxi‐ dants such as flavonoids in order to keep a normal the normal homeostasis of the plant cells. Flavonoids are known to form specific union with heavy metals, providing a great adapta‐ tion method to heavy metals toxicity autoxidation [7]. Based on these characteristics, some applications derived from these studies have been proposed to improve survival of plants in hostile environments, for example, increasing flavonoid synthesis to allow plant growth in the presence of heavy metals, so soil detoxification can be achieved by phytoremediation [50].

#### **4.2. Biotic stress**

present in rings A and B; they also interfere over the enzymatic systems composed of cyclo‐ oxygenase (in animals only), lipoxygenase, glutathione S-transferase, and xanthine oxidase, which is the other system in charge of ROS removal, together with the SOD-APX and the ascorbate-glutathione cycle enzymes that contribute to ROS control [39]. Hence, these com‐ pounds are involved in fine tuning of defensive and adaptive metabolisms, integrating all the

Flavonoids are known to be nonessential regulators auxin transport, modulating different transporters such as PIN proteins, and the transporter superfamily (ABCB) [40–42] proteins involved in their transport along the plant. It has been demonstrated that changes in flavonols accumulation lead to changes in auxin transport, therefore changes in auxin distribution [43],

Abiotic stress is defined as the negative impact caused by the nonliving factors in the plant. Under adverse conditions, like intense UV radiation, heat, drought, and salt stress, presence of heavy metals, etc., there is a high increase of the reactive oxygen species (ROS) that lead to signal transduction to activate plant defense or to oxidative damage, as described above.

The UV radiation causes a stressful situation for plants [44], which is handled in two ways. First of all flavonoids and other pigments present mainly in the outer parts of the plant (epi‐ dermis and mesophyll tissues) absorb and considerably reduce the amount of radiation; the second one would consist in decreasing the effect of ROS caused by the radiation by scaveng‐ ing of ROS [7]. Among flavonols, the main compound related to light absorption is kaemp‐ ferol 3-O-glucoside because of its monohydroxy B-ring, and the flavonol with the greatest antioxidant properties is quercetin 3-O-glucoside, because of its dihydroxy B-ring. It has been shown that upon different UV exposure, synthesis of phenolic compounds is increased [45]. This may be the primary mechanism of response, which can be followed by others such as accumulation of pigments or lignification processes. Hence, flavonoids and anthocyanins are

The impact of drought and salt stress on flavonoid biosynthesis has been studied in *A. thaliana* [46, 47]. An increase of glycosides of quercetin, cyanidin, and kaempferol during drought stress has been reported, being kaempferol glycosides the most significantly increased [47]. Although the behavior of flavonoids during these types of stresses is still not well docu‐

These studies carried out in *A. thaliana* are very convenient to elucidate the mechanism of action of these flavonoids and to see the flavonoid profile. However they cannot be directly extrapolated to evaluate behavior in fruit production, fruit quality, or fruit endurance, as *A. thaliana* does not have edible fruits. For these purposes, other model plants are used,

Concerning fruit quality, there is a great concern in the endurance of the fruit after harvest; it is one of the most important traits for commercial value and economic profit. The relationship

external information, to optimize plant energetic resources for survival.

involved in protection against oxidative stress due to high UV radiation.

mented, this evidence their role against salt and drought stress.

such as tomato or strawberry among the berries.

and the corresponding changes in plant physiology.

**4.1. Abiotic stress**

138 Flavonoids - From Biosynthesis to Human Health

Flavonoids are important molecules for plant adaptation under adverse conditions, among which defense to biotic stress is included. These molecules have a nonspecific mechanism of action; their effect is partly derived from their antioxidant properties, because of the ROS generated by plants when they are attacked by some pathogen. Flavonoids are involved in the earliest defense mechanism and the programmed cell death, and they have been found in necrotic and adjacent cells to pathogen invasion in the hypersensitive response [46, 51].

Their role in defense is not limited to the hypersensitive response, since consistent with their ability to chelate metals, they are able to inhibit some pathogen enzymes, mainly those involved in digesting the cell wall by chelating metals, blocking, or retarding pathogen inva‐ sion [52]. Different studies have shown that there are different mechanisms of action against pathogen infection, inhibition of cellulases, pectinases, and xylanases, chelation of metal ions that belongs to cell membranes and enzymes, and more general detoxifying cells of ROS [45].

They can also affect bacterial DNA synthesis, by interacting with DNA gyrases, as the B ring of flavonoids can form hydrogen bonds with nucleic acid bases, or by direct interaction with the ATP binding site of the gyrase, leading to an inhibition of the synthesis of new DNA. This may be their method of protection against virus [45].

Antifungal properties have also been proved for flavonoids; these properties depend on their structure, for example dihydroquercetin has proved to be much more active against *Fusarium* sp. infections than other types of flavonols, and it is believed that is due to the hydroxyl groups [53]. In addition to the antifungal effects reported in plant, some have shown that certain compounds like phenols, phenolic acids, flavonoids, and isoflavonoids inhibit pathogen proliferation in the rizhosphere, preventing root infections.

#### **4.3. Other functions**

Flavonoids play a very important role in symbiotic bacteria relations. Bacteria belonging to the family Rhizobiaceae include several genera, each of them specific to a legume species. Rhizobiaceae are capable of fixing nitrogen for the plant; in exchange they obtain photosyn‐ tates. First, they need to establish the symbiotic relationship and form the nodule; in this pro‐ cess, flavonoids are key since these bacteria are attracted by these flavonoids that are specific signals for each rhizobia-legume couple. There are studies of different plants growing in soils with low nitrogen concentration that induces the accumulation of flavonoids [54]. Based on this fact, knowledge of the specific flavonoids that enhance symbiosis establishment could be applied to field production of legumes, in low productivity soils, to enhance nodulation, which in turn, will enhance yield in developing areas. This goal could be achieved at a low cost and easily implemented in local areas therefore contributing to food security, as marked by the FAO.

Connecting with this improvement in production and also with their natural physiological role, flavonoids provide color, taste, and fragrance to the fruit and seeds, and also play an important role in pollination, because these characteristics attract insects [45]. Although these characteristics may attract some organisms can also deter some others, in the cases of herbi‐ vores and some nematodes, avoiding to be eaten by these living beings.
