**2. Studies results**

#### **2.1 Studies place, objects, and methods**

A vegetative experiment was conducted with amaranth species *A. tricolor* L. cv. Valentina (vegetable, red-colored cultivar, with the leaves and seeds having nutritional value for human organisms) and *A. cruentus* L. cv. Krepysh (grain, greencolored cultivar with the seeds having nutritional value for human organisms) in 2020–2021 in the department of gene pool and plant bioresources of the Federal Horticultural Research Center for Breeding, Agrotechnology and Nursery (FHRCBAN), Moscow. The plants of both species were placed outdoors with artificial protection from precipitation. The climate of the study site is moderately continental, the height above sea level is 168 m, the coordinates are 55° 7 ́27 ̋north latitude, 37° 56 ́ 55 ̋. The amaranth plants were grown by seedlings and transplanted into plastic pots (250 and 175 mm in diameter and height, respectively) one plant per a pot. Totally 40 pots were planted, 20 pieces of each species: 10 control plants and 10 drought-affected ones.

The pots were filled with a mixture of peat and sand (5:1) with a drainage layer at the bottom. In the pots with the control samples, the humidity of the substrate for the plants was maintained at the level of 45–50%. Soil moisture was determined using soil moisture meter MC-7828 SOIL. All the plants were grown for 2 months in wellwatered conditions in natural light (**Figure 1**). The average day/night temperature,

*Metabolite Profile of* Amaranthus tricolor *L. and* Amaranthus cruentus *L.… DOI: http://dx.doi.org/10.5772/intechopen.102375*

#### **Figure 1.**

*General view of control plants* A. tricolor *L. cv. Valentina and* A. cruentus *L. cv. Krepysh and drought-prone plants.*

relative humidity, and the day length during the experimental period were 17.2°C/ 11.7°C, 64%, and 17 h, respectively. After 2 months of growth, the degree of stress from drought was determined according to the moisture content in the soil. The watering of the experimental plants was stopped until the signs of wilting. The duration of the soil drought period was 7 days. The plants were examined when the soil moisture dropped till 20–25%.

The biochemical research studies were held in the Laboratory of Physiology and Biochemistry of FHRCBAN.

The understudied parameters included the laboratory studies of the leaves (microscopy of cross sections of the leaf blade, photosynthetic pigments content, antioxidant activity, phenolic compounds sum, ash composition seeds, and quality content of the leaves' main metabolites). The leaves' microscopy and ash composition were determined on analytical REM JEOL JSM-6010 LA (JEOL Ltd., Japan). Photosynthetic pigments Chl a and b and total carotenoids (Car) were studied on spectrophotometer Helios Υ UV–vis (USA) in accordance with the method [12]. Total phenolic amount was determined with Folin–Ciocalteu reagent in accordance with the method [13] and tоtal antioxidant capacity, the scavenging activity for the 2, 2 dipheny l-1-picrylhydrazyl (DPPH) radical was determined in accordance with the method [14].

Metabolites quality composition contained in leaf extracts was analyzed on JEOL JMS-Q1050GC (JEOL Ltd., Japan) via the method of gas chromate-mass-spectrometry in accordance with the method [15].

### **2.2 Biomineralization of amaranth leaves**

An important morphological feature of *A. tricolor* L. cv. Valentina is biomineralization—the presence of multiple crystals in the leaf tissue. In the leaves of *A. cruentus* L. cv. Krepysh, the crystals were not found. The round spiked crystals (metabolic products) are often located on the transverse sections of the leaf of *A. tricolor* L. cv. Valentina between the adaxial and abaxial sides (**Figure 2**).

The local energy dispersive spectrometry (EDS) analysis showed that the inclusions contained four elements. The main element was Ca (5.9–8.3 mass %); K (0.34– 0.38 mass%); Mg and P—0.03–0.07 mass %. The combination of scanning electron microscopy (SEM) and EDS was a convenient method for determining the microstructure in the cross section of the leaves of *Amarantus* L. X-rays showing the location of all the elements are presented in **Figure 3**. K, P, and Mg are evenly distributed around the mineral inclusions. Ca is concentrated in the crystal. The SEM/ EDX results allowed determining the concentration and distribution of elements in the mineral inclusions in the leaves of *A. tricolor* L. cv. Valentina. Calcium oxalate (Ca) crystals are found in many plant species and in most organs and tissues of photosynthetic organisms [16–19]. The modern research studies say that Ca2+ is a key element of signaling pathways and is mobilized during the adaptation process to biotic and abiotic stresses [20–22].

Calcium is involved in regulating metabolic processes, plant growth and development [23]. Under drought stress, Ca is an integral part of the recovery process after stress exposure, regulating the plasma membrane enzyme adenosinetriphosphatase, which is required to pump back nutrients lost during cell damage [24].
