Keywords

1 Introduction

Secondary metabolites in plants are defined as low molecular weight by-products of primary metabolism that are usually not directly involved in the primary growth of plants but play an immense role in crop protection or yield improvement by interacting with several biotic and abiotic factors. Secondary metabolites are produced from primary metabolites via various metabolic pathways under certain physiological conditions (Ahmed et al. 2017). According to Delgoda and Murray (2017), there are about 2,00,000 secondary metabolites of diverse chemical nature produced by different plants, though not all of them are biologically active. Their expression patterns are also differentially regulated by complex signaling pathways involving several enzymes and genes. Depending on their biological functions, secondary metabolites are grouped into several major classes. Plant growth regulators or phytohormones occupy an important group of secondary metabolites that can regulate growth and development and also aid in defense against a wide range of biotic and abiotic stresses (Erb and Kliebenstein 2020). Other major classes include nitrogen-containing compounds (alkaloids, cyanogenic glycosides and non-protein amino acids) terpenoids, phenolic compounds and sulphur-containing compounds (glucosinolates, defensin, phytoalexins, etc.) that directly or indirectly help in promoting crop improvement under several unfavourable environmental conditions. Diversity of plant secondary metabolites is also correlated with their multifaceted functional attributes, viz. protection against herbivores, pathogenic microorganisms and weeds, facilitating pollination and fertilization by attracting pollinators, establishing symbiotic association with mycorrhizal fungi and providing tolerance against environmental stress factors (Zhang et al. 2020a). Plant secondary metabolites also serve as excellent sources of pharmaceuticals, agrochemicals, food additives, flavouring agents, cosmetics and many other industrial products due to their effective antimicrobial, antioxidant, insecticidal and other beneficial properties (Tiwari and Rana 2015). The basic skeletons of all the secondary metabolites are synthesized via three major metabolic pathways, viz. shikhimic acid pathway, isoprenoid pathway or mevalonic acid pathway and the polyketide pathway, which are further modified by series of addition, deletion and substitution depending on the specific requirement (Teoh 2016). In general, plants produce secondary metabolites in very low concentrations, however, production increases under the adverse environmental condition that is attributed to increased stress tolerance. The complex interaction between several genes and transcription factors is known to be responsible for regulating the levels of secondary metabolite production in plants (Jan et al. 2021).

Crop production is influenced by a range of external and internal factors, viz. soil condition, temperature, light intensity, humidity, nutrient status, gene expression and regulation, signaling pathways, etc. Slight variation in any of these factors can cause significant alterations in the growth and yield of crops. Many advanced strategies have been adopted in the recent past to optimize crop yield throughout the year. Targeting secondary metabolite biosynthetic pathways is one such tool for yield improvement. Advancement in genetic engineering and biotechnological tools are being utilized to dissect the secondary metabolite biosynthetic pathways for decoding their specific functions in plants both in laboratory and field conditions. This review attempts to present an overview of major secondary metabolites in plants and their functions in plant growth and development. It also focuses on the advanced metabolic engineering techniques for their increased production and the problems for the implementation of these techniques in the agricultural sector.

2 Role of Secondary Metabolites in Plant Growth and Development

2.1 Phytohormones

Phytohormones play a significant role in plant growth and development (Table 1). They act as chemical messengers and target specific plant tissue to elicit certain physiological responses under several environmental conditions. For example, ethylene triggers the signaling pathway and interacts with other hormones for eliciting developmental responses. Ethylene is a gaseous hormone with a simple structure that regulates leaf development, flower development, fruit ripening, seed germination, etc. Dubois et al. (2018) showed that mutation in positive and negative regulators of the ethylene signaling pathway showed altered growth patterns. For example, the mutation in ethylene receptor proteins, viz. ETR1 and ERS1 showed decreased leaf growth, whereas overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) and ARGOS-LIKE (ARL) caused a negative feedback regulation of ethylene response and stimulated growth in Arabidopsis (Shi et al. 2015). Several reports are available depicting the complex interaction of ethylene with other phytohormones during growth and development. According to Iqbal et al. (2017), auxin stimulates endogenous ethylene biosynthesis and thereby regulates shoot apical meristem and leaf development in tomato and Arabidopsis. However, auxin-independent ethylene responses are also observed in common beans (Phaseolus vulgaris) (Keller et al. 2004). Ethylene also influences ABA sensitivity and acts synergistically with gibberellin during various stages of seed germination. Post-germination developmental changes, viz. apical hook formation, hypocotyl growth, root initiation, etc. are also found to be regulated by the combined interaction of phytohormones, viz. auxin, cytokinin, ethylene, jasmonic acid, salicylic acid and brassinosteroids via regulation of several genes and transcription factors like YUCCA1, YUCCA5, HLS1, HY5, MYC2, DELLA, etc. (Ahammed et al. 2020). Sami et al. (2019) reported the crosstalk between the phytohormones with glucose in modulating plant developmental responses via hexose-dependent and hexose-independent signaling pathways. ABA, BR, SA, auxin, ethylene and GA help to minimize the adverse effects of salinity stress by interacting with signaling molecules like NO, ROS and Ca+2 and stimulate plant growth by maintaining ion homeostasis, reducing oxidative damage, upregulation of genes for secondary metabolite biosynthesis, accumulation of osmoprotectants, increased biomass production under several abiotic stress condition (Amir et al. 2019). Interaction of NO with phytohormones like cytokinin, ethylene and auxin resulted in increased salinity tolerance in Lactuca sativa by regulating Na+ accumulation, antioxidant activities, mineral uptake and photosynthetic rate (Campos et al. 2019). Crosstalk between strigolactones and other phytohormone signaling pathways also led to plant growth promotion under normal and abiotic stress conditions by the formation of adventitious roots, regulation of shoot branching, inhibition of bud growth, promotion of secondary growth, leaf senescence, seed germination, internode elongation and nutrient uptake, etc. (Yang et al. 2019). Kumari and Parida (2018) showed that molecular crosstalk between ABA and JA brought about stomatal closure, reduced transpiration, regulation of major metabolic pathways, viz. glycolysis, pyruvate metabolism, TCA cycle, shikimic acid pathway, gluconeogenesis, alkaloid biosynthesis, etc. under saline condition. Exogenous application of gibberellic acid showed growth promotion in wheat cultivars by stimulating higher accumulation of proline along with the increased activity of Rubisco and antioxidant enzymes (peroxidase and superoxide dismutase) under saline conditions (Manjili et al. 2012).

Table 1 An overview of the role of plant secondary metabolites in plant growth and development
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2.2 Terpenoids

Terpenoids are the largest group of plant secondary metabolites and confer important roles in plant defense and crop vigour. Yu et al. (2018) reported the functional attribution of Terpenoid Indole Alkaloids (TIAs) during the process of photomorphogenesis in Catharanthus roseus. They observed the light stimulated activation of TIA biosynthetic enzymes and TIA accumulation (tabersonine, catharanthine, vindoline, vinblastine and vincristine) at the time of cotyledon opening. The combined interaction of auxin and cytokinin with terpenoid biosynthetic pathway in the morphogenesis and structural organization of chloroplasts in Artemisia alba has also been described by (Danova et al. 2018). Terpenoids were also known to improve the rate of plant reproduction and stimulate crop protection by increasing the rate of insect pollination, decreasing the spread of weeds and invader plants and reducing damages caused by pests and microbes in several agriculturally important crops, viz. apple, blueberry, tomato, etc. (Abbas et al. 2017). Terpenoid phytoalexins (zealexins and kauralexins) were known to induce root growth and biomass accumulation along with prevention of oxidative damage under various biotic and abiotic stress conditions (Akhi et al. 2021). Terpenes may also influence the expression of genes involved in plant defense mechanisms by acting as chemical messengers and regulating the adaptive features of plants under various biotic and abiotic stress (Zwenger and Basu 2008). For instance, 7-epizingiberene and R-curcumene were reported to increase insect resistance in tomatoes (Bleeker et al. 2011). β-caryophyllene could also induce resistance against microbial pathogens via jasmonic acid signaling, whereas isoprene and α- and β-pinene confer resistance via salicylic acid signaling in Arabidopsis thaliana (Frank et al. 2021). Wang et al. (2020) showed that sesquiterpenes secreted from glandular trichomes of wild tomato (Solanum habrochaites) could be responsible for conferring repellence against the potato aphid Macrosiphum euphorbiae. They highlighted the role of β-caryophyllene, α-humulene, α-santalene, α-bergamotene and β-bergamotene in affecting the survivorship and feeding behaviour of the aphid population. Capsidiol was known to be accumulated in Nicotiana attenuata in response to Alternaria alternata infection, and confer resistance against this fungal pathogen independent of jasmonic acid and ethylene signaling pathways (Song et al. 2019). Habash et al. (2020) evaluated the impact of a sesquiterpene, nootkatone against Heterodera schachtii parasitism on A. thaliana. They pointed out that nootkatone specifically decreased the number of nematodes and upregulated the defense-related genes involved in salicylic acid, jasmonic acid and ethylene biosynthetic pathways. The significance of terpenoids in plant growth and development is summarized in Table 1.

2.3 Phenolic Compounds

2.3.1 Coumarins

Coumarins are produced via phenylpropanoid pathway and involved in plant defense against pathogens, management of abiotic stresses and hormonal regulation. Coumarins along with reduced glutathione helped to ameliorate salinity stress and improve crop production in tomatoes by detoxifying ROS and methylglyoxal via enhancing the activities of glyoxalase enzymes (Table 1). Significant reduction in lipid peroxidation, membrane destabilization and Na+ toxicity was also observed (Parvin et al. 2020). Sultana et al. (2020) also observed that the exogenous application of coumarin effectively enhances vegetative growth and antioxidant enzyme activities (catalase, ascorbate peroxidase and guaiacol peroxidase) in sorghum seedlings on exposure to different concentrations of NaCl. Conversely, regulation of seed germination by coumarins was also observed in Brassica parachinensis via the reduction in ROS accumulation and decreased GA biosynthesis (Chen et al. 2021). Coumarin accumulation was also shown to confer increased resistance in A. thaliana against soft rot causing bacteria Dickeya spp. (Perkowska et al. 2021). Sarashgi et al. (2021) showed that coumarins present in root exudates of Brassicaceae plant species (Brassica napus, Raphanus sativus and Sinapis alba) play an important role in iron accumulation. Overexpression of genes involved in the coumarin biosynthetic pathway helped to mitigate the mycorrhizal incompatibility in A. thaliana. It was also experimentally proved that root secreted coumarins, especially scopoletin can improve pre-penetration signaling and established chemical communication between the host and the arbuscular mycorrhizal fungus Rhizophagus irregularis (Cosme et al. 2021). Coumarins were also known to promote lateral root growth in A. thaliana by inhibiting basipetal transport of auxin and altering microtubule cortical array organization (Bruno et al. 2021). Scopoletin exuded from roots could improve plant growth by establishing an association with rhizospheric microorganism Pseudomonas simiae and Arabidopsis thaliana (Stringlis et al. 2018). In vitro experiments with plant-derived coumarins showed improved soil characteristics and also stimulated the growth of beneficial soil microflora (Niro et al. 2016). Seed priming of Vicia faba with different concentrations of coumarin showed improved vegetative growth by the enhanced accumulation of primary and secondary metabolites, viz. carbohydrates, proteins, phytohormones, phenolics, etc. (Saleh et al. 2015). Abenavoli et al. (2004) showed that different concentrations of coumarin could affect root growth parameters in different types of maize seedlings in hydroponic cultures by regulating lateral root formation, root length and branching patterns. Coumarins also facilitate nitrate uptake from the soil, increased accumulation of nitrate in root cells and translocation of nitrates from root to shoot in durum wheat seedlings. The diameter of the xylem vessels of the root cells and the increased respiration rates were also found in response to coumarin treatment (Abenavoli et al. 2001).

2.3.2 Flavonoids

Flavonoids play a major role in plant growth in several adverse environmental conditions (Table 25.1). Liang and He (2018) experimentally demonstrated the protective role of nine anthocyanins, viz. pelargonidin 3-robinobioside, pelargonidin 3,5-di-(6- acetylglucoside), pelargonidin 3-(600-p-coumarylglucoside)-5-(6000-acetylglucoside), pelargonidin 3-(600-malonylglucoside)-7-(6000-caffeylglucoside), cyanidin 3-rutinoside, cyanidin 3-O-[b-DXylopyranosyl-(1->2)-[(4-hydroxybenzoyl)-(->6)-b-D-glucopyranosyl-(1->6)]-b-D-galactopyranoside], cyanidin 3-lathyroside, cyanidin 3-[6-(6-sinapylglucosyl)-2-xylosylgalactoside] and cyanidin 3,5-diglucoside (600,6000-malyl diester) during low nitrogen stress condition by enhancing seed germination rate in A. thaliana. Apigenin helped to mitigate the adverse effects of salinity stress by improving the activities of enzymatic (catalase and ascorbate peroxidase) and non-enzymatic antioxidants (carotenoids and flavonoids) and maintaining K+/Na+ homeostasis in rice seedlings (Mekawy et al. 2018). Li et al. (2019) observed that the MYB- stimulated enhanced synthesis and accumulation of flavonoids help to increase plant growth in A. thaliana under saline conditions. They elucidated that the overexpression of MYB transcription factor (MYB111) during salinity stress condition, in turn, activate the major enzymes of flavonoid biosynthesis, viz. chalcone synthase, flavanone carboxylase and flavanol synthase 1. Caliskan et al. (2017) have revealed the significance of phenolic compounds in salinity tolerance in Hypericum pruinatum. Their study showed increased accumulation of phenolics, viz. chlorogenic acid, rutin, hyperoside, isoquercetine, quercitrine and quercetine in salt-stressed plants. Similar enhancement in flavonoid biosynthesis was also observed in Camellia sinensis subjected to drought stress. The activation of enzymes, viz. chalcone synthesis1, cinnamate-4-hydroxylase, flavonoid 3ʹ5ʹ hydroxylase and flavanone-3-hydroxylase lead to increased accumulation of a good number of flavonoids (myricetin, quercetin and kaempferol) that successfully ameliorate the adverse effects of drought and promote plant growth by maintaining cellular antioxidative status (Sun et al. 2020). Increased expression of three key enzymes of phenolic metabolism, viz. Shikimate Dehydrogenase (SKDH), Cinnamyl Alcohol Dehydrogenase (CAD) and Polyphenol Oxidase (PPO) associated with enhanced accumulation of phenolics could alleviate the adverse effects of Zn and Cd toxicity in Kandelia obovata (Chen et al. 2019). Phenolic acids and flavonoids (quercetin, catechin, apigenin, o-coumaric acid, luteolin, etc.) showed enhanced scavenging of free radicals like superoxide and peroxide anions, that in turn provide better insect resistance and antibiosis in winter triticale (Czerniewicz et al. 2017). Anthocyanin accumulation in leaves of Euphorbia pulcherrima showed higher antioxidant activities associated with tolerance against photo-oxidative stress (Moustaka et al. 2020). Oleuropein was known to protect salt-stressed olive plants by accelerating biomass production via increased antioxidative defense and osmoregulation (Petridis et al. 2012). Munné-Bosch and Alegre (2003) showed that the synergistic action of carsonic acid and α-tocopherol prevent oxidative damages and promote the growth of rosemary and sage plants under drought stress. A similar accumulation of anthocyanins and flavonols was observed in A. thaliana, which might be responsible for increased antioxidant activities in response to drought (Nakabayashi et al. 2014). UV-B LED light pre-treatment in lettuce showed enhanced accumulation of flavonoids that in turn helped to confer resistance against downy mildew (McLay et al. 2020). Constitutive expression of a UDP-dependent glycosyltransferase (OsUGT706C2) stimulated flavonoid biosynthesis in rice, which in turn contributed to UV-B tolerance and crop improvement (Zhang et al. 2020b). Li et al. (2021) showed that increased flavonoid content helped to enhance antioxidant activity and crop yield in drought-affected maize plants. Overexpression of GSA1 that encodes a UDP-glucosyltransferase which helped to regulate flavonoid accumulation, grain size and abiotic stress tolerance in rice (Dong et al. 2020).

2.4 Nitrogen-Containing Compounds

2.4.1 Cyanogenic Glycosides

Cyanogenic glycosides are by-products of amino acid metabolism and can produce toxic hydrogen cyanides by enzymatic hydrolysis. Siegień et al. (2021) showed that a higher accumulation of two major cyanogenic glycosides, namely linamarin and lotaustralin could function as nitrogen reservoirs in flax leaves under low nitrogen conditions. According to the studies of Sohail et al. (2020), dhurrin stimulated the rapid growth of germinating seedlings of sorghum and helped to adapt to dry climatic conditions by serving as a nitrogen source. In another experiment, Myrans et al. (2021) also showed that cyanogenic glycosides play a divergent role in the allocation of nitrogen according to their availability in wild and cultivated sorghum species that eventually contribute to plant defense against environmental stresses and also improve crop growth. However, according to Cuny et al. (2019), two cyanogenic glycosides (limanarin and lotaustralin) played a significant role in defense against herbivores rather than in growth promotion in Phaseolus lunatus.

2.4.2 Non-protein Amino Acids and Derivatives

Non-protein amino acids are generally referred to the amino acids other than the proteinogenic amino acids. γ-aminobutyric acid (GABA) is a well-known amino acid that rapidly accumulates in plant tissues under stress and known to regulate plant growth and development. GABA has been known to alleviate oxidative damage and denaturation of chlorophyll molecules under high-temperature stress by stimulating activities of antioxidant enzymes (SOD, CAT, POX, APX and GR), overexpression of heat-shock proteins, accumulation of osmolytes and by lowering electrolyte leakage and lipid peroxidation in Agrostis stolonifera (Zeng et al. 2021). Similar heat tolerance was also conferred by exogenous application of GABA in wheat seedlings by regulation of amino acid metabolism and maintenance of redox homeostasis that in turn help to grow the plants under such adverse environmental conditions (Wang et al. 2021). GABA signaling could modulate stomatal movement in leaves of A. thaliana and suffice optimum water resilience under drought stress. Overexpression of Glutamate Decarboxylase 2 gene (GAD2) in turn was found to be responsible for increased biosynthesis of GABA via Ca+2/calmodulin signaling (Xu et al. 2021). Wu et al. (2020) also found that exogenous application of GABA into tomato plants helped to reduce Na+ uptake and accumulation in roots and leaves, prevent ROS formation and lipid peroxidation and also improve plant growth under NaCl stress. Sita and Kumar (2020) pointed out the role of GABA in the alleviation of multiple abiotic stress factors in leguminous plants by modulating carbohydrate and amino acid metabolism and maintaining antioxidative mechanisms and membrane stability. In this context, Priya et al. (2019) also elaborated the thermo-protective effect of GABA on Vigna radiata by enhancement of reproductive function under high-temperature stress. Post-harvest exogenous application of GABA could mitigate the toxic impacts of low-temperature storage by reducing the activities of lipooxygenases and phospholipases and accelerating antioxidative activities of SOD and CAT in cucumber fruits (Malekzadeh et al. 2017). Dopamine, an amino acid derivative, could act as a signal molecule in plant growth and development and protect against various abiotic stresses by regulating the expression of major stress-responsive genes involved in senescence, chlorophyll degradation, nitrate transport, etc. (Liu et al. 2020). Dopamine application could increase biomass production of apple seedlings by enhancing the rate of photosynthesis and chlorophyll content and decreasing the accumulation of ROS under alkali stress (Jiao et al. 2019). Exogenous application of β-Aminobutyric Acid (BABA) has been shown to improve Relative Water Content (RWC), photosynthetic efficiency and antioxidant activities in Vicia faba through the over-expression of stress-responsive genes, viz. VfGST, VfMYB, VfDHN, VfLEA, VfERF, VfNCED, VfWRKY, VfHSP and VfNAC under drought stress (Abid et al. 2020). Kim et al. (2013) also studied the effect of BABA on seedling growth of Kimchi cabbage by inducing resistance against Alternaria brassicicola and Colletotrichum higginsianum (Table 1).

2.5 Sulphur-Containing Compounds

2.5.1 Glucosinolates

Glucosinolates are a group of plant secondary metabolites with S-β-d-glucopyrano unit anomerically connected to an O-sulphated (Z)-thiohydroximate (Blažević et al. 2020). Glucosinolates are found to be indirectly involved in crop improvement by increasing resistance against ZnO nanoparticle toxicity in A. thaliana (Tao et al. 2021). Tao and co-workers also showed that treatment with ZnO nanoparticles promoted increased accumulation of glucosinolates that in turn helped to mitigate the adverse effect of the nanoparticles on root growth, chlorophyll content and plant biomass. Glucosinolates like isothiocyanates, glucobrassicin, sinigrin, glucoiberin, etc. were known to confer greater resistance against a wide range of plant pathogens (Rhizoctonia solani, Fusarium sp., Alternaria solani, Aspergillus flavus, etc.) and indirectly involved in crop protection and improvement (Poveda et al. 2020). Eom et al. (2018) conducted a transcriptome analysis study to elucidate the role of glucosinolate metabolism in Chinese cabbage (Brassica rapa ssp. pekinensis) in response to drought stress. Differential expression of several drought-responsive genes, viz. AP2/ERFs, bHLHs, NACs, bZIPs and particularly, BrbZIPs were found to be involved in improving plant tolerance by increased accumulation of glucosinolates in leaves and preventing water loss by inducing stomatal closure. In this connection, a marked increase in glucosinolate accumulation was also observed under the influence of drought and salinity stress in different Brassicaceae crops, viz. Brassica rapa, B. juncea, B. oleracea, B. napus, etc. (Essoh et al. 2020). It was also shown that the glucosinolates confer abiotic stress tolerance by the overexpression of MYB genes (especially MYB28 and MYB29), involved in increased aquaporin synthesis and osmoregulation (Essoh et al. 2020) (Table 1).

3 Strategies to Regulate Secondary Metabolite Production

Several strategies have been adopted to increase the production of secondary metabolites beneficial for crop growth and improvement (Fig. 1). A detailed account of these strategies is discussed in the following sub-sections and the major studies depicting the use of these elicitors in the improvement of crop plants are also enlisted in Table 2.

Fig. 1
figure 1

Commonly known elicitors that are being used for the engineering of secondary metabolites in plants for the improvement of crop production

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Table 2 Studies depicting the strategies to engineer secondary metabolite production in plants for crop improvement
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3.1 Chemical Elicitors

Exogenous application of plant growth regulators (salicylic acid and 24-epi-brassinolide) showed improved secondary metabolite production in Brassica nigra under salinity stress (Ghassemi-Golezani et al. 2020). Application of Gibberellic Acid (GA3) in hairy root cultures of Echinacea purpurea showed enhanced accumulation of caffeic acid derivatives and lignin (Abbasi et al. 2012). Indole Butyric Acid (IBA) and Benzyladenine (BA) also showed elevation in the accumulation of phenolics and flavonoids along with increased antimicrobial activity in Ocimum basilicum and Origanum vulgare (Karalija et al. 2016). 2,4-D and kinetin also showed increased essential oil synthesis in Cuminum cyminum (Farvardin et al. 2017). Exogenous application of melatonin has been shown to stimulate biomass production and accumulation of many Amaryllidaceae alkaloids (especially lycorine and galanthamine) in in vitro cell cultures of Leucojum aestivum (Ptak et al. 2019). Melatonin could also enhance phenolics and flavonoid content and antioxidant activity by upregulation of the STS gene and ethylene signaling in grape berries (Xu et al. 2017). Foliar application of vitamin B1 (thiamin) also stimulated secondary metabolite production, antioxidant activity and growth promotion in Brassica rapa, subjected to drought stress (Jabeen et al. 2021). Polysaccharides such as dextran, pectin and chitin also acted as elicitors for the biosynthesis of phenolics, flavonoids and napthodianthrones (hypericin and pseudohypericin) in Hypericum perforatum shoot cultures (Simic et al. 2014). Exogenous application of essential fatty acids (linoleic acid and α-linolenic acid) also helped to elicit the production of phenolics, flavonoids and ginsenoside along with increased biomass production and antioxidant activity in Panax ginseng, cultured in bioreactors (Wu et al. 2009). Immobilization of cells of Solanum chrysotrichum within the calcium-alginate gel matrix showed significant enhancement in the production of antimycotic saponin––spirostanol (Charlet et al. 2000). Salicylic acid, jasmonic acid and methyl jasmonate also act as regulators of secondary metabolite biosynthesis in Withania somnifera, Gymnema sylvestre, Panax ginseng, etc. (Chodisetti et al. 2015; Sivanandhan et al. 2013; Thanh et al. 2005). According to Gantait and Mukherjee (2021), inhibition of spindle fibre formation or induction of polyploidy can also be considered effective in manipulating secondary metabolite production in several plants. They showed that treatment with colchicine and oryzanol in different culture conditions can significantly increase biosynthesis of alkaloids (capsaicin, vincristine, etc.), terpenoids (andrographolide, α-gualene, limonene, etc.), glycosides (stevioside, rebaudioside, bacoside etc.), lactones (artimisinin, wedelolactone, etc.) total phenolics, flavonoids (quercetin, kaempferol, chlorogenic acid, etc.) in several polyploid plant species––Catharanthus roseus, Capsicum fruitescens, Aquilaria malaccensis, Sphagneticola calendulacea, Andrographis paniculata, Stevia rebaudiana, Bacopa monnieri, Eucommia sp., Citrus limon, etc.

3.2 Physical Elicitors

Induction of light stress by exposure with LED lights of different wavelengths (blue, red, blue-red 30%:70% and white; λ = 430 nm, 670 nm, and λЄ = 430–670 nm) showed significant enhancement in the biosynthesis of phenolic and flavonoid derivatives (chlorogenic acid, caffeic acid, salvianolic acid, apigenin p-coumarylrhamnoside, methyl rosmarinate, etc.) in in vitro shoot culture of Dracocephalum forrestii (Weremczuk-Jeżyna et al. 2021). UV-B radiation was also found to induce biosynthesis and accumulation of flavonoids, tannins, isoprenoids, glucosinolates in Glycyrrhiza uralensis, Tropaeolum majus, Ocimum basilicum and many others (Yavas et al. 2020). Gamma irradiation also showed similar enhancement in biosynthesis and accumulation of psoralen in Psoralea corylifolia, capsaicinoids in Capsicum annum, stevioside in Stevia rebaudiana, ginsenoside in Panax ginseng by activating the key enzymes of secondary metabolism, viz. phenylalanine ammonia-lyase, chalcone synthase, squalene synthase, etc. (Vardhan and Shukla 2017). Ye et al. (2004) elucidated the efficiency of pulse electric field for stimulation of secondary metabolite biosynthesis in Taxus chinensis. Regulation of photosynthetic photon flux density and root temperature was shown to stimulate the biosynthesis and accumulation of total flavonoids, chlorogenic acid, rutin, trans-2-decenal in addition to increased antioxidant activity and crop yield in coriander (Nguyen et al. 2019). Caser et al. (2019) reported the implication of controlled drought conditions could modulate terpenoid, phenolics and flavonoid biosynthesis in Salvia dolomitica. Narayani and Srivastava (2017) reported similar elicitation of secondary metabolite production by several abiotic (heat shock, osmotic stress, ultrasound, ozone exposure, etc.) and biotic factors (extract of algae, fungi, bacteria, microbe-derived chitosan, pectin, cyclodextrin, etc.) in in vitro cell culture.

3.3 Microparticles and Nanoparticles as Elicitors

Sustainable utilization of chitosan-coated microcapsules combined with calcium and copper ions and Trichoderma viridae on the enhanced production of secondary metabolites and significant increments in antioxidant activities was observed in Lactuca sativa (Jurić et al. 2020). Application of silver and gold nanoparticles in callus culture of Prunella vulgaris showed enhanced production of phenolics and flavonoid content along with increased antioxidant activity (Fazal et al. 2016). Seedling treatment of Tanacetum parthenium with zinc oxide nanoparticles showed improved crop yield, mineral absorption along with the biosynthesis of essential oil and sesquiterpene lactones (parthenolide) (Shahhoseini et al. 2020). Nano ZnO also helped to increase root growth coupled with increased phenolic content, antioxidant activity and increased accumulation of tropane alkaloids (hyoscyamine and scopolamine) by upregulation of hyoscyamine-6-beta-hydroxylase (h6h) gene in Hyoscyamus reticulatus (Asl et al. 2019). Application of different concentrations of ZnO nanoparticles and microparticles on the Echinacea purpurea callus extract showed enhanced accumulation of flavonoids, which could be correlated with its anticancer activity (Karimi et al. 2018). Biofabrication of silver nanoparticles with the leaf extract of Euphorbia helioscopia showed enhanced growth parameters and secondary metabolite production in Helianthus annuus, in different developmental stages (Batool et al. 2021). Polyvinyl Pyrrolidone (PVP) coated cobalt nanoparticles also stimulated alkaloid biosynthesis in cell suspension culture of Catharanthus roseus under oxidative stress conditions (Fouad and Hafez 2018).

3.4 Biotic Elicitors

Elicitation of secondary metabolite production with the help of microbial extract was found to be useful in Hypericum perforatum (Shakya et al. 2019). The addition of cell culture filtrates of Colletotrichum gloeosporioides, Aspergillus niger, Fusarium oxysporum, Saccharomyces cerevisiae, and Botrytis cinerea have shown increased accumulation of xanthones, flavonoids and phenolic substances in shoot and suspension culture of H. perforatum. Apart from the fungal elicitors, several bacterial species, viz. Agrobacterium tumefaciens, A. rhizogenes and Stenotrophomonas maltophilia also showed the enhanced synthesis of flavonol, flavanol, lignin and other phenolics in H. perforatum (Shakya et al. 2019). A Co-cultivation system using an endophytic fungus Colletotrichum gloeosporioides stimulated the biosynthesis of asiaticoside content in Centella asiatica (Gupta and Chaturvedi 2019). Many cyanobacterial genera, viz. Synechocystis, Synechococcus and Anabaena are successfully transformed with plant-derived genes to produce important secondary metabolites like- limonene, p-coumaric acid, caffeine, carotenoid, etc. (Xue and He 2015). Viral infection often acted as elicitors for secondary metabolite production in the host plants. For example, cucumber mosaic virus elicits the production of polyphenols and flavonoids in Passiflora edulis, Grapevine red blotch-associated virus elicits flavonoid and anthocyanin synthesis in Vitis vinifera, poppy mosaic virus elicits alkaloid production in Papaver somniferum, etc. (Mishra et al. 2020).

3.5 Biotechnological Approaches

3.5.1 Regulation at DNA Level

Epigenetic regulation (DNA methylation, histone modification, RNAi transcription) of selected metabolic pathways was also found to be significant for modulation of secondary metabolites production in the plant cell (Brzycki et al. 2021). Yang et al. (2018) have confirmed the role of a DNA methylation inhibitor (5-azacytosine) in increasing the expression of major genes involved in phenolic acid biosynthesis in Salvia miltiorrhiza. However, the opposite result was observed when a donor for DNA methylation, SAM (S-adenosyl methionine), was added. Similarly, treatment of suspension culture of Bambusa multiplex cells with two histone deacetylase inhibitors (SBHA and trichostatin A) showed enhanced biosynthesis of cryptic secondary metabolites (Nomura et al. 2021). Gene silencing approaches through RNA interference are also extensively used to enhance the production of plant secondary metabolites for crop improvement (Rajam 2020). The silencing of a novel gene (MSYABBY5) showed increased terpene biosynthesis in the peltate glandular trichome of Mentha spicata. On the contrary, heterologous expression of MSYABBY5 repressed secondary metabolite production in Ocimum basilicum and Nicotiana sylvestris (Wang et al. 2016). Attempts have also been made through gene silencing via RNA interference by topical application of dsRNA (Deguchi et al. 2020).

3.5.2 Transcriptional Regulation and Transgenesis

Transcriptional regulation of MYB and bHLH protein-encoding genes (C1 and R), AP2/ERF-domain transcription factor ORCA3, DREB2A and DREB2B proteins are also being successfully implemented in Catharanthus roseus, Arabidopsis thaliana and several other plants for engineering biosynthesis of anthocyanins, flavonoids, terpenoid indole alkaloids, etc. (Memelink et al. 2001). Overexpression of genes of secoiridoid pathway (geraniol synthase and geranyl diphosphate synthase) stimulated the accumulation of commercially important monoterpene indole alkaloids (vindoline and catharanthin) in transgenic C. roseus (Kumar et al. 2018). Similar overexpression of ornithine decarboxylase (ODC) gene showed increased biosynthesis of tropane alkaloids, viz. putrescine, N-methyl putrescine, hyoscyamine and anisodamine in Atropa belladonna hairy root cultures. Transcriptional and post-transcriptional regulation of several transcription factors, viz. WRKY, MYB, bHLH, APETALA2/Ethylene Responsive-Factor (AP2/ERF), Jasmonate-responsive ERF (JRE), Basic Leucine Zipper (bZIP), SQUAMOSA Promoter-binding protein-Like (SPL), etc. could help modulate the biosynthesis of several classes of terpenoids and flavonoids—monoterpenes, sesquiterpenes, diterpenes, triterpenes, steroidal lactones, etc. in several crop species (Nagegowda and Gupta 2020). Similarly, homologous and heterologous overexpression of class I TGA transcription factor from Tripterygium wilfordii stimulated the biosynthesis of sesquiterpene and pyridine alkaloids (Han et al. 2020). Agrobacterium-mediated transgenesis of cannabinoid-synthesizing genes was also successfully applied for increased metabolite production in hemp (Cannabis sativa) in tissue culture. Manipulation of the shikonin biosynthetic pathway with the introduction of the 4-hydroxybenzoate-3-polyprenyltransferase (ubiA) gene from E. coli had successfully enhanced shikonin production in Lithospermum erythrorhizon (Boehm et al. 2000). Activation of cryptic gene clusters by co-cultivation or epigenetic modification of several endophytes (Fusarium mairei, Trichoderma atroviridae, Enterophosphospora sp., etc.) also helped in large-scale production of high-value plant secondary metabolites like taxol, berberine, camptothecin, vincamine (Venugopalan and Srivastava 2015).

According to Chandra and Chandra (2011), the formation of hairy root lines by the transformation of T-DNA from Agrobacterium rhizogenes could offer a useful strategy for the increased production of secondary metabolites. For instance, incorporation of the chalcone isomerase (chi) gene from Saussurea medusa into the genome of Saussurea involucrata showed increased production of naringenin, apigenin and total flavonoid content. Moreover, binary vectors formed by combining the T-DNA of the Ri-plasmid and putrescine N-methyltransferase (pmt) gene showed enhanced production of scopolamine and hyoscyamine by upregulation of tropane alkaloid biosynthetic pathway in Datura metel and Hyoscyamus muticus (Moyano et al. 2003). Transformation of Rubia cordifolia hairy root cultures with rol genes boosted the plant cells for increased production of secondary metabolites (Bulgakov et al. 2010). Transgenic A. belladonna also showed enhanced biosynthesis of hyoscyamine and anisodamine due to overexpression of the ODC gene (Zhao et al. 2020). Transgenic mimicry of pathogen attack could also lead to increased secondary metabolite production in several crop species, suggesting a suitable method for eliciting defense responses in plants. A. rhizogenes mediated transfer of fungal β-cryptogein gene (crypt) resulted in improved growth and enhanced accumulation of calystegine, polyphenols, withaferin, tylophorin, etc. in Convolvulus sepium, Withania somnifera, A. thaliana and Tylophora tanakae (Chaudhuri et al. 2009). Genetically modified potato cultivars constructed using Ribosome-Inactivating Protein-coding gene (RIP) from maize, showed enhanced synthesis and accumulation of sesquiterpenes and glycoalkaloids under a range of biotic and abiotic stress conditions (Matthews et al. 2005). Adventitious root culture using bioreactors is considered useful for large-scale production of plant secondary metabolites, viz. ginsenoside, resveratrol, camptothecin, vindoline, etc. (Kumar 2015). Catalytic synthesis of glycoconjugate involving the multigene superfamily of glycosyltransferases can also be employed in the production and modification of plant secondary metabolites. For instance, reduced expression of Tobacco Glycosyltransferase (TOGT) in transgenic tobacco plants showed decreased glycosylation of scopoletin that impaired resistance against TMV. Whereas overexpression of the TOGT1 gene led to increased resistance against Potato Virus Y by enhancing scopoletin synthesis (Wang and Hou 2009). Transgenic overexpression of 1-deoxy-D-Xylulose-5-phosphate Synthase (DXS) gene from Pelargonium spp. showed enhanced production of secondary metabolites in both homologous (essential oil in Pelargonium spp.) and heterologous conditions (withanolide in W. somnifera) (Jadaun et al. 2017). Heterologous expression of cinnamate 4-hydroxylase gene from Ipomoea batatas (IbC4H) showed enhanced drought tolerance associated with increased polyphenol biosynthesis and antioxidative activities in transgenic tobacco (Wang et al. 2017). Liu et al. (2019) showed that overexpression of a jasmonate ZIM-domain gene from a moss Pohlia nutans (PnJAZ1) in A. thaliana regulated ABA signaling pathways and induced seed germination and seedling growth under salinity stress. Overexpression of an anthocyanin-producing gene (production of anthocyanin pigment 1, PAP1) showed enhanced accumulation of anthocyanin by regulating the enzymes of flavonoid biosynthetic pathways in Nicotiana tabacum and A. thaliana (Gantet and Memelink 2002). Ma et al. (2019) reported the efficient application of a fungal host Yarrowia lipolytica for the heterologous synthesis of plant terpenoids by the introduction of terpene synthase and modifying the mevalonate pathway. Nascimento and Fett-Neto (2010) pointed out the effective transformation of entire plant-derived metabolic pathway genes from one species to another. For instance, complete pathway transfer of cyanogenic glycoside dhurrin from Sorghum bicolor to Arabidopsis thaliana was carried out successfully for increased herbicide resistance (Tattersall et al. 2001). Zhang et al. (2021) reported that overexpression of a UV-B responsive UDP-sugar glycosyltransferase gene from Carex rigescens (CrUGT87A1) showed increased salinity tolerance in A. thaliana by stimulating flavonoid biosynthesis and antioxidative activity. Bleeker et al. (2012) successfully transfer the biosynthetic pathway to produce a sesquiterpene (7-epizingiberene) from the wild tomato into a greenhouse cultivated variety for enhanced herbivore resistance.

3.5.3 Molecular Engineering Techniques

Several reports are depicting the significant contributions of molecular engineering approaches in the modulation of secondary metabolite biosynthesis for crop improvement. Sabzehzari et al. (2020) have demonstrated the significance of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-mediated transcriptional regulation of secondary metabolite production in several crop plants by silencing the enzymes of biosynthetic pathways. In this context, genome editing of 4′OMT2 (3′-hydroxyl-N-methylcoclaurine 4′-O-methyltransferase) gene by CRISPR/Cas9 system helped in mass production of bioactive benzylisoquinoline alkaloids (papaverine, codeine, thebaine, laudanosine, noscapine, s-reticuline and morphine) in Papaver somniferum by converting them into biofactories (Alagoz et al. 2016). Genome editing of SmRAS (rosmarinic acid synthase) gene through CRISPR/Cas9 helped in regulating the biosynthetic pathway in Salvia miltiorhiza (Zhou et al. 2018). Genetic manipulation of Morphogenic Regulator (MR) gene during somatic embryogenesis was found to be helpful in increased production of cannabinoids in vitro. Targeting Induced Local Lesions In Genomes (TILLING) mutation technique of cation/H+ exchangers transporters showed improved salinity tolerance and crop growth in Brassica rapa by influencing phytohormone signaling and ion homeostasis (Navarro-León et al. 2020). Stable Isotope Labelling by Amino acids in Cell culture (SILAC) approach was successfully employed for enhancing the secondary metabolism pathways in plants (Martínez-Esteso et al. 2015). Multiple Reaction Monitoring (MRM) would also be an innovative approach in targeting the key enzymes and transporter proteins of plant secondary metabolic pathways for enhanced production and crop improvement (Martínez-Esteso et al. 2015). Isolation of plant extracellular vesicles and nanovesicles from different plant sources have also been proved to be useful for plant bioprocess engineering of secondary metabolite production (Woith et al. 2021). Wany et al. (2014) mentioned the significance of functional genomic approaches for enhancing secondary metabolite production via antisense- or sense gene suppression of metabolic pathways, production of novel compounds, regulation by compartmentalization in many plants (A. thaliana, Ocimum americanum, etc.). Jain et al. (2013) mentioned that cell suspension culture in bioreactors could be helpful for large-scale bioproduction of saponins from Bacopa monnieri. Biotechnological approaches were applied to identify and characterize the genes responsible for climate resilience (SiATG8a, SiASR4, SiMYB56, EcbZIP17, EcGBF3, PgNAC21, PgeIF4A, etc.) in millets and were successfully introduced into several crop plants (tobacco, rice, Arabidopsis, etc.) for improved stress tolerance and crop performance (Singh et al. 2021). Ferrari (2010) summarized the importance of the data pool retrieved from transcriptomics, proteomics and metabolomics for developing novel techniques of secondary metabolite production. Careful dissection of the signaling network could offer a detailed understanding of elicitor induction for the modulation of metabolic pathways.

4 Constraints of Plant Secondary Metabolite Production and Future Prospects

A large number of external and internal factors regulate the biosynthesis of secondary metabolites in plants. Verma and Shukla (2015) have classified the factors into four broad categories—genetic, ontogenic, morphogenetic and environmental factors. The complex interplay between these factors, in turn, affects plant secondary metabolism in several different manners. Metabolic engineering of secondary metabolite biosynthetic pathways has faced some challenges regarding the desired concentration, bioavailability, absorption criteria and bioactivity of the compounds specifically used as food products (Davies and Espley 2013). In this connection, Nascimento and Fett-Neto (2010) pointed out some major drawbacks of developing novel engineering strategies for plant secondary metabolite production. These include—lack of availability of sustainable plant sources, constraints regarding transformation and regeneration, proper evaluation of synthesis and accumulation in different developmental and environmental conditions, problems in proper identification of metabolic intermediates, difficulties in separating the actual plant metabolic reactions from that of the endophytes or plant–endophyte interaction and the lack of knowledge regarding the intracellular and intercellular transport mechanism of secondary metabolites in the plant. Plant tissue and organ culture have been successfully employed for large-scale production of medicinally important plant secondary metabolites for many years. However, the culture conditions, media requirements, cultivation techniques showed great variation in yield and quality of the final products (Isah et al. 2018). Large-scale production of secondary metabolites is still not obtained by using cyanobacterial biofactories due to the absence of specific proteins and transcription factors required for post-translational modifications of some enzymes involved in plant secondary metabolism (Xue and He 2015). Tiago et al. (2017) pointed out several limitations regarding the production of secondary metabolites in plants, viz. complex interaction of soil and environmental factors on the biosynthesis of secondary metabolites, pleiotropy of genes encoding the biosynthesis of secondary metabolites, multiple regulations of biosynthetic genes, obtaining superior genotypes, toxicity symptoms, etc. According to Brzycki et al. (2021), a combination of traditional approaches such as metabolic engineering and cellular engineering techniques with modern targeted epigenetic engineering could overcome the production deficit of secondary metabolites in plants. Recently, the extensive use of mathematical modelling approaches like Response Surface Method (RSM), Artificial Neural Network (ANN), Kriging and the ANN-RSM combined approach in plant biotechnology helped to maximize the yield of secondary metabolites by selecting high-performance cell lines, optimizing the culture conditions and improving cell permeability (Amdoun et al. 2021). Decoding the mechanism of induction of plant secondary metabolite production in response to environmental stresses and increased resistance against herbivores would help to optimize the targeted gene manipulation for achieving enhanced crop yield (Kessler and Kalske 2018).

5 Conclusion

Secondary metabolites play a significant role in plant growth and affect crop production in various ways. Biosynthesis and the function of plant secondary metabolites are tightly regulated by several genes and transcription factors that are involved in complex crosstalks between them. Biotic and abiotic elicitors stimulate the biosynthesis of secondary metabolites by different molecular mechanisms. Overexpression of these transcription factors by differential expression of these genes, modification at the transcriptional and translational level, induction and maintenance of polyploidy, production of new transgenic crops, use of nanoparticles, etc. are considered effective engineering approaches in increasing secondary metabolite production for crop improvement. Although there are some concerns regarding the sensitivity and specificity of these techniques, several reports are available citing successful implementations of genetic engineering approaches towards crop improvement. Future scope lies in a detailed understanding of the signaling cascade of the secondary metabolites and their specific molecular interaction with the other components responsible for plant growth and development.