**2. Reactive Oxygen Species (ROS)**

**1. Introduction**

thrive in extreme habitats.

Species belonging to the Amaranthaceae compose a diverse and interesting family of plants. They can develop in highly contrasting habitats, from arid and semi-arid zones, where they can survive in sandy alkaline and/ or serpentine soils, to disturbed tropical forests. A minority are found in aquatic, semi-aquatic or marine environments. Their high affinity to saline conditions stems, in part, from to the weedy nature of most of the species that constitute the *Alternanthera*, *Amaranthus*, *Celosia, Chamissoa, Froelichia, Gomphrena, Guillemi‐ nea,* and *Iresine* genera. These plants are widely distributed in tropical and subtropical zones

The *Amaranthus* genus is highly diverse, including approximately 70 species. A fraction of these may grow in saline soils; in this regard, *A. greggii* is considered to be a marker of saliferous soils. This genus includes grain-producing species, the most important being *A. cruentus*, *A. caudatus,* and *A. hypochondriacus*. Grain amaranths are valued for the high protein content and multiple nutraceutic properties of their seeds [2,3]. Moreover, they possess an inherent tolerance to high temperatures and drought, traits which have been associated with their C4 physiology, indeterminate flowering habit and superior water use efficiency due to their ability to grow long tap roots and develop an extensive lateral root system [4–6]. In concordance with related species within the Amaranthaceae, grain amaranths can tolerate poor and saline soils conditions and erratic rains unsuitable for the cultivation of other grain crops. Besides, they are not particularly susceptible to major diseases or insect pests [5–8]. Their high tolerance to severe defoliation, produced by mechanical means [9,10] or by insect folivory [6,11], is believed to contribute to this trait. In addition, they can readily respond to chemical elicitors of defense responses, such as jasmonic acid (JA) [12-14] or benzothiadiazole (BTH) [15], to increase their resistance against highly damaging phloem-feeding insect pests, such as the tarnished bug *Lygus lineolaris*, or against potentially lethal pathogenic bacteria. However, the contribution of secondary metabolites to (a)biotic stress tolerance in grain amaranth has been barely explored.

In this chapter, we shall first review the role played by phenylpropanoid pathway in the amelioration of both biotic and abiotic stress in plants. Then, we shall proceed to de‐ scribe the state of the art with regard to the phenylpropanoid pathway in *Amaranthus*, mostly in relation to stress, but also in relation to the their role in the biosynthesis of compounds with proposed nutraceutic properties. Finally, a thorough description of the progress we have achieved so far with respect to the genomic characterization of this particular secondary metabolic pathway in grain amaranth, mostly in terms of stress tolerance and/ or resistance will be made. The information gathered as a result of these efforts will expand the knowledge, perhaps into unknown territory, about the chemical diversity in plants, particularly in grain amaranth and related species, many of which can

due to their ability to colonize or invade diverse habitats [1].

186 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

#### **2.1. Brief description of their ubiquitous and malignant role in plant stress and the antioxidant defense mechanisms induced for their control — What is known regarding amelioration of ROS-related damage in amaranth plants during stress?**

The abundance of reactive oxygen species (ROS) tends to increase in the tissues of plants exposed to diverse environmental challenges including contact with heavy metal contami‐ nants in soil, water or salinity stress, which is often accompanied by high light and tempera‐ ture, and nutrient deficiency [16]. Most stress conditions in plants cause an accumulation of ROS, such as superoxide ion, hydrogen peroxide, oxygen-containing radicals, and others. These chemical entities can produce extensive oxidative damage in the apoplastic compart‐ ment and may also harm cellular membranes by lipid peroxidation. Additionally, they can have an impact on ion homeostasis mechanisms, which are crucial for many stress tolerance mechanisms, by interfering with ion fluxes [17]. ROS detoxification frequently involves the combined action of both antioxidant metabolites such as ascorbate, glutathione, tocopherols, and ROS-detoxifying enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) [18,19]. In this sense, overproduction of antioxidants in response to drought-induced oxidative stress has been frequently found to be associated with the drought stress tolerance of different plant species [20,21]. Also, enhanced drought tolerance has been generated in several different transgenic plants transformed with genes encoding diverse types of antioxidant-related enzymes or metabolites (e.g., SOD, APX, monodehydroascorbate reductase, and tocopherol cyclase, a key enzyme of tocopherol biosynthesis, [22]). Regarding grain amaranth, a series of proteomic studies performed in plants exposed to drought and saline stress detected the accumulation of various antioxidant enzymes, similar to those mentioned above [23,24]. In addition, the participation of antioxidant genes, such as *AhCAT*, *AhAPX*, and *AhSOD*, was implied by findings showing their induced-up-regulation in grain amaranth plants primed to resist infection by pathogenic bacteria [15]. However, a further characterization of the additional six *CAT*, four *APX,* and three *SOD* genes identified in grain amaranth [12] remains to be performed.

Glycine betalaine (GB) is a quaternary ammonium compound that acts as an osmolite with protective functions in plants subjected to the osmotic stress normally produced under drought, high temperature and/ or excessive salinity conditions. GB accumulation in the cytoplasm reduces ion toxicity, and ameliorates the highly damaging effects caused by the usually simultaneous presence of dehydration, salinity, and extreme temperature stresses. The protective effect is proposed to be exerted by the stabilization of macromolecular structures, and/ or by the protection of chloroplasts, particularly the photosystem II complex. The latter is believed to involve the thermodynamic stabilization of the indirect interaction of extrinsic photosystem II complex proteins with membrane phosphatidylcholine moieties [25]. GB is synthesized by the two-step oxidation of choline. The first step is catalyzed by choline monooxygenase, followed by the action of betaine aldehyde dehydrogenase. Both genes have been described in different amaranth species [12,26,27], whereas the presence of GB has been reported in all Amaranthaceae species examined so far, with the exception of *Chenopodium quinoa* and *Noaea mucronata*. However, the GB concentration needed to exert a protective effect in plants of this family has been found to vary widely in a species-specific manner [25]. On the other hand, GB-accumulating transgenic plants usually show a significant improvement in their tolerance to different abiotic stress conditions [28].

#### **2.2.** *Betacyanins* **in amaranth: More than pigments?**

Betalains are water-soluble, nitrogen-containing pigments that are found only in one group of angiosperms, the Caryophyllales. For reasons that remain unresolved, they have never been found jointly with anthocyanins in the same plant [29,30]. This particular trait has been employed for chemo-taxonomical purposes. These pigments can be divided into the red-violet betacyanins and the yellow betaxanthins. Both are immonium conjugates of betalamic acid covalently bonded with cyclo-dihydroxyphenylalanine (cDOPA) glucosides, which can undergo further acylations [31–33]. These pigments are possibly needed for the optical attraction of pollinators and seed dispersers. Regarding stress, a protective role against accumulating ROS has been inferred from a number of studies [34–36] whereas the betacyanin amaranthine has also been proposed to exert protective effects against photo-oxidative damage in *A. tricolor* [36].

Recently, an analysis of key genes/ enzymes of the betacyanin biosynthetic pathway in *A. hypochondriacus* was performed. The study included genes coding for cyclo-DOPA glycosyltransferase (*AhcDOPA5-GT*), two 4, 5-DOPA-extradiol-dioxygenase isoforms (*AhDODA-1* and *AhDODA-2*, respectively), a betanidin 5-glycosyl transferase (*AhB5-GT*), and an ortholog of the cytochrome P-450 R gene (*CYP76AD1*). The expression pattern of these genes, together with DOPA oxidase tyrosinase assays, was determined in both green and red tissues. The results obtained suggested that two apparently independent glycosyla‐ tion pathways leading to betanins could be operating in *A. hypochondriacus* to synthesize amaranthine. In addition, these genes/ enzymes were shown to be induced differentially in a tissue-specific and genotype-specific manner in response to different stimuli, including water- and salt-stress and insect herbivory. The results obtained from the abiotic stress assays suggested that genes other than those examined in this study were probably contributing significantly to pigment content in tissues of stressed *A. hypochondriacus* plants. They also offered the possibility that these genes, due to their high induction by insect herbivory, particularly in acyanic plants, could function in defense responses against chewing insect pests by still undetermined mechanisms [37].

In addition to pigments, diverse phytochemical studies have shown that amaranth plants are capable of synthetizing a notable diversity of secondary metabolites [3,38,39]. Although many of these compounds are not considered to be essential for the primary needs of the plant, they are certainly required for survival in and/ or adaptation to challenging environmental conditions. Many of these compounds may be employed in amaranth and other plants as signaling compounds, in defense and/ or for communication with other organisms, such as pollinators [40–44].

The most commonly found secondary metabolite families found in amaranth and related species are phenylpropanoids, including flavonoids, phenolic acids, and their related amides, followed by alkaloids and terpenoids. From an anthropocentric perspective, some of these chemicals, including betacyanins, flavonoids polyphenols, and phenolic acids, are responsible for conferring amaranth and quinoa tissues with the bioactive antioxidant activity associated with their well-documented health benefit effects [3,38,45–49].
