Progesterone and Steroids In/On Plants

Abstract

Plants and animals contain many steroid compounds that act as signaling molecules during complicated growth and development processes. Mammal sex hormones (MSHs), such as progesterone, estrogen, and testosterone, are another class of steroids. These hormones play an important role in regulating the mammals’ growth and reproduction processes as well as organic and inorganic metabolism. Steroid sex hormones, such as progesterone, beta-estradiol, and testosterone, support plant life processes including callus expansion, cytokinesis, root and shoot enlargement, and pollination in plants and have appropriate effects on handling abiotic stresses. An interesting impact of MSH is its capability in improving plant resistance to various abiotic stresses. MSH treatment extensively can reduce the adverse effects of environmental stress by promoting the activity of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT), and improving proline production.

1. Introduction

Mammalian sex hormones, including estrogens, androgens, and progesterone, are classified as steroids due to their tetracyclic triterpanes (sterane) structure. The diversity of steroids within animals and plants is dictated by the arrangement and kinds of functional groups linked to the sterane. In mammals, these steroid sex hormones play a crucial role in regulating development, reproduction, as well as mineral and protein metabolism [1]. The discovery of estrogen in plants dates back to 1926 when Dohrn et al. [2] first identified it. However, these early findings were considered preliminary due to the imperfect detection methods available at the time. Subsequent to these discoveries, a plethora of studies analyzing human and animal sex hormones in plants were published between the initial findings and the 1980s.

Certain analytical techniques, such as the Kober color reaction, have been subject to critical assessment. For instance, Van Rompuy and Zeevaart, in 1979, highlighted the need for more sensitive investigative methods for detecting estrogen-like substances in plants. The continuous evolution of detection methods has been crucial in advancing our understanding of the presence and role of sex hormones in plant biology [1]. Extensive research on the presence of mammalian steroids in plants (128 species from over 50 families) was conducted through radioimmunoassay in 1989 [3]. Steroids such as androsterone, progesterone, testosterone, dihydrotestosterone, estrone, and 17β-estradiol have been found in a significant number of plant species. In fact, over 80% of the studied species contained androsterone and progesterone, while 70% contained androgens, and 50% contained estrogens. The levels of steroids can vary significantly throughout plant growth and are influenced by species, cultivar, and plant organ [1]. During the latter part of the twentieth century, scientists worldwide actively sought to confirm the existence of animal steroid hormones in plants. Indeed, all animal steroid hormones or their analogs were discovered in plants. Moreover, the external application of animal hormones was shown to influence growth, development, and sexual characteristics in plants [4, 5]. These findings regarding the growth-regulating impact of animal steroid hormones in plants spurred the exploration of naturally occurring steroidal compounds that regulate growth. These investigations ultimately led to the identification of brassinosteroids, the sixth category of plant hormones found universally in plants.

2. Brassinosteroids: a novel phytohormone

Brassinosteroids (BRs) are a class of growth-promoting steroidal phytohormones. BRs are a distinct group of plant polyhydroxysteroids that bear a striking resemblance to cholesterol-derived animal steroid hormones. These compounds are found throughout the plant kingdom and have the ability to induce significant physiological changes in various plant species when applied externally. Surprisingly, despite their discovery in rapeseed pollen, BRs did not gain recognition as crucial plant hormones for more than 25 years. The delayed acknowledgment of the importance of BRs highlights the complexity of plant hormone research and the intricacies of understanding plant physiology. As scientists continue to delve deeper into the role of BRs in plant growth and development, it becomes increasingly clear that these compounds play a significant role in regulating various physiological processes [6].

Brassinosteroids (BRs), a class of steroid plant hormones, play a crucial role in regulating various developmental processes in plants, such as root and shoot growth, vascular differentiation, fertility, flowering, seed germination, and response to environmental stresses. Over the past 40 years, research has extensively delved into the BR biosynthetic pathways using forward- and reverse genetics approaches. These pathways have shed light on the structure of free BRs, which typically consist of 27, 28, or 29 carbons. These carbon structures bear resemblance to sterols, as they share common alkyl substituents. BRs are derived from sterols with similar side chains. The structural differences among various BRs, specifically C27-BRs, C28-BRs, and C29-BRs, are indicative of their distinct origins from different sterols such as cholesterol, campesterol, 24-epicampesterol, 24-ethylenecholesterol, and sitosterol. Furthermore, variations in substituents at specific carbon positions further differentiate BRs derived from these sterols. For instance, the classification of C27-BRs without a substituent at C-24 as derivatives of cholesterol, and C28-BRs with specific substituents as originating from campesterol, 24-epicampesterol, or 24-ethylenecholesterol, along with C29-BRs with an α-ethyl group being derived from sitosterol, underscores the intricate biochemical pathways involved in the synthesis of sterols, whereas those with a methylene group at C-24 and an additional methyl group at C-25 are derived from 24-methylene-25-methylcholesterol. Commonly, brassinosteroids are synthesized through pathways dependent on cycloartenol and cycloartanol. Notably, more than 17 compounds have been identified as inhibitors of BR biosynthesis, with specific target reactions within the pathway elucidated for nine inhibitors like brassinazole and YCZ-18 [7].

Brassinosteroids (BRs) have been identified as the 6th group of phytohormones, with approximately 70 naturally occurring compounds belonging to this group. These compounds, including brassinolide (BL), are structurally similar to androgens, estrogens, corticoids, and ecdysteroids. BRs are found in both lower and higher plants, particularly in angiosperms, and are present in various plant organs. These hormones are essential for the development and growth of plants, as they trigger various morphological and physiological responses. Among these hormones, brassinosteroids (BRs) play a crucial role in enhancing plant tolerance against abiotic and biotic stressors. BRs help plants adapt to adverse environmental conditions, allowing them to thrive and survive in challenging situations. Their importance in plant biology cannot be overstated, as they contribute significantly to the overall health and resilience of plants. In conclusion, understanding the role of hormones such as BRs is vital for improving crop productivity and sustainability in agriculture [7, 8, 9].

2.1 BRs biosynthesis

BRs, a group of phytohormones, are categorized as C27, C28, or C29 steroids based on their C-24 alkyl substituents. Among them, brassinolide, a C28 brassinosteroid, has been found to possess the most potent biological activity [10]. The discovery of two distinct biosynthesis pathways (the early and late C-6 oxidation pathways) for brassinolide in cultured Catharanthus roseus cells has provided valuable insights into the production of this important plant hormone and the complex mechanisms governing plant growth and development [11, 12]. The validation of each step within these pathways was achieved through the conversion of labeled brassinosteroids. Research indicates the occurrence of cross-talk between these parallel pathways, underscoring the complexity of brassinosteroid biosynthesis. Additionally, an early C-22 oxidation pathway has been identified to occur at the initial stages of biosynthesis. These intricate biosynthetic pathways form a network that appears to be prevalent across the plant kingdom, with similar pathways observed in a diverse range of plants, including Arabidopsis, rice, pea, and zinnia [11]. In tomato and tobacco plants, the late C-6 oxidation pathway is the primary route for the synthesis of brassinosteroids (BRs). This is due to the fact that the endogenous BRs found in these species consist solely of members from the late C-6 oxidation pathway. Unlike other plants where both early and late pathways are involved in BR synthesis, tomato and tobacco plants rely exclusively on the late C-6 oxidation pathway. This unique characteristic sets them apart from other plant species and highlights the importance of understanding the specific pathways involved in BR synthesis in different plants. Further research into the regulation and significance of the late C-6 oxidation pathway in tomato and tobacco plants could provide valuable insights into their growth and development processes [11].

The biosynthesis of brassinosteroids (BRs) involves three distinct pathways that result in the production of C27-, C28-, or C29-type BRs (Figure 1). The initial steps of synthesis are shared among these pathways and can occur through either the mevalonate (MVA) or non-MVA pathway. However, the later stages of biosynthesis, which are cycloartenol- and cycloartanol-dependent, differentiate the BR biosynthesis pathways. The C28-BR biosynthesis pathway, primarily studied in Arabidopsis thaliana, has provided significant insights into the reactions, enzymes, and genes involved in BR production. This pathway involves the synthesis of campesterol and 22a-hydroxycampesterol. C27-BRs are derived from cholesterol (CR) and culminate in the production of 28-norBL, while C29-BRs originate from b-sitosterol and result in 28-homoBL. Despite the progress made in identifying compounds along these pathways, not all indirect compounds have been fully characterized yet (Figure 1) [7].

Figure 1.

Brassinosteroids biosynthesis and their sterol biosynthetic precursors [7].

In the realm of plant biology, the oxidation/hydroxylation steps in the BR biosynthetic pathway play a crucial role, being catalyzed by cytochrome P450 enzymes. Traditionally, it was thought that brassinolide is synthesized from campesterol through campestanol (CN) in the initial BR biosynthetic pathway [13]. P450s catalyze the stereospecific oxidation of unactivated hydrocarbons. They are found in over 5000 species. In plants, Arabidopsis thaliana has 245 P450 genes, Oryza sativa has 334, Vitis vinifera has 316, Glycine max has 332, Physcomitrella patens has 71, Chlamydomonas has 40, and Volvox has 19. The number of plant P450s surpasses that of Drosophila (87 genes) and humans (56 genes). P450 is a hemoprotein featuring heme iron at its active center, along with a cysteine-derived thiolate anion coordinated with the heme iron. The maximum absorption band (Soret band) of reduced P450 is approximately 420 nm. Upon carbon monoxide binding to the heme iron Fe (II) in reduced P450, the Soret band shifts to 450 and 380 nm (the origin of the name “pigment 450 nm”). Plant P450s are located in the endoplasmic reticulum membrane and facilitate substrate oxidation by activating molecular oxygen with NADPH-P450 reductase. Notably, P450 serves as a common target for fungicides and plant growth substances [13, 14, 15].

2.2 BRs biosynthesis inhibitors

The discovery of brassinazole as a potential BR-biosynthesis inhibitor marked a significant advancement in the field of plant research. Through investigations on small molecules that induce BR-deficiency-like phenotypes in Arabidopsis, researchers were able to pinpoint brassinazole as the primary candidate for inhibiting BR biosynthesis. Studies have shown that treatment with brassinazole effectively reduces BR content in plant cells by binding directly to the DWF4 protein, a cytochrome P450 monooxygenase responsible for catalyzing 22-hydroxylation of the side chain of BRs. The findings strongly indicate that brassinazole acts as a potent inhibitor of the natural synthesis of BRs, with effects resembling conditional mutations in BR biosynthesis. The identification of brassinazole and other known inhibitors represents a crucial step forward in understanding the intricate mechanisms governing plant growth and development [11, 16, 17].

Today, seventeen inhibitors (KM-01, brassinozole (Brz), Brz2001, Brz220, propiconazole, YCZ-18, yucaizol, fenarimol, spironolactone, triadimefon, imazalil, 4-MA, VG106, DSMEM21, finastride, AFA76, and brassinopride) have been known (Figure 2); however, the activity position of just 9 inhibitors is recognized [7].

Figure 2.

Inhibitors of biosynthesis of sterol and BR. Numbered compounds have a recognized activity site [7].

The following are the action sites of inhibitors:

• campestanol—6-deoxoCT for brassinazole, Brz2001, Brz220, triadimefon, and spironolactone;

• 6-deoxoCT—6-deoxoTE for brassinazole, Brz2001, Brz220, propiconazole, and fenarimol;

• 6-deoxoTE—6-deoxo-3DT for YCZ-18, yucaizol, propiconazole, and fenarimol;

• 6-oxocampestanol—CT for brassinazole, Brz2001, Brz220, and triadimefon;

• CT—TE for brassinazole, Brz2001, Brz220, propiconazole, and fenarimol;

• TE—3DT for YCZ-18, yucaizol, propiconazole, and fenarimol (Figure 1) [7, 18].

The first stated BR inhibitor, i.e., KM-01, was sequestered from a microbiological media. KM-01 disabled BR activity in a rice lamina. Regardless of the uncertain location of activity, KM-01 displays extremely strong activity. But, brassinazole (Brz) represents the primary particular BR synthesis inhibitor, which inhibits the exchange of campestanol to 6-deoxoCT, 6-deoxoCT to 6-deoxoTE, 6- oxocampestanol to CT, and CT to TE in the BR biosynthetic same reactions [7, 16, 17, 18]. Brz and Brz2001 can induce morphological changes, including dwarfism, altered leaf color, and curling in de-etiolated barley. Brz reduced the amount of BRs in the shoots of barley, but not in roots. The inhibitory impact of Brz on plant growth is retreated by exogenous BR. Propiconazole, a triazole compound, also affects similar to Brz. New triazole-type BR biosynthesis inhibitors, YCZ-18 and yucaizol, bind to the CYP90D1 enzyme and prevent the BR-induced cell growth. On the other hand, just BL denies the inhibition influence of YCZ-18 or yucaizol [7, 11, 18].

3. Estrogens and androgens in plants

Steroid hormones in mammals are categorized into five classes according to their construction and natural functions (Figure 3). These groups include androgens, estrogens, which are male and female sex hormones, mineralocorticoids and glucocorticoids, necessary for regulating the body’s homeostasis, and progestins, with progesterone playing a crucial role in the onset and maintenance of pregnancy [19]. Estrogen is a group of sex hormones that play a role in developing and controlling the female reproductive system and secondary sexual traits. Three main natural estrogens with hormonal effects are estrone (E1), estradiol (E2), and estriol (E3). Estetrol (E4), another estrogen, is only produced during pregnancy. An androgen is a natural or synthetic steroid hormone that controls the development and upkeep of male traits in vertebrates by binding to androgen receptors. The primary androgen in males is testosterone. Dihydrotestosterone (DHT) and androstenedione are equally significant in male development.

Figure 3.

Constructions of steroids which are main groups of mammalian steroid hormones: 1, testosterone (androgens), 2, estradiol (estrogens), 3, progesterone (progestins), 4, cortisol (glucocorticoids), 5, aldosterone (mineralocorticoids) [19].

The presence of endogenous estrogens and androgens may be questioned due to their small amounts in plants. Additionally, various metabolites in the plant extract can impede analysis and result in inaccurate outcomes. According to this subject, in more progressive analytical procedures, emphasis should be placed on appropriately cleaning the sample. These compounds (androgens and estrogens) are found in plants at the pg. and ng levels [20].

Khaleel et al. [21] found that 17β-estradiol varied in concentration, from 14 pg/g F.W. × 10⁻¹ in a bisexual tree (branch 1, November) to 2624 pg/g F.W. × 10⁻¹ in the generative buds of a bisexual tree Populus tremuloides (March). They observed that in catkins, hormone concentrations were higher before anthesis, peaked during flowering, and then decreased as the flowers matured. These increases were associated with sporogenesis and gametophyte development. Seasonal variation in 17β-estradiol concentration was noted, along with the effect of radiation circumstances and the specific plant tissue being investigated. Dormant winter parts had minor 17β-estradiol than spring active organs, and twigs developing in more strong radiation contained further 17β-estradiol content. Fluctuations in 17β-estradiol and testosterone were also found in Actinidia pollen by researchers [22].

Hormone levels rose during pollen germination, specifically during tube organization, emergence, and elongation phases. The process of pollen germination in kiwifruit involves fluctuations in hormone levels, particularly 17β-estradiol and testosterone, during key phases such as tube organization, emergence, and elongation. The presence of 17β-estradiol in kiwifruit pollen can rise from nearly undetectable levels in ingeminated pollen to around 4 ng/mg pollen after 90 minutes of germination, while testosterone levels can vary from 0 to 2.5 ng/mg pollen. Furthermore, the introduction of bisphenol A has been shown to elevate the levels of both 17β-estradiol and testosterone. Excessive concentrations of exogenous 17β-estradiol and testosterone have been found to hinder kiwifruit pollen germination, suggesting that environmental contamination by bisphenol A could potentially disrupt plant fertility by affecting steroid content. Some studies have indicated that the presence of estrogens and androgens in plants may pose risks to both humans and animals [20, 21, 22, 23]. For instance, research by Lu et al. [24] revealed varying levels of 17β-estradiol in vegetables and fruits, with concentrations ranging from 1.3 to 2.2 ng/g F.W. across different species, while estrone was detected in smaller amounts. Notably, the intake of 17β-estradiol in children from plant-based foods could exceed recommended daily limits, highlighting the importance of monitoring hormone levels in food sources as discussed by Palacios et al. [25].

Zeitoun and Alsoqeer [23] studied the sex steroid hormones in alfalfa and their following impacts on camel reproduction. Testosterone was recognized in Cakile arabica (3.69 ng/g D.W.) and Dwarf papyrus sedge (2.97 ng/g D.W.) but not in Greater plantain, Arfaj, Buffel grass, and Alfalfa. Additionally, 17β-estradiol was reported in prickly lettuce (379 pg/g D.W.), Rocket (247 pg/g D.W.), and Heliotropium bacciferum (229 pg/g D.W.) but not in Broom bush, Arfaj, and plumose needlegrass. Based on the results, due to the existence of these compounds, among others, camels may be affected by cystic ovarian syndrome, leading to delayed pregnancy [20, 23]. Milanesi et al. [26] have identified the presence of estrogens, specifically 17β-estradiol and estrone, in various parts of Solanum glaucophyllum, including the seeds, leaves, flowers, and calli. The concentration of these steroids varied depending on the specific tissue or organ being examined. For instance, 17β-estradiol was detected in all the mentioned tissues, with the highest levels observed in the seeds at 120 ng/kg F.W. On the other hand, estrone was found in the calli and seeds, albeit in much lower quantities (a few ng/kg F.W.), and was not present in the aerial organs. Additionally, Milanesi and Boland [27] have also noted the existence of estrogen-like metabolites in the shoots of tomatoes.

Seasonal variations in steroid levels in plants are influenced by various factors, with growth temperature being a significant contributor. Research by Janeczko et al. [28] highlighted the impact of temperature on androgen levels in wheat, showing a notable decrease in androstenedione content when plants were exposed to colder temperatures. This phenomenon was also observed in other plant species like Nicotiana tabacum and Inula helenium, albeit with varying concentrations. Interestingly, Digitalis purpurea did not exhibit the presence of androstenedione. Additionally, Tarkowská’s study [29] on Tribulus terrestris revealed the detection of testosterone and androst-4-ene-3, 17-dione within a specific concentration range. These findings underscore the intricate relationship between growth conditions and steroid levels in plants, shedding light on the dynamic nature of plant physiology in response to environmental cues. In Table 1, Janeczko [20] has presented the presence of estrogens and androgens in plants over the 20 years leading up to 2021; definitely, the highest concentrations of androgen and estrogen were found in pollen.

Plants	Steroids	Content
Populus tremuloides	17β-estradiol	14 pg/g F.W. × 10^−1 in a bisexual tree; (branch 1, November); 2624 pg/g F.W. × 10^−1 in the reproductive; buds of a bisexual tree (March)
Kiwifruit	17β-estradiol	up to 4,000,000 pg/g pollen (dependent on; stage of germination)
Kiwifruit	Testosterone	0–2,500,000 pg/g pollen (dependent on; stage of germination)
Lettuce, pumpkin, potato, carrot, citrus, apple	17β-estradiol	1300–2200 pg/g F.W.
Pumpkin, potato, carrot, citrus, apple	Estrone	Less than 800 pg/g F.W.
Cakile arabica; Cyperus conglomerates	Testosterone	3690 pg/g D.W.; 2970 pg/g D.W.
Lactuca serriola; Eruca sativa; Heliotropium bacciferum	17β-estradiol	379 pg/g D.W.; 247 pg/g D.W.; 229 pg/g D.W.
Solanum glaucophyllum	17β-estradiol	120 pg/g F.W. (seeds); 4–10 pg/g F.W. (calli, leaves, flowers)
Solanum glaucophyllum	Estrone	3–6 ng/kg F.W. (calli and seeds)
Winter wheat	Androstenedione	6215 pg/g F.W.; (leaves of seedlings growing at 20°C)
Nicotiana tabacum; Inula helenium	Androstenedione	2177 pg/g F.W. (leaves); 3202 pg/g F.W. (leaves)

Table 1.

Presence of estrogens and androgens in plants (F.W.: fresh weight; D.W.: dry weight) [20].

Steroid hormones have long been associated with the endocrinology of animals, leading to reluctance to acknowledge their presence in higher plants, particularly in the case of testosterone (4-androsten-17-ol-3-one; TS) and its derivatives. This hesitance may stem from the common perception that the effects of steroid hormones are exclusive to animal physiology. However, emerging research suggests that these hormones play a significant role in plant biology as well.

TS, along with epitestosterone and androstenedione, was first isolated from plant sources in 1971. The researchers utilized Scotch pine Pinus silvestris pollen and later found these substances, along with progesterone (PRG), in Pinus nigra pollen [29]. Pine trees have been identified as a significant source of testosterone, a hormone that plays a crucial role in various physiological functions. Research has shown that testosterone is present in species such as Pinus tabulaeformis and Pinus bungeana, as well as in the reproductive organs of other plants like ginkgo and lily [30]. Some literature suggests that testosterone and dihydro-testosterone are present in 20 species, such as Zea mays, Hordeum vulgare, and Rheum rhabarbarum [29]. Additionally, Hartmann et al. [31] discussed the normal presence of these phytohormones in diet, highlighting the occurrence of testosterone in Solanum tuberosum, Glycine max, Phaseolus vulgaris, and Triticum aestivum, with amounts ranging from 0.02 to 0.2 μg kg⁻¹. The researchers also noted the presence of this androgen in native oils used in human nutrition, such as olive oil, corn oil, and safflower seed oil. Safflower seeds have relatively high phytosterol content, ranging from 2000 to 4500 μg g⁻¹, with β-sitosterol constituting the majority (50–70%) of the total amount of plant sterol [20, 32].

From a biological perspective, the synthesis and biochemical role of testosterone in plants appear to mirror that in animals [29]. This C₁₉ steroid is produced through the MVA pathway in the cytosol of plant cells from cholesterol, through a series of enzymatic reactions. These reactions involve the breakdown of the cholesterol side chain into the C21 steroid pregnenolone, then its conversion to androstenedione, and ultimately to TS (Figure 4). The conversion to TS was validated through experiments involving feeding 14C-androstenedione, which was transformed into TS in pea and cucumber seedlings, as well as in cultured cells of Nicotiana tabacum [29]. Unlike in the animal realm, where TS and other androgens function solely as sex hormones, research has indicated that in plants, they influence not only reproductive development (particularly flowering and floral sex determination) but also vegetative growth [1, 29].

Figure 4.

A simplified biosynthetic pathway of testosterone in plants [29].

Some literature reported the endogenous formation of weak androgenic substances, such as Boldenone and boldione, from phytosterols in plants. These substances may be produced naturally in plants [33]. Androsta-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AED) are intently correlated compounds. Plant sources containing AED and/or ADD have been identified, with AED found in pine pollen of Pinus sylvestris (0.59 μg g⁻¹) and Pinus nigra (0.08 μg g⁻¹) as early as 1971, 1979, and 1983 [29]. In 1998, notable levels of AED were also detected in wheat (0.48 ng g⁻¹) and potato (0.05 ng g⁻¹) [31]. Additionally, trace amounts were observed in soybeans, haricot beans, mushrooms, olive oil, safflower oil, wine, and beer. AED was also found in tobacco (Nicotiana tabacum; 2.20 ng g⁻¹ F.W.) and elfdock (Inula helenium; 3.20 ng g⁻¹ F.W.) [33]. Furthermore, AED, ADD, PRG, and TS were conclusively identified in Tribulus terrestris [29]. Endogenous steroidal estrogen levels peak in reproductive plant parts like flowers, pollen, fruits, and seeds, while vegetative organs (stem, leaves, roots) have lower concentrations [29, 34]. Estrogens were found in 50% of the 128 species tested, indicating their widespread presence in nature [3].

4. Progesterone in plants

Progesterone, a vital gonadal steroid hormone crucial for the maintenance of early pregnancy and various reproductive processes in mammals, has been found in diverse sources such as false rubber tree shoots and apple seeds [35]. While C21 pregnane steroidal hormones play pivotal roles in mammalian reproductive functions and hormone synthesis, there is a scarcity of information about these compounds in phytochemical literature. The discovery of progesterone in plants [3] showcases how a well-known compound can be identified in natural sources, even in trace amounts, without being previously isolated [36]. Progesterone (PRG) is a C-21 steroid (pregn-4-ene-3, 20-dione).

Progesterone regulates pregnancy progression and menstruation in humans, in addition to serving as a precursor for androgen (C19) and estrogen (C18) production. It also functions as a crucial neurosteroid for brain activities [37]. Studies have revealed the presence of progesterone in various plant species, with levels varying between species and different plant organs. The concentration of progesterone in plant tissues generally remains around 1 μg or less per kg of fresh weight. Research has identified progesterone in a wide array of plant organs, including shoots, roots, tubers, inflorescences, and seeds, with reproductive tissues containing higher levels of the hormone. Furthermore, genome DNA databases of higher plants have indicated the existence of genes similar to mammalian progesterone-binding proteins, with membrane steroid binding protein (MSBP1) identified in Arabidopsis as a negative regulator of cell elongation [35, 38]. Progesterone has been detected not only in animal-derived products like meat and eggs but also in plant-based foods such as wheat meal, steamed potatoes, and various oils. Simons and Grinwich [3] noted the presence of PRG in a range of plant species (80% of plants from 50 families). Progesterone is naturally present in wheat seedlings in its conjugate form (as glycosides) [38]. According to Iino et al. [35], progesterone was detected in a variety of dicot and monocot species by GC/MS (Table 2).

Plants	Organ	PRG amount (ng kg^−1 F.W.)
Dicotyledons
Thale cress	Foliage	160
	Flowers	400
Pea	Foliage	190
	Root	260
	Ripe seed	410
Common bean	Etiolated seedling	48
Mung bean	Etiolated seedling	21
Tomato	Leaves	25
	Unripe tomato	280
	Red tomato	6
Potato	Tuber	25
Apple	Flesh	150
	Seed	430
Monocotyledons
Rice	Foliage	1540
	Ear	440
Onion	Bulb	68

Table 2.

Endogenous amounts of PRG in several plants [35].

Shiko et al. [39] believed that progestogens and androgens are widespread steroids within the plant kingdom. Several researches utilizing 3H and 14C-labeled precursors have indicated that sitosterol, a prevalent sterol in higher plants, along with the less common sterol cholesterol, can act as precursors of progesterone in plants [29, 40]. Some research also proposes that campesterol and stigmasterol (C₂₉, i.e., 24-ethyl Δ^5,22 sterol) could potentially serve as precursors of PRG. The majority of these experiments involve the exogenous application of PRG to various plant systems, such as seedlings or plant cell cultures of different plant species. These studies suggest that PRG plays a role in regulating plant growth and development, impacting both vegetative and generative processes. For example, studies on the model plant Arabidopsis thaliana [35] and Helianthus annuus have shown that PRG can influence shoot and root growth in a dose-dependent manner [29].

5. Physiological effects of steroids on plants

Brassinosteroids, as highlighted in various studies, play a crucial role in regulating cell growth and differentiation at nano- to micro-molar concentrations. They exhibit diverse regulatory activities such as stimulating cell enlargement and division, inducing leaf bending at joints, altering membrane potentials, regulating gene expression, and influencing nucleic acid and protein metabolism. Notably, these compounds have shown significant promise as plant growth regulators in agriculture [41, 42]. The physiological effects of brassinosteroids are manifold, with exogenous application leading to a wide array of changes in plants. These effects are thought to occur through two main pathways: direct action on the genome and an extra-genetic route, both involving secondary messengers. Studies have demonstrated that brassinosteroids can promote elongation of plant stems when applied externally, with radiolabeling experiments suggesting their movement from roots to shoots via the xylem [42, 43]. Furthermore, research on brassinosteroid-deficient mutants has shed light on the role of these compounds in signal transduction and light-regulated plant development. Mutants such as BRI1, identified through studies on Arabidopsis, have provided insights into the molecular mechanisms underlying brassinosteroid responses. The discovery of BRI1’s homology to leucine-rich receptor kinases has further deepened our understanding of how brassinosteroids function in plants [44, 45].

The impact of mammalian sex hormones on callus induction has been documented to have various effects. These include the promotion of epinasty, an increase in sugar and protein content, stimulation of reproductive growth and flowering, enhancement of flower number, modulation of the ratio of female to male flowers, as well as improvements in pollination and fertilization processes [1, 46]. The study conducted by Ahmadi-Lashaki et al. [47] found that the application of progesterone did not have a specific effect on the physiological and growth traits of Petunia hybrida, Tagetes erecta, and Calendula officinalis. Despite the potential benefits of progesterone in other plant species, the results of this study suggest that its application may not be effective in enhancing the growth and development of these particular plants. The growth-promoting activity of progesterone was much lower than brassinolide as shown by Li et al. [48].

Recent studies have shed light on the growth-promoting effects of estrone and 17b-estradiol on dwarf pea plants (var. Cud Kelwedonu). Interestingly, it has been discovered that while estrone had a positive impact on mutants and wild-type plants, 17b-estradiol was found to be inactive in both cases. This finding raises questions about the mechanisms by which these hormones exert their effects in plants. It is known that in mammals, progesterone can be converted to estrogens, which then trigger biological responses. However, the results of this study suggest that progesterone may have biological activity in plants even without being transformed into estrogens [35].

Several studies have demonstrated the involvement of brassinosteroids in light-mediated plant development, suggesting a potential mediating role in phytochrome regulatory functions [42, 49, 50]. Additionally, steroids have shown efficacy in enhancing plant resistance against various stressors such as chilling, pathogens, herbicides, and saline condition [51]. Recent findings have highlighted the significant impact of brassinosteroids on modulating plant immunity [42]. Research by Filek et al. [52] revealed that PRG binds to protein receptors in cell membranes, thereby enhancing wheat’s tolerance to chilling. Furthermore, the work of Shpakovski et al. [53] emphasized the fundamental interplay between steroid biosynthesis and regulatory systems in both plants and animals. Notably, studies have successfully elevated endogenous progesterone levels in transgenic tobacco and tomato leaves, resulting in positive hormonal effects on plant growth, development, and stress tolerance. Moreover, Pauli et al. [36] definitively identified the presence of PRG in Persian walnut and discovered five other mammalian-type steroids in Adonis aleppica.

An interesting impact of MSH is its ability to improve plant resistance to various abiotic stresses [54]. Erdal and Dumlupinar [55] reported that mammal sex hormones (MSHs) mitigate the adverse effects of salinity stress by enhancing the activity of antioxidant enzymes, including SOD, POX, and CAT, as well as increasing proline content. Research has also demonstrated that MSH treatment can stimulate antioxidant process and biosynthesis reactions, leading to a reduction in reactive oxygen species (ROS) in chickpea seedlings [56]. Studies have indicated that the application of PRG and salicylic acid (SA), either individually or in combination, can enhance nutrient (N, Ca, K) uptake and improve plant resilience against injurious elements like Cl and Na in salinity-affected plants [51]. While grasses can still be cultivated in saline conditions, their growth and development may be hindered. However, the adverse effects can be significantly alleviated through the use of PRG, SA, and their mixture [51, 57]. Brassinosteroids have the ability to counteract the inhibitory effects of salinity on seedling growth in groundnut [58]. 24-epibrassinolide and 28-homobrassinolide also alleviated the stress of saline condition and improved Oriza sativa germination in saline conditions[59]. Seed treatment with very dilute solutions of BRs has shown significant improvements in the growth of Oriza seedlings under salinity [42, 59]. Brassinosteroids have been found to possess stress-protective properties against heavy metals, with 24-epibrassinolide demonstrating a reduction in heavy metal uptake in mustard. When applied to wheat seeds under environmental stress, epibrassinolide led to enhanced germination rates and increased protein content by 15–30%, albeit with a decrease in starch content by 6–19%. Among the two methods of brassinosteroid application, soaking seeds proved more effective than spraying plants. Additionally, brassinolide treating of rice plants was effective in mitigating injury caused by certain herbicides. Studies on oxidative lipid degradation in pea plants indicated that 24-epibrassinolide not only reduced breakdown product levels in normally aerated tissue but also under conditions of hypoxia and elevated CO₂ levels. Compared to kinetin, 24-epibrassinolide treatment was established to be more efficient in various aspects [42].

Immunomodulation is a manner for modifying the immune response that involves inhibiting or modulating the roles of specific antigens in vivo through the intracellular ectopic expression of particular antibodies. This modulation occurs through the interaction of antibodies and antigens, leading to the formation of antigen-antibody complexes. The modulatory impacts of BRs in thale cress seeds highlight their significant function in plant immunomodulation. The less explored trait of immunomodulation by BRs, when administered in precise amounts, has paved the way for new avenues in research on plant growth regulation. Furthermore, it holds the potential for the development of environmentally friendly substitutes for conventional pesticides that are both harmless and biodegradable [42].

Brassinosteroids have demonstrated a practical utility in augmenting the yield of ornamental plants. Significant improvements have been noted particularly in the yield and quality of bulbs and bulbils. Moreover, the application of BRs has been found to boost crop productivity in potatoes, leading to enhanced starch and vitamin C levels in the produce. Additionally, the foliar application of epibrassinolide during the budding or flowering phases has been observed to reduce the susceptibility of plants to fungal infections [42, 60].

The impact of exogenously applied mammal sex hormones (MSH) on plant growth stages, from germination to flowering, has been a subject of research interest [1, 56, 61]. Numerous studies have explored the effects of MSH on various morphological and biochemical parameters, including root and shoot length, enzyme activities, protein, sugar, nucleic acid, and chlorophyll content. These investigations have highlighted the significant stimulation of plant growth and development by MSH, particularly at low concentrations. Elements play crucial roles in the metabolic activities of living organisms, serving structural, electrochemical, and catalytic functions. They are essential for the formation of organic substances and maintaining ion balances, as well as contributing to enzyme formation [61]. Limited studies, such as those by Dogra and Thukral [62, 63] on wheat and maize plants, have examined changes in inorganic constituents like N, P, Fe, Na, and K following MSH application. Additionally, Erdal et al. [64] have reported alterations in inorganic element concentrations in germinating chickpea seeds exposed to progesterone and estradiol.

The impact of various hormones on the growth of sunflower and tomato seedlings has been a subject of recent research. In sunflower seedlings, 17β-estradiol and progesterone at specific concentrations have shown to affect shoot and root growth differently. While they promoted shoot growth, they inhibited root growth, except for progesterone at a lower concentration which actually promoted root elongation. Testosterone, on the other hand, facilitated cotyledon axillary bud formation at certain concentrations [1]. In tomato seedlings, estrone and 17β-estradiol, when administered as sulphate derivatives in the nutrient solution, led to reduced root growth and root number in shoot cuttings. Interestingly, in Medicago sativa L., watering with nutrient solutions containing estrone and 17β-estradiol had varying effects depending on the concentrations used. Lower concentrations favored growth and increased dry weight of shoots and roots, whereas higher concentrations inhibited plant growth. The authors highlighted the potential impact of estrogen levels found in sewage water on the vegetative growth of alfalfa plants. Recent data also suggest that estrogens and progesterone can stimulate winter wheat seedling roots and leaves when grown in vitro, but higher concentrations of these steroids can lead to a slight inhibition in seedling growth [1, 65].

Bonner et al. [66] conducted a study using a steroid biosynthesis inhibitor (SK & F7997) to halt the blossoming progression in an SD plant rough cocklebur. However, this compound also interfered with the production of membrane sterols. In non-vernalized plants of chicory, flowering was induced by estrone and 17β-estradiol, with 55 and 85% of plants flowering, respectively, while control plants remained in a vegetative state. Androgens like TS were found to be ineffective in reproductive development in scarlet sage [67]. In a separate experiment by Biswas et al. [68], androstane and androsterone did not promote flowering in chrysanths.

The application of exogenous progesterone significantly affects plant shoot and root growth, seed germination, and reproductive development (Figure 5). Progesterone, a hormone primarily associated with female reproductive functions in animals, shares striking similarities with plant hormones in terms of its role in mediating growth and development processes, as well as regulating responses to environmental stresses. While progesterone is not traditionally classified as a plant hormone, its functions parallel those of known plant hormones such as auxins, cytokinins, and abscisic acid. Plant hormones play a crucial role in coordinating various physiological processes in plants, including seed germination, root and shoot growth, flowering, and fruit development. Similarly, progesterone has been found to influence cell division, elongation, and differentiation in animal cells, suggesting a conserved mechanism of action across kingdoms. Furthermore, both plant hormones and progesterone are involved in regulating responses to biotic and abiotic stresses. Plant hormones help plants adapt to challenging environmental conditions such as drought, salinity, and pathogen attacks. Similarly, progesterone has been shown to modulate immune responses and stress tolerance in animals and plants, indicating a shared function in enhancing resilience to adverse conditions. In conclusion, although progesterone is not officially recognized as a plant hormone, its functional similarities with plant hormones highlight the interconnectedness of biological processes across different organisms [69].

Figure 5.

The summary of plant progesterone regulation during growth, development, and biotic/abiotic stress responses [69].

Li et al. [69] fully summarized the regulation of progesterone on plant growth and development and the alleviating effects of progesterone on biotic and abiotic stresses in plants in two (Tables 3 and 4).

Plant growth and development	Progesterone (PRG) role	Plants	Ref
Foliage and root growth	PRG controlled plant growth in a dosage-related manner.	Thale cress	[35]
		Sunflower	[70]
		Chickpea	[55]
Tissue culture	PRG enhanced foliage and callus production.	Sainfoin	[71]
	PRG regulated responded embryogenic callus and regenerable callus induction.	Wheat	[72]
Seed germination	PRG improved seed germination.	Chickpea	[54]
		Common bean	[56]
		Maize	[73, 74]
Generative progress	PRG augmented increasingly with pollen germination.	Kiwifruit	[22]
	PRG enhanced pollen germination and tube enlargement.	Tobacco	[75, 76]
	PRG encouraged plant flowering and induced generative growth.	Wheat	[77, 78]
		Thale cress	[79]

Table 3.

The effects of PRG on plant life process [69].

Biotic/abiotic stress	Progesterone role	Plants	Ref
Salt	PRG induced enzymatic and non-enzymatic antioxidant systems and improved the amounts of osmoprotectants.	Wheat	[80]
	PRG increased SOD, POX, and CAT actions and alleviated the salt-reduced K/Na ratio.	Bean	[54]
	PRG improved antioxidant activity and osmoprotectant accumulation.	Maize	[81]
	PRG enhanced salinity tolerance and augmented pigments and antioxidant enzyme activities.	Kentucky bluegrass	[82]
Chilling	PRG stimulated relative leaf water content, chlorophyll content, and antioxidative activity.	Chickpea	[83]
	PRG stimulated the mitochondrial respiratory pathway and upregulated the transcript level and protein accumulation of alternative oxidase (AOX).	Maize	[84]
	PRG encouraged AOX and enhanced enzyme and non-enzymatic antioxidant protection systems.	Dwarf banana	[85]
	PRG improved the transcription level of IbAOX1 and the activity of AOX, prevented the creation of chilling damage, decreased membrane penetrability, MDL and ROS levels, and improved the antioxidant defenses.	Sweet potato	[86]
	The increase in area per lipid molecule by PRG led to the formation of more flexible surface structures in monolayers.	Wheat	[52]
Drought	Drought caused to increase PRG-binding sites on the cell membrane of Katoda (drought-sensitive cultivar) but not in Monsun (drought-tolerant cultivar), while this stress caused to augment of PRG-binding sites in the cytoplasm of Monsun, but not in Katoda.	Wheat	[87]
	More-expressing animals’ CYP11A1 in Solanum lycopersicum can considerably improve tolerance to water deficit and prolonged dehydration.	Tomato	[53]
High temperature and high irradiance	PRG improved overheating-induced H2O2, MDA, and ionic leakage, increased the production of SOD, CAT, POX, and reduced photosystem II damage by stimulating D1 protein phosphorylation.	Wheat	[88]
	PRG improved antioxidant resistance system and simplified D1 protein strength under temperature and high irradiance stress.	Wheat	[89]
Biological stresses	PRG reduced the necrosis and the ion leakage, and enhanced the efficacy of PSII affected by pseudomonad	Thale cress	[90]
	CYP11A1-overexpressing transgenic tobacco exhibited resistance to contamination by fungal pathogens Botrytis cinerea.	Tobacco	[53]

Table 4.

The positive properties of progesterone (PRG) on environmental stresses in plants [69].

Türkoğlu et al. [91] studied the effect of different mammalian sex hormones (17 β-estradiol, estrogen, progesterone, and testosterone) in several concentrations on genetic or epigenetic levels in bean plants and found that genetic strength is decreased. It was found that the CRED-iPBS profile highlighted a significant increase in methylation levels associated with DNA cytosine nucleotide when exposed to 10⁻⁴ mM of estrogen hormone. Notably, polymorphism was evident across all hormone administrations in comparison to the control group (without hormone), signifying a reduction in genomic stability at higher concentrations. These findings collectively suggest that 17 β-estradiol, estrogen, progesterone, and testosterone impact genomic stability in bean plants, leading to epigenetic modifications that play a crucial role in regulating gene expression.

6. Conclusions

Numerous studies have been conducted to investigate the impact of steroid hormones on the growth and development of plants. The findings have revealed that steroid sex hormones, including progesterone, estrone, beta-estradiol, and testosterone, play a significant role in stimulating growth and development, callus development, cell division and elongation of roots and stems, and enhancing pollination in plants. Moreover, these hormones have been found to have a positive effect on plant stress response. Steroid hormones are considered secondary metabolites that are synthesized under specific conditions, such as exposure to stress. Among these compounds, progesterone has been shown to enhance the antioxidant properties of plants, thereby aiding them in coping with environmental challenges. Overall, the research suggests that steroid hormones can have beneficial effects on plant physiology and resilience.

Acknowledgments

The authors would like to thank colleagues for their assistance.

Conflict of interest

The authors have no conflict of interest to declare.

Notes/thanks/other declarations

This research received no external funding.