*Utilization of Secondary Metabolites in Cotton Production DOI: http://dx.doi.org/10.5772/intechopen.114098*



**Table 1.**

*List various flavonoids reported in cotton.*

#### *2.1.2 Tannins*

Tannins are polyphenolic substances that have molecular weights ranging from 500 to 3000 [47]. They can cause proteins to precipitate. Tannins possess numerous phenolic hydroxy groups that enable them to establish multiple hydrogen bonds with proteins and other large molecules with -NH-, -NH2, or -OH groups [47]. This interaction results in the formation of complexes that can dissociate under normal pH conditions and are resistant to enzymatic degradation. The term "tannins" initially emerged from the leather industry, referring to substances capable of converting animal hides into leather [48]. Tannins can be categorized into two groups based on their biological origin. Hydrolyzable tannins are complex polyphenols that can be broken down through hydrolysis to yield gallic or ellagic acid and a sugar unit, typically glucose [47].

On the other hand, nonhydrolyzable or condensed tannins are formed by the polymerization of hydroxy flavans, resulting in the formation of dimers, trimers, or oligomers. Certain condensed tannins can become insoluble during extraction due to strong interactions with proteins, nucleic acids, or polysaccharides, leading to precipitate formation. Condensed tannins are also procyanidins since they produce anthocyanidins upon acid heating [48].

Together with phenolic acids [49, 50], gossypol [51–54], flavonoids [27], and tannins [47] are also listed in the groups of compounds that have been investigated and utilized in host resistance-breeding programs in cotton.

Condensed tannins have been documented as host plant resistance mechanisms for cotton bollworm (*Helicoverpa zea* Boddie) and tobacco budworm (*Heliothis virescens* F.). As a cotton resistance mechanism, condensed tannins were also found to antagonize the effects of the B.t. toxin [55]. The resistance imparted by condensed tannins deterred feeding by the larvae, which ultimately reduced the amount of B.t. toxin ingested by the insect; as a result, larvae feeding on high-tannin, B.t.-toxin-containing diets had lower mortality rates than insects feeding on diets containing condensed tannins or B.t. toxin alone. Condensed tannins could have also directly reduced the availability and activity of the B.t. toxin protein since tannins can precipitate proteins. Hence, the report proposed that insect management strategies utilizing B.t. toxin, whether as a topical insecticide or in genetically modified cotton crops, may not be suitable when combined with high-tannin plants.

Therefore, the report suggests that insect control tactics employing the B.t. toxin, either as a topically applied microbial insecticide or in transgenic cotton plants, may not be compatible with plants with high tannin concentrations.

Ref. [25] reported that condensed tannins in cotton leaves were up to 20% of dry weight. Their concentrations in individual leaves increased successively until about the tenth leaf. The upper leaves maintained the concentrations until early fall. The tannins resisted spider mites and could be hydrolyzed into digests consisting of one cyanidin and four delphinidin.

Tannins contribute to disease resistance. Resistant young leaves of cotton contain condensed tannins in higher concentration than do older susceptible leaves [56].

#### *2.1.3 Lignins*

Besides the above bioactive secondary metabolites, some structural metabolites such as lignins and cellulosic also belong to secondary metabolites [57], constituting a significant part of the biomass. For example, the cotton plant stalk comprises about 68% holocellulose and 26% lignin. These two classes of secondary metabolites also combine to be called lignocellulosic [1]. Lignins have protective functions, such as preventing lodging, etc.

#### *2.1.4 Terpenoids*

Terpenoids, or isoprenoids, are the largest group of plant secondary metabolites derived from the five-carbon compound isoprene, and its derivatives called terpenes. Terpenoids contain additional functional groups, usually oxygen, such as terpene aldehyde. Compared to flavonoids, cotton terpenoids do not have so many compounds, but the naked eye can see their existence as pigment glands. Cotton plants produce a variety of monoterpene and sesquiterpenoids. Monoterpenes include α-pinene, β -pinene, limonene, β-ocimene, β-myrcene, α-terpenene, γ-terpenene, etc. They are volatile oils, often as semiochemicals, attracting or deterring insects or parasitoids [34, 58]. Cotton plants produce both volatile and nonvolatile sesquiterpenoids. The former includes α-humulene, α-copaene, β-carophyllene, β-carophyllene oxide, α-aromadendrene, α-selinene, β-selinene, γ-bisabolene, β-bisabolol, δ-cadinene, 12-hydroxy-β-caryophyllene, gossonorol, spathulenol, 12-hydroxy-β-caryophyllene acetate, 12-hydroxy-β-caryophyllene oxide acetate, and guaia-1(10), 11-diene, etc. The latter includes hemigossypol, hemigossypolone, desoxyhemigossypol, gossypol, and heliocides H1, H2, H3, and H4 [59], as well as 6-methoxy and 6, 6<sup>0</sup> -dimethoxy gossypol derivatives [60]. They are nonvolatile. Almost all sesquiterpenoids and part of monoterpenes (the remaining are released to the air) are stored in the pigment glands, which appear oval and spherical and as black, orange, yellow-brown (yellowish-brown), green (red-brown), or purple depending on the species in all the tissues of cotton plants except pollen and seed coat [61]. Some monoterpenes (β-ocimene and myrcene) and sesquiterpenoids (hemigossypolone) form heliocides (C-25) H1, H2, H3, and H4) by direls alder reaction in the glands. Different genotypes impacted the distribution of the pigment glands [62, 63]. **Figure 2** shows the structures of some common terpenoids in cotton plants.

Gossypol is a dimeric sesquiterpenoid stored in the pigment glands of cotton plants. Gossypol was first extracted from cotton seeds by Marchlewski in 1899 and characterized by Adams et al. in 1938 [64, 65]. It constitutes 20–40% of the pigment gland weight and accounts for 0.4–1.7% of the whole cotton seed kernel. From the gossypol history, gossypol was first discovered from cotton seeds (cotyledons) because the toxicity of gossypol to humans and animals blocked the efficient use of cottonseed oil and protein. However, [66] found the importance of cotton roots in

#### **Figure 2.**

gossypol production in cotton plants. Recent progress [61] showed that cotyledons and roots are primary sources of gossypol in cotton plants. These two plant parts contain higher gossypol than other parts. The former is responsible to provide gossypol during germination, after which the developing roots become the primary source for gossypol production in cotton plants.

The defensive role of cotton pigment glands and gossypol was first recognized by Cook [67] in 1906. It turned out that gossypol and other terpenoids in the cotton pigment glands are associated closely with resistance mechanism to insects and pathogens [68, 69], including monoterpenes, sesquiterpenoids, and heliocides.

Despite that the pigment glands are distributed almost throughout the whole plant, the gland content compositions are different from different parts. The glands of cottonseeds contain mostly gossypol with traces of deoxyhemigossypol, and the glands of cotton leaves contain hemigossypolone, a gossypol derivative, and heliocides [70]. On fresh weight basis, cotton seed (cotyledons) usually has similar gossypol content of around 1% in roots at the harvesting stage (Yue, unpublished data); on dry weight basis, cotton root usually has the highest gossypol content [66] among different plant parts, which is consistent with the recent progresses that cotyledon (seeds) and roots are the source of gossypol for the whole plants [61, 71].

## *Utilization of Secondary Metabolites in Cotton Production DOI: http://dx.doi.org/10.5772/intechopen.114098*


**Table 2.** *Phenolic acids in cotton.*

#### *2.1.5 Phenolic acids*

Phenolic acids in cotton include benzoic acid, cinnamic acid, and their derivatives (**Table 2**). They are water-soluble. They include vanillic acid, benzoic acid, ferulic acid, sinapic acid, cinnamic acid, syringic acid, p-coumaric acid, gentisic acid, caffeic acid, chlorogenic acid, gentisic acid, p-hydroxybenzoic acid, p-hydroxybenzoic acid, 3, 4-dihydroxybenzoic (protocatechic) acid, etc. Their functions in cotton plants were thought to be related to resistance mechanism to pests and pathogens [50]. They also played a role in cotton allelopathy [74].

#### *2.1.6 Fatty acids*

Cotton fatty acids are mainly in the seeds. Cottonseed oil has a fatty acid profile that is composed of 52.89% linoleic acid, 25.39% palmitic acid, 16.35% oleic acids, together with small amounts of 2.33% stearic acid, 1% myristic acid, and 0.6% palmitoleic acid, as well as 0.17% linolenic acid [75]. Ref. [76] reported malvalic, sterculic, and dihydrosterculic acids in the hypocotyl of both glanded and glandless cotton seeds. Malvalic and sterculic acids represent 0.3% and 0.5% of the total fatty acids in the analyzed varieties.

## *2.1.7 Miscellaneous secondary compounds*

Beyond these main classes of secondary metabolites, the following secondary compounds were also identified in the cotton plant (**Table 3**).


**Table 3.**

*Additional secondary compounds identified in the cotton plant.*

#### **2.2 Secondary metabolite compositions of different cotton species**

Compositions of secondary metabolites vary among cotton species. From 1-weekold roots of *Gossypium hirsutum* and *G. barbadense*, the disesquiterpenoid aldehydes, gossypol, 6-methoxygossypol, and 6, 6'-dimethoxygossypol, and the sesquiterpenoid aldehydes, hemigossypol and methoxyhemigossypol were isolated and identified. While the compounds 6-methoxygossypol and 6, 6<sup>0</sup> -dimethoxygossypol constituted 30% of the total terpenoid aldehydes in the seeds of the cultivar of *G. barbadense* but occurred only in trace quantities in those of *G. hirsutum* [60].

As mentioned above, the cotton genus contains around 50 species, and four are domesticated independently [2, 78]. These different cotton species have other secondary metabolite profiles, which are representative of biodiversity. For example, myrcene is a major monoterpene presented in *G. hirsutum* but not present in *G. barbadense*. In contrast, *G. barbadense* contains copaene, β-carophyllene oxide, and (�)-δ-cadinene at 14.3, 8.0, and 7.8% in leaf oil compared to only 0.7, 0.4, and 0.3 respectively in oils from *G. hirsutum*. The variations between the two species could potentially impact insect-host relationships [79].

Different biotypes in the same species also have different secondary metabolite compositions. Ref. [80] found high levels of intraspecific diversity in the terpene profiles of the plants. Two distinct chemotypes were identified: one chemotype contained higher levels of the monoterpenes γ-terpinene, limonene, α-thujene, αterpinene, terpinolene, and p-cymene, while the other chemotype was distinguished by higher levels of α- and β-pinene. The distribution of chemotypes followed a geographic gradient from west to east with an increasing frequency of the former chemotype. Such differences in secondary metabolites are the foundation for a cotton breeding program for a high level of one or several secondary metabolites.

#### **2.3 Inductive vs. constitutive secondary metabolites**

A secondary metabolite can be constitutive or inducive. A constitutive secondary metabolite means the plant produces the secondary metabolite as a routine without environmental influence. An inductive secondary metabolite is produced or increased under one or more environmental factors, such as pest infestation or abiotic stresses. Most secondary metabolites are both constitutive and inductive. For example, cotton flavonoids and gossypol are constitutive as they are routinely produced, and they are inductive as their production increases when there are abiotic stresses, such as salinity stress [81]. In addition, increased terpenoid accumulation in cotton foliage was observed as a general wound response upon mechanical damage, *Spodoptera littoralis* caterpillar infestation, and jasmonic acid treatment [82] generally increased secondary metabolites production with E-β-ocimene, heliocide H1 and H4, showing the highest increases. In response to aphid attack, cotton plants synthesized and released callose outside the cell plasma membrane [83].

#### **2.4 Cotton secondary metabolites and abiotic and biotic stresses**

#### *2.4.1 Abiotic stresses*

Cotton production can be affected by various abiotic stresses, which include drought, salinity, nutrient deficiency, chemical burns, and ultraviolet radiation stress. They all turned out to have interactions with secondary metabolites. Abiotic stresses can cause secondary metabolite content and pest resistance change, influencing fiber and seed quality.

Ref. [84] reported total phenol and tannin content under salinity stress increased significantly after third water stress exposure. Evidence also showed that secondary metabolite increase resulted from abiotic stresses, increased pest resistance, and led to higher fiber quality. Such cotton traits can be applied in cotton production directly.

Nutrient deficiency is another kind of abiotic stress, and Ref. [85] showed that K deficiency reduced the antioxidant capacity of cotton seedlings and resulted in a metabolic disorder characterized by elevated levels of primary metabolites and reduced production of secondary metabolites. The results were from an analysis of xylem sap. How much degree the xylem sap represents the defense response of cotton plants to K deficiency is of concern. We compared root gossypol concentrations of cotton seedlings from irrigated under tap water and Hoagland solution for three weeks after emergence. The root gossypol concentrations of tap water irrigated seedlings (subjected to nutrient deficiency) were higher than the Hoagland solution irrigated seedling (Yue, unpublished data).

Cotton plants suffer from ultraviolet radiation damage, but many cotton secondary metabolites can protect against ultraviolet radiation. This partly originates from the fluorescence properties of some secondary metabolites that can convert ultraviolet radiation to lower-energy light. It partly results from the solid antioxidative properties of cotton secondary metabolites [86].

#### *2.4.2 Biotic stresses*

Biotic stresses in cotton production include insects and other herbivores, pathogens, weeds, etc. As discussed in 2.3, the secondary metabolite production was a defense response to the insects and salinity stresses; pathogen inoculation/ infection also induced secondary metabolite biosynthesis. Ref. [28] used dimethoxybenzaldehyde (DMB) histochemical reagent to reveal induced biosynthesis of flavanols (catechin, gallocatechin, and their condensed proanthocyanidins) as red color in the cotton stem section upon *Verticillium dahliae* inoculation. The *Verticillium* wilt-resistant cultivar showed much more intense flavanol synthesis than the susceptible cultivar.

Among the different stresses for cotton plant growth, development, and reproduction such as insects, pathogens, and abiotic stresses (drought, salinity, and nutrient deficiency, etc.), only weed infestation has not been reported to induce plant defense or immune response or change of secondary metabolites concentrations, which is the basis for potential utilization. Phenolic acids have been explored for their weed-suppressive effects in cotton [74]. Flavonoids, however, have not been examined. Cotton has a rich array of flavonoids, some of which, such as quercetin, have been reported to be allelopathic [87].

#### **2.5 Glandless cotton**

Since McMichael bred a glandless cotton cultivar in 1959 [88], which was devoid of toxic gossypol, the utilization of cotton secondary metabolites has another aspect: reduction of secondary metabolites so that cotton seeds can be less toxic and more valuable. Recently, Ref. [89] reported the use of RNAi to selectively silence the δcadinene synthase gene to reduce gossypol levels in cotton seed by 97% while not affecting levels of gossypol and other terpenoids in the rest of the plants.
