1. Introduction
Manganese (Mn) is the active center ion in at least 35 kinds of enzymes, including catalase (CAT) and Mn superoxide dismutase (Mn-SOD) [
1,
2]. They perform critical functions in plant photosynthesis, respiration, protein synthesis, and hormone activation [
1]. Although Mn is an essential mineral for plant growth and development, excessive Mn may be hazardous to plants [
3]. Plants only require 20–40 mg/kg Mn (dry weight) to sustain normal growth and development, whereas the Mn concentration in most plants is typically 30–500 mg/kg (dry weight) [
4]. Therefore, the Mn level in many plants is above the typical requirement, and excess Mn may impair plant development [
5]. Mn toxicity has become a major problem in many regions of the world, thus restricting crop development and decreasing agricultural output in acidic soil [
3,
4].
Plants have suffered from Mn toxicity stress in recent years as a result of soil acidification and Mn pollution, and the Mn content stored in most plants has considerably surpassed their physiological needs [
6]. Excessive Mn buildup in plants may have harmful effects. When plants are subjected to Mn poisoning, visible signs appear on the plant leaves. Plants with brown Mn oxidation patches on their leaves include
Stylosanthes guianensis, barley (
Hordeum vulgare), and
Vigna unguiculata [
7,
8]. Mn poisoning slows plant development, decreases the number of lateral roots, and reduces root vigor. It also blocks the activities of several enzymes and affects chlorophyll synthesis, resulting in a reduction in the amount of chlorophyll and the effectiveness of photosynthesis in plants [
9]. It affects the production of numerous hormones [
10]. When crops are subjected to Mn toxicity, the concentration of indoleacetic acid in the body decreases, thus inhibiting leaf growth and stomatal opening. This process subsequently affects the CO
2 assimilation response in crop photosynthesis, resulting in a considerable loss in crop production and quality [
11,
12]. Furthermore, excessive Mn deposition in food crops might impair the health of humans via the food chain [
13] through conditions such as Parkinson’s syndrome. It can also impair the regular functioning of the digestive, cardiovascular, immunological, and reproductive systems [
14]. Consequently, the harm caused by Mn toxicity stress to crops is very direct and severe, thus affecting crop growth and development.
Plants have developed several adaptive methods against Mn toxicity, including absorption control, differential isolation of Mn in subcellular structures, activation of the antioxidant enzyme system, and increased production and secretion of acidic organic compounds to change Mn to the inactive state [
1,
2,
8,
15]. Mn toxicity, for example, enhances the activity of peroxidase to promote Mn compartmentation in the apoplast of cowpea (
Vigna sinensis) and induces excess Mn oxidation [
7,
16]. Sequestering excess Mn into vacuoles may play a vital function in plant responses to Mn poisoning in leaves [
17]. Furthermore, numerous Mn transporters that transport Mn into vacuoles have been identified, including
Oryza sativa OsMTP8.1,
Arabidopsis thaliana AtMTP11, and
Cucumis sativus CsMTP8, indicating that plant Mn tolerance may be mediated by Mn transporters [
17,
18,
19]. Increased organic acid exudation in roots improves tolerance to excess Mn via chelating excessive Mn [
8,
20]. Furthermore, increased oxalate and citrate root exudates in ryegrass (
Lolium multiflorum) can limit Mn absorption, thus increasing tolerance to Mn toxicity stress [
20]. And yet enhanced malate secretions have a significant impact on the tolerance of Mn for
Stylosanthes guianensis [
8]. Furthermore, roots can improve tolerance to Mn toxicity stress by controlling the uptake of mineral nutrients, including magnesium (Mg), calcium (Ca), and iron (Fe) [
21,
22,
23].
Peanut (
Arachis hypogaea L.) is a significant cash crop and oil crop that offers edible oil and protein to people all over the world [
24,
25]. Peanuts, as agricultural crops, frequently confront numerous metal stressors during their life cycle, thus restricting productivity and endangering human health owing to hazardous metal buildup [
26,
27]. Peanuts are extremely vulnerable to Mn poisoning. Peanut seedling development is impeded when the concentration of soluble (Mn
2+) exceeds 150 μM. [
28]. Mn stress can impair plant ion absorption and transport, resulting in visible Mn oxidation spots on leaves, which can lead to reduced chlorophyll synthesis, decreased photosynthetic rate, reactive oxygen species buildup, and disturbance of hormone balance in leaves [
9,
10]. Therefore, Mn toxicity is among the major variables that influence peanut growth and limit peanut yield. A method for enhancing peanut tolerance against Mn toxicity stress needs to be determined.
In recent years, high-throughput transcriptome sequencing technology has been extensively employed to study the response mechanisms of plants to toxic stresses of heavy metals, such as copper, lead, aluminum, and cadmium, including
Citrus grandis,
Raphanus sativus,
O. sativa, and
Saccharum officinarum [
29,
30,
31,
32]. However, limited studies have focused on manganese-induced stress in peanut plants, and the molecular regulatory mechanism of peanut plants in response to Mn stress remains unknown. Mn toxicity response genes in roots and leaves were compared using high-throughput deep sequencing technologies. Furthermore, the physiological parameters of the appearance of Mn oxidation spots and the concentration of metal ions (Mn, Mg, and Fe) in roots and leaves were both studied at different concentrations of Mn treatment conditions. Our findings will reveal more information on the unique molecular regulatory pathways that underpin root and leaf response to Mn toxicity. The results of this study may provide a preliminary basis for additional research into the specific functions of genes that are sensitive to Mn toxicity.
3. Discussion
Excess of available Mn is hazardous to crops and a constraint for agricultural development, particularly in acid-soil [
1,
14,
33]. Generally, various types of crops have varying levels of toleration to Mn poisoning. For instance, soybean is more susceptible to Mn poisoning than
Stylosanthes guianensis [
8,
33,
34]. Nevertheless, the distinct response at the molecular level of different parts of crops to Mn poisoning remained unknown. In the present study, the fresh weight of peanut shoots and roots remarkably reduced while the concentration of Mn enhanced to 300 μM. Excess Mn has an inhibitory effect on cucumber (
Cucumis sativus) growth, resulting in a significant reduction in above- and below-ground dry weight [
35]. Under Mn toxicity stress, the biomasses of the above-ground part and underground part of soybean are severely decreased, and the development of roots is impeded in some ways [
14]. This study showed the aboveground and root biomass were also decreased observably, and the growth of roots was restrained to some degree when the peanut plant was subjected to Mn toxic stress. This phenomenon occurred, possibly because the excessive Mn accumulation of plants may result in severe injury of cells [
1,
36], ultimately influencing the normal growth and development of the plant.
In the present study, Mn toxicity caused Mn spotting and wrinkling of peanut leaves, which correlates with previous related reports in
O. sativa,
Stylosanthes guianensis, and soybean [
14,
37,
38]. The generation of Mn oxide spots is primarily attributed to the increasing amount of Mn compounds or oxidized phenol-like substances in the outside cell wall of the leaf epidermis of these plants under Mn-toxic conditions [
1,
39]. In this study, under Mn toxicity stress, peanut leaves showed obvious Mn oxidation spots, indicating that peanut leaves also accumulate large amounts of Mn oxides or oxidized phenol-like substances.
Mn can aid in the maintenance of the chloroplast membrane’s normal shape by taking part in the systems of photosynthesis electron transfer and photolysis of water [
39]. Fe is participated in the process of photosynthesis and the electron transfer system in respiration and influences the development of chloroplast, which is required for the formation of chlorophyll [
2]. Mg is required for the production of plant chlorophyll and is vital for the metabolism of plants [
2,
21]. As a result, the balance and stability of the relative levels of Mn, Mg and Fe might be critical for chlorophyll production and the process of photosynthesis. In this study, when peanuts suffered from Mn poisoning stress, although the accumulation content of Mn in both peanut roots and leaves was higher, the Mn content in the roots was notably more than that in peanut leaves, and the transfer or enrichment of superabundant Mn in the roots might be a crucial mechanism for peanut to mitigate its poisoning effect. Therefore, Mn might have some distribution mechanism in the roots and leaves, but this molecular mechanism has not been fully understood.
Unlike the varied pattern of Mn content in plants, Mg content in peanut roots and leaves declined with increasing exogenous Mn concentration, demonstrating that Mn and Mg absorption had antagonistic effects. The absorption of Mg in
S. guianensis,
Solanum lycopersicum,
Sorghum bicolor, and other plants was blocked, and the Mg concentration in plants was dramatically lowered, similar to earlier study results [
40]. In this study, Mn toxicity stress considerably lowered Mg concentration in peanut roots and leaves, indicating that Mn toxicosis stress primarily impeded Mg absorption by peanut roots and leaves. Reduced Mg concentrations in the roots and leaves might play an important role in reducing Mn poisoning effectiveness and preserving normal root and leaf function, which might be one of the accommodation processes for peanuts suffering from Mn poisoning.
Furthermore, boosting Fe concentrations and improving Fe absorption will be beneficial to plants to accommodate responses to Mn poisoning [
1]. Fe concentrations in cotton varieties of Mn-resistant strains were greater than those of cotton varieties of Mn-intolerant strains [
41]. When peanuts were subjected to Mn poisoning, the contents of Fe in roots and leaves changed distinctly and maintained high concentrations. Therefore, peanut roots and leaves could keep a higher content of Fe to relieve the effect of Mn poisoning, which may serve as a physiological mechanism for peanut plants to adapt to Mn poisoning.
As a normal secondary metabolite in plant cell metabolism, ROS (reactive oxygen species) has a beneficial effect on plant response to environmental stress, depending mainly on whether the delicate balance between ROS production and bursting is disrupted [
42]. SOD, APX, and POD are the main antioxidants responsible for eliminating ROS in plants [
43,
44]. Stress tolerance in plants can be improved by enhancing the vitality and content of antioxidases to reduce the accumulation of ROS in cells [
43,
44]. In the present study, the activities of POD, SOD, and APX in peanut roots and leaves remarkably increased in responding to Mn poisoning. Therefore, the antioxidant defense system of peanut plants was activated in response to Mn poisoning, and the activities of different enzymes in different parts of the plant were remarkably different. POD primarily took charge of eliminating the oxygen free radicals in peanut leaves, while SOD and APX were mainly responsible for the scavenging of reactive oxygen radicals in peanut roots and leaves. Furthermore, the MDA content is a crucial referent for the level of lipid peroxidation of the membrane in plant cells in a comprehensive manner [
45]. In this study, the degree of membrane lipid peroxidation in stressed peanut leaves was less than that in the experimental control group under the joint protection of various protective enzymes, while no significant difference was observed in the roots. High Mn stress may have broken the metabolic balance of intercellular ROS, causing differences in the MDA content in different parts.
Proline has a vital function in osmoregulation, maintenance of plant cell strength, and maintenance of osmotic pressure in the cytoplasm, contributing to the stability of cellular proteins and membranes [
46]. Proline also acts as a scavenger of ROS and works synergistically with antioxidant enzymes to reduce ROS in plants [
47]. In this study, the contents of proline significantly elevated in both leaves and roots of peanut plants suffering from Mn poisoning, indicating positive regulation in responding to Mn poisoning, which decreased the osmotic pressure of cells and required more proline to maintain osmotic pressure. Soluble proteins can participate in osmoregulation as osmotic regulators and reflect the degree of damage to plant organs [
48]. Plant biosynthesis of soluble proteins is affected by abiotic stress [
49]. In our study, soluble proteins showed an obvious increase in peanut roots and a significant decrease in leaves, which could be a response of soluble proteins to Mn stress. In addition, considering that soluble protein degradation produces a large amount of free amino acids, proline is one of the first rapidly increasing amino acids in various crops, and the increase in proline content may be related to soluble protein breakdown [
50].
Although genome-wide identification of DEGs in different types of plants in response to stressors of heavy metal ions has been examined, only one study in grape (
Vitis vinifera) roots in terms of Mn toxicity has been published [
51,
52,
53,
54]. A total of 2629 and 3278 DEGs were discovered in response to Mn poisoning in grape roots of two distinct varieties, namely, Combier and Jinshou, separately, indicating that Combier and Jinshou have differing tolerances to Mn toxicity [
54]. Limited genome-wide investigations have focused on leaf responses to Mn toxicity [
2,
55], but a comparison analysis of roots and leaves under Mn toxicity stress has not been carried out. In the present work, RNA-seq was used to conduct a whole genome investigation of the responding of DEGs to Mn poisoning stress on peanut roots and leaves, and 749 and 4589 DEGs were discovered from the peanut roots and leaves separately. Therefore, peanut roots and leaves might have very distinct molecular pathways.
Plant respiratory action, oxygenic photosynthesis, and activities of various secondary metabolism are all influenced by Mn poisoning [
7,
56]. In this study, a significant number of DEGs were functionally enriched involving MF, BP and CC in peanut leaves and roots, demonstrating that complicated metabolic alterations might exist in peanut leaves and roots in responding to Mn poisoning.
Excess Mn translocation is a mechanism employed by many plants to adjust to Mn poisoning, and this process is primarily adjusted and controlled by metal-ion transporter [
23,
57]. For instance, genetically modified rice with down-regulated expression of
a metal-nicotianamine transporter named
YSL (
yellow stripe-like member) showed a substantial drop in Mn content in rice grains, suggesting that it acts in managing the long-distance transmission of Mn-nicotianamine in plants [
57]. In our study, five
metal-nicotianamine transporters were upregulated in peanut leaves in response to Mn poisoning, intensely indicating that transporters of metal-nicotianamine might participate in Mn transporting in peanut leaves in Mn poisoning circumstances. The activities of
oligopeptide transporters are comparable to those of Mn recombination transporters in
Arabidopsis thaliana [
1,
58]. In the present work, seven
oligopeptide transporters were found in peanut roots and leaves in responding to Mn poisoning. Therefore, altering the transshipment of excessive Mn from peanut roots and leaves via changed transcriptions of
metal-nicotianamine transporters and
oligopeptide transporters might be essential for peanut tolerance to Mn poisoning.
Mn subcellular compartmentalization has an important effect on plants’ resistance to Mn poisoning [
36]. AtECA1 and AtECA3 are localized respectively in the endoplasmic reticulum and Golgi body, which are both belonged to calcium ATPases, direct adjusting excessive Mn transporting into the endoplasmic reticulum and Golgi body in
A. thaliana, separately [
59,
60]. Under Mn toxicity circumstances, the mutations of
AtECA1 or
AtECA3 can impede root development and cause serious chlorosis on the leaves of
A. thaliana [
59,
60]. In the present work, six
calcium-transporting ATPases were upregulated in both peanut leaves and roots in responding to Mn poisoning, indicating that the family genes of
Ca2+-ATPase might participate in Mn detoxifying in peanut roots and leaves. Mn toxicity upregulated two
vacuolar iron transporters (AH05G36140 and AH15G37400) in both roots and leaves, which were anticipated to be situated in the Golgi, indicating that it might participate in regulating the delineation and compartmentalization of excessive Mn transferring into the Golgi body.
Moreover, members of the family of MTP are important Mn transporters that govern Mn uptake and transshipment in plants [
61,
62]. CsMTP8.2 operates as an Mn-specific transporter in the tea plant (
Camellia sinensis) that contributes to the outflow of excess Mn
2+ from plant cells [
62]. OsMTP11 takes part in Mn remobilization in the cell cytoplasm and vacuolar membrane and may have an important effect on the transshipment of Mn and other heavy metals in
O. sativa [
61]. In this study, four
AhMTPs were discovered in response to Mn poisoning in roots or leaves in our investigation. Mn toxicity upregulated two
AhMTPs (AH10G30290 and AH13G56980), AH16G14430, and AH09G00220 in roots and leaves, suggesting that the four
AhMTPs might participate in peanut acclimatizing to Mn poisoning by adjusting Mn enrichment and isolation. The findings might supply molecular support for the evident Mn poisoning phenotype discovered in peanut root and foliage.
Homeostasis, or the regulation of the uptake and transshipment of other metallic elements such as Mg or Fe, is a key approach for coping with Mn poisoning [
2,
21]. In the present work, treatment with 300 μM Mn resulted in the decreased expression of a
magnesium transporter (AH16G35450) and a substantial drop in Mg content in peanut leaves, demonstrating that Mn poisoning might influence the expression of
magnesium transporter and, consequently, Mg aggregation in peanut leaves. Moreover, an increase in Fe effectiveness alleviates Mn poisoning in
Hypogymnia physodes [
21]. Besides Fe, Mg aids in enhancing wheat (
Triticum aestivum) tolerance to excessive Mn poisoning [
63]. In the present study, Mn poisoning increased the Fe concentrations in both roots and leaves. Furthermore, reduced expression of a
magnesium transporter (AH16G35450) was detected in peanut leaves exposed to Mn toxicity, suggesting that the gene of
magnesium transporter might control Mg balancing in peanut leaves in responding to Mn poisoning.
Considering that Mn poisoning may cause an irritable oxidation reaction, controlling the activity of antioxidative enzymes is often regarded as one of the essential Mn poisoning toleration methods [
38,
64]. In
Pennisetum purpureum, the expression levels of
PpSOD are significantly higher in the Mn-tolerance variety with quite high SOD activity, whereas this was not seen in the Mn-intolerance variety, indicating that
SOD may control tolerance against Mn poisoning stresses in plants [
64]. Mn toxicity stress increases POD activity and gene expression in cowpea (
Vigna sinensis) and stylosanthes (
Stylosanthes guianensis), respectively, which both have a significant role in adaptation to Mn poisoning for plants [
38,
39]. In this study, One
SOD and 24
PODs were all upregulated exclusively in the peanut leaves of this research but not in the roots. Therefore, the upregulation expression of one
SOD gene and 24
PODs might be beneficial to the increased Mn toleration of the roots in comparison with the peanut leaves.
Finally, several genes of
transcription factors were found in this investigation. In brief, 147
DEGs of transcription factor were differently sensitive to Mn poisoning stress in peanut leaves and roots. Three of the 16
bHLH transcription factors were up-regulated of expression in roots to make the response to Mn poisoning. Already there is evidence that one gene belongs to the family of the
bHLH transcription factor, namely,
AtNAI1, influencing
AtMEB1/2 expression levels and governing Mn poisoning toleration in
A. thaliana [
65]. However, the activities of
bHLH transcription factors family in peanut leaves and roots in response to Mn poisoning were still unclear. Therefore, intricate regulatory systems in peanut roots and leaves did not respond to Mn poisoning.