**1. Introduction**

Comprehensively, biotic and abiotic stresses negatively affect crop production and cause a marked decrease in annual crop yield, i.e., qualitative and quantitative [1,2]. Recently, biologists, especially agriculturists, need to find an alternative way to deal with biotic and abiotic stresses such as herbivores, insects, and pathogens, as well as salinity,

**Citation:** Salam, U.; Ullah, S.; Tang, Z.-H.; Elateeq, A.A.; Khan, Y.; Khan, J.; Khan, A.; Ali, S. Plant Metabolomics: An Overview of the Role of Primary and Secondary Metabolites against Different Environmental Stress Factors. *Life* **2023**, *13*, 706. https://doi.org/ 10.3390/life13030706

Academic Editors: Othmane Merah, Kousuke Hanada and Balazs Barna

Received: 8 December 2022 Revised: 2 January 2023 Accepted: 28 February 2023 Published: 6 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

trace metals (TM) contamination, drought, and extreme temperatures [3,4] respectively. All these stresses affect the physiological and morphological aspects, such as the hindering of the functional groups of important molecules, e.g., enzymes, polynucleotides, transport systems for substantial ions and nutrients, as well as the growth and metabolic activities of plants [5,6]. However, to cope with these stresses, plants adopt several mechanisms, including metabolomics, transcriptomics, proteomics, and genomics, individually or in combination. The plant metabolome consists of the following two kinds of metabolites: primary and secondary metabolites. Primary metabolites are essential for the proper growth and development of plants and microorganisms. On the contrary, secondary metabolites are formed near the stationary phase of growth and have no direct role in growth, reproduction, and development. The metabolic profiling of primary and secondary metabolites provides extensive knowledge of biochemical processes that occurs in plant metabolism [7].

Modern research endorsed the purpose of several important genes, metabolites, proteins, and molecular systems that induced plant reactions to drought, salt stress, cold, TM, heat, and certain other biotic and abiotic factors [8,9]. Metabolomics analyses have become an influential tool to monitor plants' responses to different environmentally stressed conditions [10]. Therefore, the findings of such studies give an understanding of the working of plants in definite circumstances, which are considered an important part of enlightening the molecular processes in responses to various stress conditions [11]. An appropriate data analysis, detection, identification, and evaluation of these metabolites are possible with the help of advanced metabolic tools such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) nuclear magnetic resonance (NMR) [12].

Furthermore, it is estimated that biotic and abiotic stresses are responsible for more than 50% of crop losses in the world [13,14]. The findings of Bayer in 2008 demonstrated that crop losses caused by abiotic stressors were significantly higher than by biotic factors [15]. However, the exact loss of crop yield depends on the plant's developmental stage and the intensity and duration by which various stresses occur [16]. Among other stresses, salinity affects more than 800 million hectares of land—nearly 50% of the total irrigated area, which provides about 33% of the world's food [17,18]. In the same way, drought also causes a loss of more than 50% of the average yield of crops [19]. Subsequently, other studies indicated that abiotic factors, such as temperature (low or high), salinity, and drought, significantly decreased plant production if existing alone or in combination [20]. Interestingly, another concern is the aggregation of reactive oxygen species (ROS), which is produced by excessively stressed accumulators of cadmium (Cd), chromium (Cr), lead (Pb), zinc (Zn), and copper (Cu) that can cause oxidation and dysfunction of biological molecules, hence disturbing certain physical and biological processes in plants [3,21]. Optimizing metabolic flux by the organellar electron transport chain (ETC) is essential in reducing oxidative stress [3]. Consequently, keeping the redox state of a cell is another essential issue that provides the decreasing power necessary for the foraging of ROS [22].

Therefore, there is a need for novel, easy, inexpensive, ecologically friendly, and robust crop types that can be conceived by cross-breeding or genetic engineering [23]. For example, recently, different wheat, rice, barley, maize, and other economically crucial varieties of crop plants have been considered very necessary than model plants [24,25]. However, the development of some modern 'omics tools, such as genomics, proteomics, transcriptomics, and metabolomics, has rationalized the research of crop plants and abetted the complete study of acquaintances concerning biological components and plant breeding [26]. In this concern, metabolomics gives the possibility to accelerate the selection of superior breeding stock and the screening of elite crop types [27]. Primary and secondary metabolites, with their functional diversity, play an important role in fine-tuning the environmental stress tolerance and productivity in crops. Understanding plant behavior under multiple environmental stressors is one of the ways to deal with agricultural sustainability [20]. In this piece of work, more than 200 published works were considered to provide an overview of the role of primary and secondary metabolites against several abiotic and biotic stressors.

#### **2. Instrumentation Applied in Metabolomics Studies**

The identification of different classes of metabolites in plants is largely based on using hyphenated mass spectrometric methods to chromatographic equipment and electrophoretic approaches [28]. Choosing an appropriate ionization technique and analyzer type for metabolite analysis is important in a mass spectrometer [29]. Through the study of mass spectrometry (MS), ionized molecules are calculated. Similarly, mass-to-charge ratio values (*m*/*z*, m-mass, or z-charge) of the produced ions are assessed with the precision of one mass unit and to the fourth decimal point, small or high-resolution mass spectra, after elimination in the MS analyzer. The use of a high-resolution mass analyzer permits the accomplishment of the elemental composition of the identified ions existing in mass spectra. At first, it is probable to estimate the elemental composition and molecular mass of the molecules from enumerated *m*/*z* values for protonated [M + H]+ and deprotonated molecules [M − H]−. The clear documentation of compounds is highly dependent on the applied MS system. MS machines designed with electrospray ionization (ESI) and matrixassisted laser desorption or ionization (MALDI) source could be utilized. The ionization of MALDI can be joined to one or two unified times of flight analyzer (TOF and TOF/TOF). The source of ESI works well through quadrupole (Q), ion trap (IT), time of flight (TOF) analyzer, and a mixture of them. The maximum resolution in the mass analyzer could be attained by ion cyclotron resonance through Fourier Transformation Instruments (FT ICR MS) when the ESI is employed as an ionization system.

Moreover, designing the experiment according to the Metabolomics Standard Initiative (MSI) is also crucial, which endorses defined measures for the right biological materials preparation, procedures of metabolite extraction, and analytical protocols [30]. Following the regulations that have been stipulated, a sufficient number of sample replications and the conditions under which plant development should occur to be investigated and defined [31]. Similarly, the control of MS parameters in mass spectra registration is necessary. Such data deliver environments for suitable documentation and quantification of metabolites and consistent statistical quantification [32].

After employing this method, different statistical calculations could be performed to determine the metabolites' capacities that allow the defining changes of a specific compound in definite situations [33]. The number of primary and secondary metabolites in a single organism may range from several hundred to tens of thousands, with little variation across orders of magnitude in concentration. Some strategies developed for metabolites analysis include metabolic profiling, metabolic fingerprinting, and target analysis [34].

Metabolic profiling is expected a simultaneous measure of a set of metabolites in a sample. Several analytical techniques can be used for metabolic profiling, such as (GC-MS), (LC-MS), and (NMR). To date, GC-MS is the most advanced analytical approach to metabolic profiling in plants [35]. Using GC-MS, it is possible to recognize several hundred compounds belonging to various classes, including sugar, organic acids, amino acids, alcohols, amines, and fatty acids. Similarly, LC-MS provides a better alternative for nonvolatile compounds. The importance of LC-MS is increasing in metabolomics, especially after the adoption of ultra-performance liquid chromatography technology that can increase separation efficiency and decrease analysis time [36]. Substantially, NMR spectroscopy offers an entirely different analytical technique compared to MS-based approaches. The sensitivity of the NMR technique is much lower than MS-based techniques; however, the structural content information, reproducibility, and computable aspect could be superior to them [37]. Moreover, the preparation of the sample is simple, more convenient, and non-destructive measurement may possible. These properties of NMR make it an ideal tool for the identification of metabolites through metabolic profiling [38].

#### **3. Workflow of Plant Metabolomics Analysis**

The metabolomics of plants is very complex and varied in their chemical structure. Extensive identifications and a wide range of metabolic depictions could be attained with the arrangement of two or more metabolomics approaches and analytical methods, with the

difference in extraction protocols [39]. Metabolomics analyses comprise the following three key tentative methods: (1) sample preparation, (2) data gaining, and (3) the identification of compounds by using the statistical analysis of the data. The preparation of the sample is a key step because it can contribute to the identification of a wide range of metabolites, which is comprised of tissue collecting, drying, or quenching, and metabolite extraction for analysis (derivatization) [40]. Thus, care should be taken in this step to avoid engaging in undesirable variation that can significantly disturb the analysis results. Many methods of enzyme quenching, such as drying, enzyme inhibitors and acids, and high meditations of organic solvents, could also distress the analysis and identification [41].

Plant metabolites are structurally different with high complications, such as dissimilar size, solubility, explosive nature, separation, amount, and stability [42,43]. The extraction method of metabolites relies on varied factors such as the type of plant organs, physical and chemical properties of the targeted metabolites, chemical structure, and the solvent used [44]. Generally, metabolite extraction methods include solvent extraction, supercritical fluid extraction, solid-phase extraction, and sonication [45]. Moreover, other methods are used to extract the essential oils, such as hydrodistillation, vapor-hydrodistillation, vapordistillation, hydro diffusion, organic solvent extraction, and cold pressing [46]. Though, it is critically essential to evaluate metabolite extraction methodologies for a precise metabolite extraction study because a solvent composition that is good for one chemical class may not be suitable for another chemical class. Moreover, this could not be appropriate for extracting large numbers of metabolites from a specific tissue. So, it is important to understand and monitor the effects of the applied solvent treatment on the sample's metabolic content and profile obtained [47].

The measurement of complex metabolites needs an advanced analytical platform for sample analysis. Every platform's range has a particular constraint, maybe in selectivity or sensitivity [48]. The selection of the analytical platform relies on the study initiated, the group of compounds, and their physiochemical properties, such as polarity, solubility, volatility, and concentration levels [49]. Additionally, one issue is that metabolites occur in a wide dynamic range of concentrations such as nanomolar and millimolar in the plant body. Subsequently, another problem is that not every metabolite is present in each tissue [50].

However, the most applied metabolomics approaches in analytical studies are liquid or gas chromatography synchronized with mass spectrometry (LC/GC-MS) and nuclear magnetic resonance spectrometry (NMR) [51]. Subsequently, another report [52] demonstrated an integrated technique that combines metabolites extraction and analysis with proteomic and RNA from a single sample that permits the immediate inquiry of all molecular levels and examines their interrelation and co-variance structure [53]. Consequently, biochemical regulation could result in the co-variance design of molecular dynamics in a cellular system [54]. In the context of metabolomics, the block diagram (Figure 1) of a typical experiment shows the following key steps:


**Figure 1.** Respective illustration of the processes involved in plant metabolomics analysis of GC–MS, LC-MS, CE-MS, and NMR-based chromatography.

#### **4. Metabolomics for Plant Stress Responses**

Metabolomics is the scientific study of the set of metabolites present within an organism, plant cell, or tissue [55]. However, plant stress is any amendment in the growth and developmental conditions that distracts metabolic homeostasis and needs to modify the metabolic pathways in a process generally designated as acclimatization [35]. Over the last decade, metabolomics has developed promptly and is recognized as the prevailing technology in changing climatic conditions and assessing or elucidating testing phenotypes in assorted living systems [56]. Substantially, it may contribute to studying stress biology in plants or other organisms by recognizing various molecules, such as by-products of stress metabolism and compounds of stress signal transduction and related to the plant acclimation responses [52]. Their application has been driven in several fields, including medicinal, imitation biology, or analytical molding of plants, animals, and microscopic organisms [57].

Additionally, to the applicability of other fields, nowadays metabolomics could also be used on a large scale in the assortment procedure of plants and resistant to the varying environmental states. Different findings revealed that drought stress, salinity, extreme temperature, and soil flooding could cause significant instabilities in the pattern of plant metabolome [22]. Metabolomics signifies the ultimate omic's level in a living system or reveals modifications in the traits of an organism or function. Different findings show the study of metabolomics under several environmental abiotic stresses, such as temperature [58], salinity and drought [59], and soil flooding [60]. In the same way, various metals and metalloids including, sulfur [61], phosphorus [62], oxidative stress [63], TM [64], and the combination of other several stress factors [65] in plants (Table 1). Various environmental factors that could negatively disturb the homeostasis and growth of plants are shown below (Figure 2).

**Figure 2.** Environmental stresses of biotic and abiotic factors affecting the growth and homeostasis of plants.

#### *4.1. The Response of Primary Metabolites to Abiotic Stresses*

Plants established several adaptive mechanisms to endure abiotic factors, containing variations of metabolism in various directions, to confirm their existence in combative environmental situations [66] (Table 2). Several plant metabolites could assist and reduce the effect of the harsh stress of salt, drought, and water by acting as osmolytes and osmoprotectants [67]. Examples of such metabolites include dimethylsulfoniopropionate (DMSP) and glycine betaine; sugars, such as sucrose, trehalose, and fructan; amino acids, such as proline and ectoine, as well as some metabolites of polyols, sorbitol, and mannitol [68,69]. In plants, a wide range of waxy layers known as epicuticular wax keeps water balance during water shortage and acts as a mechanical stoppage to encounter disease-causing agents. Additionally, ascorbic acids, glutamine, alpha-tocopherol, anthocyanins, and carotene shield plant tissues by foraging the intermediates of bustling oxygen produced during oxidative stress [70]. Similarly, several other smaller compounds guard plants against oxidation damage related to various constrictions [65].

Besides, the plant's defense system is related to generating phytoalexins, stimulating the common phenylpropanoid pathway and producing lignin biosynthesis [71]. Further, phytochemicals and hormones such as salicylic acid and methyl salicylate, methyl jasmonate and jasmonic acid, as well as other small molecules formed due to stress, play a significant role against environmental stresses [72–74]. All of these may also function as signaling compounds by stimulating the resistance system and reactions of acclimation [75]. Among the defense systems of plants, osmotic regulation is one of the broadly pronounced responses to the water shortage that needs the accretion of harmonious solutes, such as sugars, amino acids, polyols, and glycine betaine [76]. These chemical compounds do a significant job in sustaining cell turgor and stabilizing cell membranes and protein. Moreover, other studies designate the importance of these compounds in rehabilitating redox stability through the scavenging of ROS, which could adversely affect cellular structures and metabolism [68,77].


**Table 1.** List of species, various metabolomics approaches, and applications cited in this review under diverse abiotic stresses.

#### 4.1.1. Amino Acids

Amino acids are considered a precursor for protein and other organic molecules, e.g., nucleic acids, which designate an active part in the responses of a plant under several stress factors. Amino acids could also play a significant role in signaling and controlling molecules [92]. Various studies showed that many amino acids stored in plants are apparent to different abiotic stresses [93,94]. Moreover, the exposure of plants to such stresses appearance an accumulation of proline and other amino acids. In plants, the role played by stored amino acids differs after acting as an osmolyte to adjust ions passage, reducing stomatal opening and reclamation of TM [95]. Moreover, amino acids can also disturb the synthesis and activity of several enzymes, gene expression, and redox state of homeostasis [96]. The accumulation of proline and ectoine is considered the most extensively dispersed osmolytes, as they act as osmoprotectants to protect plants from harmful effects and exciting environmental stresses, including low and high temperature, salinity, UVr, water, and osmotic stresses [68,97].

Primarily, proline is produced from a glutamate and proline metabolizing enzyme, pyrroline-5-carboxylate synthetase (P5CS), which reduces glutamate to pyrroline-5-carboxylate (P5C). At last, from the reduction of P5C, this stress-responsive amino acid forms by pyrroline-5-carboxylate reductases (P5CR) [98]. In transgenic plants, the significant role of proline was established during osmotic stress. For example, overexpression of the *P5CS* gene in soybean increased proline content and, thus, tolerance to salt stress in transgenic plants [99]. Besides osmolytes, proline is thought to accomplish many other important functions related to plant resistance, e.g., ROS scavenging, redox balancing, cytosolic pH buffer, molecular chaperon, and a stabilizer of protein structure [98]. Subsequently, in response to abiotic factors, the enlarged levels of proline were observed for several years to be the stress-responsive feature in plants. The relationship between the accumulation of proline as osmolytes and stress tolerance had a great share because of its applicability to different crops [100,101].

Remarkably, some of the metabolites were related to drought resistance and drought vulnerability of the considered hybrids [102]. Additionally, studies on drought responses at metabolomics levels indicated that Andean potatoes with a phenotype designating greater stress exposure have more proline related to the genetically assembled plant that was a higher dearth-tolerant [103]. It was established that the cultivar with a sensitive phenotype has high-level certain amino acids, containing proline and Gamma-aminobutyric acid (GABA) when barley exposed to salinity stress [104]. It may well advocate a greater liability of these plants to such stress. According to [96], this accretion could be associated with the deterioration of the leaf and slowing the development of a more subtle genotype. Furthermore, studies on Arabidopsis revealed that proline could be a lethal compound under heat stress [105], while Charlton et al. found that water deficiency was the cause of the decrease in isoleucine concentration in *Pea* and *Arabidopsis* plants [106].
