Abstract

   The relationship between metabolite changes and biological endpoints in
   response to cadmium (Cd) stress remains unclear. Fountain grass has
   good Cd enrichment and tolerance abilities and is widely used in
   agriculture and landscaping. We analyzed the metabolic responses by
   detecting the metabolites through UPLC-MS and examined the
   relationships between metabolite changes and the characteristics of
   morphology and physiology to different Cd stress in fountain grass. Our
   results showed that under Cd stress, 102 differential metabolites in
   roots and 48 differential metabolites in leaves were detected, with 20
   shared metabolites. Under Cd stress, most of the carbohydrates in
   leaves and roots decreased, which contributed to the lowered leaf/root
   length and fresh weight. In comparison, most of the differential amino
   acids and lipids decreased in the leaves but increased in the roots.
   Almost all the differential amino acids in the roots were negatively
   correlated with root length and root fresh weight, while they were
   positively correlated with malondialdehyde content. However, most of
   the differential amino acids in the leaves were positively correlated
   with leaf length and leaf fresh weight but negatively correlated with
   malondialdehyde content. Metabolic pathway analysis showed that Cd
   significantly affects seven and eight metabolic pathways in the leaves
   and roots, respectively, with only purine metabolism co-existing in the
   roots and leaves. Our study is the first statement on metabolic
   responses to Cd stress and the relationships between differential
   metabolites and biological endpoints in fountain grass. The
   coordination between various metabolic pathways in fountain grass
   enables plants to adapt to Cd stress. This study provides a
   comprehensive framework by explaining the metabolic plasticity and Cd
   tolerance mechanisms of plants.

   Keywords: cadmium stress, fountain grass, biological endpoint, amino
   acid, metabolic profiling, purine metabolism

1. Introduction

   The development of modern agriculture and industry has given rise to
   serious heavy metal pollution in the environment, of which cadmium (Cd)
   has become a global concern [[40]1]. Cd, one of the most toxic heavy
   metals, disturbs the regular physiological metabolism of plants and
   animals, threatening human health by delivering it into the food web
   [[41]2]. Cd stress can decrease stomatal density [[42]3], inhibit
   photosynthesis, transpiration, biomass accumulation, and decreased
   water use efficiency [[43]4], resulting in root necrosis and leaf
   chlorosis [[44]5]. These morphological and physiological changes under
   Cd treatment are all caused by variations in metabolites and metabolic
   pathways; however, the changes in metabolites and metabolic pathways
   under Cd treatment are highly uncertain [[45]6]. Metabolomics can
   effectively identify specific metabolites as biomarkers of specific
   stress responses, thereby gaining insight into the underlying metabolic
   mechanisms that counter stress to reveal cellular activities and
   organism responses to environmental alterations [[46]7]. Previous
   studies have shown that the type and relative content of primary
   metabolites such as amino acids, organic acids, sugars, and sugar
   alcohols are modulated, as these are important for heavy metal
   sequestration, detoxification, and tolerance [[47]8]. Additionally, the
   contents and varieties of secondary metabolites changed under Cd stress
   [[48]9]. For example, Cd stress increased the amino acid contents in
   the leaves while decreasing the amino acid contents and the sugar
   contents in the roots [[49]10]. However, the metabolite profile in the
   leaves of Catharanthus roseus showed that cadmium tolerance could be
   related to the differential accumulation of secondary metabolites
   [[50]11]. In addition to the metabolite changes, the alterations in the
   number and types of metabolic pathways under Cd also varied with the
   intensity of the cadmium treatment, the time of cadmium treatment, and
   plant tissue. Cadmium influenced seven metabolic pathways in the roots,
   one in the stems, and three in the leaves of pumpkins [[51]4]. In the
   roots of Brassica juncea, eight metabolic pathways were significantly
   changed at 48 h after Cd treatment, and four metabolic pathways were
   significantly changed on the seventh day after Cd treatment,
   respectively [[52]6]. Another study found that there were thirteen
   metabolic pathways in relation to the differential metabolites in the
   roots of Sedum plumbizincicola under low Cd treatment and six metabolic
   pathways related to the differential metabolites in the roots of S.
   plumbizincicola under high Cd treatment [[53]12]. The alterations in
   metabolites and metabolic pathways under Cd treatment could lead to
   changes in the plant’s internal and external biological endpoints (BEs)
   [[54]13]. However, few studies have explored the relationships between
   changes in metabolites and BEs. For example, orthogonal partial least
   squares (OPLS) analyses were applied to explore the relationships
   between metabolite changes and fresh weight in pumpkin tissues [[55]4].

   The monocots included about 70,000 species, which occupied about a
   quarter of all angiosperms. About 12,000 species of monocots belong to
   the true grasses (Poaceae), which are economically the most important
   family of monocots. Fountain grass is a fast-growing, low-cost,
   high-yielding, and adaptable monocot plant with attractive leaves and
   spikes in summer, autumn, and winter [[56]14]. Because of these
   advantages, there are many fountain grass species that are
   spectacularly and widely used as ornamental grass in landscaping
   [[57]15]. Fountain grass is also an excellent forage widely cultivated
   in tropical and subtropical areas and good for ecological restoration
   [[58]16]. Previous studies have explored the Cd enrichment ability of
   fountain grass cultivars [[59]17] and evaluated changes in the
   structural and physiological leaf traits of fountain grass under Cd
   stress [[60]3]. To the best of our knowledge, the changes in metabolic
   pathways and the relationship between the changes in metabolites and
   BEs of fountain grass is still unknown. Filling this knowledge gap
   could facilitate the control of urban cadmium pollution and the
   understanding of the adaptation mechanisms of monocots to cadmium
   stress.

   In this study, fountain grass was treated under different Cd stress
   levels to (1) assess the potential effects of Cd stress on the growth
   and physiology of fountain grass, (2) provide a comprehensive mechanism
   of Cd stress combined with metabolic profile and BEs [such as leaf/root
   length, leaf/root fresh weight, and leaf/root malondialdehyde (MDA)
   content], and (3) illustrate the metabolic pathway linking the
   metabolite changes and biologic endpoints under Cd stress.

2. Results and Discussion

2.1. Cadmium Accumulated More in Roots Than in Leaves

   As shown in [61]Table 1, the Cd content was not detected in the CK in
   leaves and roots. The Cd content of the leaves under Cd[10] was 1.75
   times that of Cd[5], and the Cd content of the roots under Cd[10] was
   2.10 times that of Cd[5]. Similar to our results, Cd accumulation in
   the roots and leaves increased gradually with the increase in the Cd
   content in the environment [[62]4]. In the Cd[5] treatment, the Cd
   content of the roots is 12.50 times that of the leaves, and the
   transfer factor is 8.34%. In comparison, the Cd content of the roots
   was 15.00 times that of the leaves, and the transfer factor was 6.77%
   under the Cd[10] treatment. Previous studies have also found that Cd
   accumulated more in the roots than in the leaves of two fountain grass
   cultivars at certain Cd stress levels [[63]17]. In addition, the
   transfer factor in our study was slightly lower than in the study of
   Chen et al. (2020); however, this was still less than one, indicating
   the Cd ions preferentially piled up in the roots [[64]18]. When Cd
   entered the plant, it first accumulated in the root tip before entering
   the xylem through the symplastic pathway or apoplastic pathway. Then,
   it was transported into the vessel by the transporters on the tonoplast
   and plasma membrane and finally transferred to the leaves with plant
   transpiration [[65]19]. Other studies have also shown that Cd ions
   mostly accumulate in the roots [[66]20], which could be attributed to
   the barrier effect of roots on the transportation of Cd to the leaves
   [[67]19].

Table 1.

   Cadmium (Cd) contents in the leaves and roots of fountain grass
   seedlings under cadmium stress.
   Treatment Cd Content in Roots
   (mg/g Dry Weight) Cd Content in Leaves (mg/g Dry Weight) Transfer
   Factor
   CK 0.00 (C, a) 0.00 (C, a) /
   Cd[5] 0.50 ± 0.06 (B, a) 0.04 ± 0.00 (B, b) 0.08
   Cd[10] 1.05 ± 0.21 (A, a) 0.07 ± 0.01 (A, b) 0.07
   [68]Open in a new tab

   Different letters indicate significant differences at p < 0.05 (n = 3).
   Uppercase letters indicate significant differences in the Cd content
   among different treatments in the same tissue; lowercase letters
   indicate significant differences in the Cd content between roots and
   leaves of the same treatment.

2.2. The Negative Effects of Cd Stress on Morphological Characteristics

   The leaf length significantly decreased by 22.10% and 47.10% under
   Cd[5]and Cd[10], respectively. Similar to the response of leaves, the
   root length also significantly decreased by 16.04% and 72.50% under
   Cd[5] and Cd[10], respectively ([69]Figure 1A and [70]Figure S1),
   illustrating how Cd stress significantly limited the elongation of the
   leaves and roots. Correspondingly, the leaf fresh weight, root fresh
   weight, and total fresh weight significantly decreased by 20.59%,
   69.13%, and 35.74%, respectively, under Cd[10] ([71]Figure 1B,C). The
   toxicity of Cd could reduce the uptake and translocation of water and
   nutrients, increasing oxidative damage, disrupting the metabolism, and
   finally handicapping the mass accumulation of leaves and roots
   [[72]21]. Some studies have also reported that leaf length and leaf
   fresh weight significantly decreased under Cd stress [[73]4]. Due to a
   larger decrease in the root fresh weight compared with the leaf fresh
   weight, the root/shoot ratio significantly decreased by 61.00% under
   Cd[10] ([74]Figure 1D), indicating that roots were subjected to more
   toxic effects than leaves. The root/shoot ratio decreased with the
   increment in the Cd concentration ([75]Figure 1D), demonstrating that
   the toxic effects of Cd on plants were concentration-dependent. The
   higher the Cd concentration, the more toxicity the Cd exerts on the
   tissues. Due to the particularly higher accumulation of Cd in the roots
   than in the leaves, the root morphological characteristics showed a
   larger decrease compared with the leaves.

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   Growth characteristics of fountain grass under cadmium (Cd) stress. (A)
   Length of leaf and root. (B) Fresh weight of leaf and root. (C) Total
   fresh weight. (D) Root/Leaf ratio. Different characters denote
   significant differences at p < 0.05 (n = 3). Vertical bars indicate
   standard error.

2.3. MDA Content, Soluble Protein Content, and SOD, POD, and CAT Activity
Changes under Cd Stress

   With the increase in Cd concentration, the contents of MDA and soluble
   proteins in the leaves and roots tended to increase. Among these, the
   MDA content of leaves under Cd[5] and Cd[10] significantly increased by
   17.25% and 65.88%, respectively. In comparison, the MDA content of
   roots under Cd[5] and Cd[10] increased by 33.17% and 81.91%,
   respectively ([78]Figure 2A). MDA is a highly reactive tri-carbon
   dialdehyde that is related to the level of plant growth inhibition and
   is recognized as a biomarker of oxidative stress. Our results are in
   parallel with the study of Leng et al. (2021), who stated that Cd
   stress elevated the content of MDA both in the roots and leaves,
   closely relating to linolenic acid metabolism and tryptophan metabolism
   [[79]22]. The MDA content of the leaf/root increased with the increment
   in Cd stress, indicating that Cd-induced lipid peroxidation was
   exacerbated as Cd toxicity was aggravated.

Figure 2.

   [80]Figure 2
   [81]Open in a new tab

   The MDA content, soluble protein content, and SOD, POD, and CAT
   activities of fountain grass under Cd stress. (A) MDA. (B) Soluble
   protein. (C) SOD. (D) POD. (E) CAT. Different characters denote
   significant differences at p < 0.05 (n = 3). Vertical bars indicate
   standard error.

   Similarly, the soluble protein content of leaves and roots
   significantly increased by 13.35% and 31.07% under Cd[10], respectively
   ([82]Figure 2B). Soluble proteins are significant components of
   numerous plant enzymes, reflecting the comprehensive metabolism of
   plants [[83]23]. Soluble proteins play a critical role in increasing
   the amount of functional protein needed to maintain normal metabolic
   activities and improve the plant’s resistance to environmental stress
   [[84]24]. According to a proteomics analysis of wheat under Cd stress,
   the number of upregulated differential expression proteins was higher
   than the downregulated [[85]25]. Similar to previous studies, the
   content of soluble proteins in the leaves of a hybrid Pennisetum
   increased with Cd stress aggravation [[86]26]. Under Cd stress, plants
   could synthesize some unique Cd-induced proteins, which occupy more
   than 10% of the total proteins [[87]27]. These could be the reasons
   that the soluble protein content increased under Cd stress.

   The SOD activity of roots under Cd[5] and Cd[10] significantly
   decreased by 9.31% and 17.47%, respectively ([88]Figure 2C). The POD
   activity of leaves and roots under Cd[10] significantly increased by
   28.61% and 53.07%, respectively ([89]Figure 2D). The CAT activity of
   the leaves and roots under Cd[10] also increased by 88.98% and 151.61%,
   respectively ([90]Figure 2E). With the increase in Cd concentration,
   the activities of SOD in the leaves and roots showed a decreasing
   trend, while the activities of POD and CAT showed an increasing trend.
   SOD dismutases O[2]¯ molecules to H[2]O[2] and hence can decrease OH¯
   formation through the metal-catalyzed Haber-Weiss reaction [[91]28].
   POD is one of the key enzymes, together with CAT, that participates in
   the breakdown and decomposition of H[2]O[2] [[92]29]. Similar to our
   results, the SOD activities of the leaves showed little change under a
   low Cd concentration; however, the SOD activities of the roots
   decreased under a high Cd concentration [[93]30]. The dominant
   antioxidant enzymes in different plants are different. Similar to our
   results, Xu et al. (2012) also found that under cadmium stress, the POD
   and CAT activities of the leaves and roots of Solanum torvum increased
   significantly, while SOD activity remained basically unchanged
   [[94]31]. The Cd content in the leaves was much lower than that in the
   roots; therefore, SOD activities in leaves were not significantly
   affected under Cd stress. However, the SOD activities in roots
   decreased under Cd treatment. Due to the higher Cd content in roots,
   the increased range of MDA, soluble protein, POD, and CAT in the roots
   was larger than in leaves under the same Cd concentrations.

2.4. Metabolite Changes under Cd Stress and Their Relationships with BEs

   Cd treatment not only altered the BEs (e.g., leaf/root length,
   leaf/root fresh weight, MDA content), but also changed the content of
   different metabolites both in the leaves and roots of fountain grass.
   There were 48 metabolites detected as differential metabolites in the
   leaves ([95]Figure 3A, [96]Table S1), and 102 metabolites in the roots
   ([97]Figure 3B, [98]Table S2) were detected as differential
   metabolites, with 20 shared in both the leaves and roots. Then, these
   differential metabolites were clustered based on their
   functions/pathways into several categories, such as phytohormones,
   carbohydrates, amino acids, organic acids, lipids, nucleotides,
   benzenoids, phenylpropanoids, and polyketides. As shown in [99]Figure
   3, the metabolites of CK and Cd stress were classified into two
   categories by hierarchical clustering.

Figure 3.

   [100]Figure 3
   [101]Open in a new tab

   Heatmap clustering of the relative contents of the differential
   metabolites of fountain grass under Cd stress (n = 3). Each row
   represents a metabolite, and each column represents a sample. The
   warmer color indicates a higher content, and the colder color denotes
   lower content. (A) Leaf. (B) Root.

2.4.1. The Changes in Differential Amino Acids under Cd Stress

   Under Cd stress, 23 differential amino acids increased in the roots. It
   has been found that the carboxylate (-COO) and amine (-NH[2]) groups of
   amino acids can form complexes primarily with heavy metal ions,
   intensifying the heavy metal tolerance of plants [[102]32,[103]33].
   Among these 23 differential amino acids, the content of arginine (FC =
   3.28), aspartate (FC = 3.23), glutamate (FC = 3.36), tryptophan (FC =
   4.14), alanine (FC = 2.55), and valine (FC = 2.05) in roots increased
   under Cd stress. Previous studies have also found that the arginine,
   aspartate, and glutamate content in the roots of B. juncea [[104]6],
   the levels of isoleucine and valine in the roots of Solanum nigrum
   [[105]34], and the content of tryptophan in the roots of Noccaea
   caerulescens increased under Cd stress [[106]35]. The accumulation of
   tryptophan is beneficial to prevent redox damage to peptides and
   proteins [[107]36]. Tryptophan can be dioxygenated by cracking the
   pyrrole ring to produce N-formylkynureine, which was found to be
   associated with 17 proteins involved in redox reactions. Therefore, the
   increase in tryptophan could contribute to the accumulation of soluble
   proteins [[108]37]. In spite of the increase in proteinogenic amino
   acids, the content of most of these non-proteinogenic amino acids also
   increased under Cd stress in the roots, such as γ-aminobutyric acid
   (GABA, FC = 2.51), O-acetyl-l-serine (FC = 2.78) and pipecolic acid (FC
   = 3.91). GABA, O-acetyl-l-serine, and pipecolic acid have been found to
   increase as a result of their exposure to heavy metal stress, which
   acts to enhance oxidative stress resistance or as an osmo-protectant
   [[109]38,[110]39,[111]40]. Interestingly, the content of
   4-guanidinobutyric acid (FC = 4.81), Gly-Leu (FC = 2.39), ornithine (FC
   = 2.17) increased, but the content of asparagine (FC = 0.5), pantethine
   (FC = 0.49), s-adenosyl-l-homocysteine (FC = 0.46),
   3-methyl-l-histidine (FC = 0.44), cysteine-s-sulfate (FC = 0.4),
   histidine (FC = 0.38), n-acetylhistidine (FC = 0.37), acetyl-dl-leucine
   (FC = 0.36), and saccharopine (FC = 0.22) decreased in leaves under Cd
   stress. Similar to our findings, the asparagine in plants under Cd
   stress was significantly lower than in the control [[112]41]. The
   content of histidine in leaves is lower than that in the control for N.
   caerulescens under Cd treatment [[113]35]. There were six shared
   differential amino acids in the leaves and roots, among which five
   showed similar change trends both in the leaves and roots. Similar
   findings have been reported in the differential amino acids shared in
   the roots and leaves of sunflowers, where the content of some amino
   acids was elevated, such as 4-aminobutyric acid and betaine; however,
   the content of some other amino acids was reduced, such as glutamine
   and pyroglutamic acid [[114]42]. Parallel to our findings, the levels
   of ornithine in the roots and leaves of Panicum maximum cv. Massai
   increased under Cd stress [[115]43]. Ornithine mainly acts on signal
   pathways controlling the synthesis of proline, arginine, putrescine,
   and GABA, conferring the importance of this amino acid to Cd
   hyperaccumulations [[116]43].

2.4.2. The Variations in Differential Carbohydrates under Cd Stress

   There were seven differential carbohydrates in the roots, among which
   the content of n-acetyl-d-glucosamine 6-phosphate (FC = 0.32),
   2-deoxyribose 5-phosphate (FC = 0.31), 2′-deoxy-d-ribose (FC = 0.29),
   1-deoxy-d-xylulose 5-phosphate (FC = 0.25), and UDP-glucose (FC = 0.22)
   decreased while ribitol (FC = 2.5) and galactinol (FC = 2.4) increased.
   Additionally, there were eight differential carbohydrates in the
   leaves, among which the content of gentianose (FC = 0.45),
   1-deoxy-d-xylulose 5-phosphate (FC = 0.44), UDP-d-glucose (FC = 0.43),
   perseitol (FC = 0.4), and n-acetyl-d-glucosamine 6-phosphate (FC =
   0.27) decreased while the content of threitol (FC = 3.59), galacturonic
   acid (FC = 2.58), and maltotriose (FC = 2.22) increased. Galactinol can
   scavenge ·OH as efficiently as glutathione to abate oxidative stress
   under Cd treatment [[117]44], while ribitol serves as a protectant
   against many abiotic stresses, such as heavy metal stress [[118]45] and
   cold stress [[119]46]. It has been reported that maltotriose could be
   an indicator of carbon pool reallocation under abiotic stress, and the
   increase of maltotriose could indicate starch hydrolysis countering Cd
   stress [[120]47]. In line with the study of Sun et al. (2020), threitol
   also accumulated in the leaves under Cd stress [[121]44]. It should be
   noted that changes in metabolites are associated with cell wall
   construction. Galacturonic acid is an important component of pectin,
   which approximately accounts for 70% of the cell [[122]48]. The
   methyl-esterification of galacturonic acid determines the negative
   charge in pectin and thus determines its metal binding quantity
   [[123]49]. Therefore, the increase in galacturonic acid in the leaves
   might enhance the content of Cd bound by the cell wall. In addition, Cd
   stress also reduced the UDP-glucose content, which is a shared
   differential product of roots and leaves. UDP-glucose, the activated
   form of glucose, serves as a precursor for the synthesis of
   UDP-glucuronic acid. The conversion of UDP-glucose to UDP-glucuronic
   acid is the first step of nucleotide sugar synthesis (UDP-apiose,
   UDP-arabinose, and UDP-xylose) [[124]50]. The UDP-glucose precursors
   were utilized by cellulose synthase to elongate the cellulose chain,
   which is the main component of the cell wall [[125]51]. Therefore, the
   decrease in UDP-glucose could limit the elongation of leaves and roots.
   Compared with CK, UDP-glucose decreased by 78.09% and 56.50% in the
   roots and leaves, respectively, indicating the greater inhibition
   effect of Cd ions on roots compared with leaves.

2.4.3. Alterations in the Differential Lipids Induced by Cd Stress

   Our study found that the content of 5-HETE (FC = 4.16), stearidonic
   acid (FC = 3.68), 4-oxoretinol (FC = 3.1), mesaconic acid (FC = 2.62),
   traumatic acid (FC = 2.25), and 3-hydroxy-3-methylglutaric acid (FC =
   2.17) increased and only linoleic acid (FC = 0.09) decreased in the
   roots. 5-HETE was also found to accumulate concomitantly with an
   oxidative burst in the roots under Cd stress [[126]52]. Parallel to our
   findings, stearidonic acid and 3-hydroxy-3-methylglutaric acid were
   found to be increased under Cd stress [[127]44,[128]53]. According to
   Wang et al. (2021), traumatic acid in the roots was found to increase
   under stress [[129]54]. In line with our results, linoleic acid has
   been reported to be downregulated in S. plumbizincicola under Cd stress
   [[130]12]. The content of 1-palmitoyl-sn-glycero-3-phosphocholine (FC =
   3.55), progesterone (FC = 2.4), and cucurbitacin d (FC = 2.23)
   increased, and the content of 11-beta-hydroxyandrosterone (FC = 0.45),
   trans-traumatic acid (FC = 0.43), oxandrolone (FC = 0.43),
   2-isopropylmalic acid (FC = 0.4), endo-borneol (FC = 0.31), and
   mesaconic acid (FC = 0.29) decreased in the leaves under Cd stress. The
   increase in abscisic acid could lead to a significant decrease in
   trans-traumatic acid content under Cd stress [[131]55,[132]56].
   2-isopropylmalic acid is the precursor of malic acid [[133]57];
   therefore, a decrease in 2-isopropylmalic acid could lead to a decrease
   in malic acid. By contrast, 1-palmitoyl-sn-glycero-3-phosphocholine and
   progesterone increased in the leaves. Progesterone, which is naturally
   present in plants, could significantly reduce the negative effects of
   adverse environments, stimulate antioxidant enzyme activity, reduce
   cell membrane permeability, and improve the efficiency of PSII
   [[134]58]. Mesaconic acid was a differential metabolite shared in both
   the roots and leaves in our results. Similar to our results, mesaconic
   acid in the leaves was significantly decreased with late exposure to
   soil imidacloprid [[135]59].

2.4.4. Most of the Differential Nucleotides in Roots Decreased under Cd
Stress

   We found nineteen differential nucleotides and derivatives in the roots
   under Cd treatment, of which twelve decreased and seven increased
   ([136]Table S2). In comparison, there were five differential
   nucleotides in the leaves, of which only guanosine (FC = 0.36)
   decreased while guanosine 5′-monophosphate (FC = 3.28), adenosine
   monophosphate (FC = 2.26), auanosine diphosphate (FC = 2.09), and
   uridine 5′-monophosphate (FC = 2.02) increased. Previous studies
   reported that the content of cytidine 5′-diphosphate decreased under
   heavy metal stress [[137]5]. Under stress conditions, plants inhibited
   the excessive accumulation of reactive oxygen species by consuming
   excessive reduction power in plant cells, thereby decreasing the NADH
   content [[138]60]. Similar to our studies, the content of flavine
   mononucleotide and 1-methyladenosine in the roots decreased
   [[139]61,[140]62]. The increase in adenosine monophosphate content
   could promote Cd transport to vacuoles and lower Cd toxicity [[141]63].

2.4.5. The Differential Organic Acid Variations under Cd Treatment

   Under Cd treatment, there were four differential organic acids in the
   roots, among which the content of glyoxylate (FC = 3.22) and glyceric
   acid (FC = 2.21) increased, while the content of 3-Hydroxybutyric acid
   (FC = 0.48) and gyruvaldehyde (FC = 0.26) decreased. In line with our
   results, glyceric acid in the roots of cucumber increased under Cd
   stress [[142]44]. Under Cd treatment, there were four different organic
   acids in leaves, among which only oxalate (FC = 3.17) increased while
   the content of citrate (FC = 0.44), malic acid (FC = 0.43), and
   glutaric acid (FC = 0.37) decreased. The increase of oxalate in the
   leaves has been reported to be beneficial for plants to counter Cd
   toxicity [[143]64]. In line with our results, the glutaric acid content
   in the leaves of cucumber decreased under Cd stress [[144]44].

2.4.6. The Differential Phytohormone Changes Induced by Cd Ions

   Our study found four differential phytohormones in the roots under Cd
   stress, among which the content of indole-3-butyric acid (FC = 5.79)
   and phenylacetic acid (FC = 5.08) increased under Cd stress, while the
   content of melatonin (FC = 0.45) and kinetin (FC = 0.21) decreased.
   Previous research found that indole-3-butyric acid could reduce Cd
   toxicity in the barley roots [[145]65], and the increase in
   indole-3-butyric acid might lead to an increase in phenylacetic acid
   [[146]66]. It has been found that exogenous kinetin could increase the
   fresh and dry weights of roots and shoots of sorghum plants under Cd
   stress [[147]67]. Therefore, the Cd-induced decrease in kinetin could
   lead to a decrease in root length and root fresh weight.
   Indole-3-acetamide (IAM) is a precursor of indoleacetic acid (IAA),
   which is a well-known growth regulator. The lower IAM content could
   lead to a decrease in IAA, therefore handicapping plant growth under Cd
   stress [[148]68].

2.4.7. The Alterations of Benzenoids, Phenylpropanoids, and Polyketides under
Cd Stress

   We found the content of hydroquinone (FC = 3.97 in leaves and FC = 3.76
   in roots) shared both in the leaves and roots was enhanced under Cd
   stress, which is in line with the study of Borgo et al. (2022)
   [[149]69]. Previous studies showed that exogenous hydroquinone
   inhibited cell growth and enhanced apoptosis gene expression [[150]70].
   Among the differential phenylpropanoids and polyketides, the
   bisdemethoxycurcumin in the roots showed a sharp increase (FC = 23.93)
   under Cd stress, which could effectively reduce oxidative stress
   [[151]71]. Neohesperidin also increased under Cd stress, indicating a
   higher heavy metal stress tolerance [[152]72]. Our results showed that
   genistein (FC = 4.83 in leaves and FC = 6.12 in roots), p-coumaric acid
   (FC = 3.00 in leaves and FC = 8.76 in roots), and
   3-hydroxy-4-methoxycinnamic acid (FC = 2.01 in leaves and FC = 9.31 in
   roots) were differential metabolites that were shared in both leaves
   and roots, which all increased more in the roots than in the leaves
   under Cd stress. Similar to Yadav et al.’s (2021) research, genistein
   has been found to increase under As stress in the roots, young leaves,
   and old leaves of rice [[153]62]. The function of some metabolites has
   been relatively clear; however, the functions of a lot of plant
   products remain unknown due to the large diversity and quantity of
   plant metabolites.

2.5. The Relationships of Main Metabolite Changes with BEs under Cd Stress

   In the OPLS analyses, Variable Importance Projection (VIP) values > 1
   indicated the important influence of one metabolite change in the BE,
   and this coefficient denoted the relationship (positive or negative) of
   one metabolite change with the BE. According to the result of OPLS,
   there were six amino acids’ VIPs > 1 in the leaves, among which five
   were positively correlated with the leaf length and leaf fresh weight
   while also appearing negatively correlated with the leaf MDA content
   ([154]Figure 4A–C, [155]Table S1). In contrast, there were 12 amino
   acids’ VIPs > 1 in the roots, among which nine were negatively
   correlated with root length and fresh weight but positively correlated
   with MDA ([156]Figure 4D–F, [157]Table S2). Under the Cd[10] treatment,
   the soluble protein content in the leaves was 2.58 times that of the
   roots, and the higher soluble protein content had certain
   detoxification effects. In addition, to maintain the higher soluble
   protein content, the leaves consumed more amino acids, which resulted
   in a reduction in the content of important amino acids and affected the
   growth of the leaves, thus showing positive relationships with leaf
   length and fresh weight. In contrast, under the Cd[10] treatment, the
   Cd content in the roots was 15.00 times that of the leaves; therefore,
   the roots were subjected to more toxicity than the leaves. The high Cd
   content could break down some proteins into amino acids while blocking
   the synthesis of amino acids into certain proteins, resulting in an
   elevated amino acid content in the roots [[158]73,[159]74]. The
   increase in the amino acid content was also needed for roots to resist
   high cadmium stress, which could lead to insufficient amino acid
   content for plant growth and ultimately inhibit plant growth, resulting
   in a decrease in the root length and weight with a negative
   correlation. The higher MDA content indicated severer oxidative stress
   caused by the Cd treatment; therefore, the increase in the amino acids
   could chelate the Cd ions and abate oxidative stress, showing positive
   relationships with the MDA content. In the leaves, there were two
   different carbohydrates’ VIPs > 1, which were positively correlated
   with the leaf length and leaf fresh weight but negatively correlated
   with MDA ([160]Figure 4A–C, [161]Table S1). There were also two
   differential carbohydrates’ VIPs > 1 in the roots, which were
   positively correlated with the root length and fresh weight while also
   appearing negatively correlated with MDA ([162]Figure 4D–F, [163]Table
   S2). Similar results were also found in organic acids such as citrate,
   malic acid, and pyruvaldehyde. It has been reported that heavy metal
   stress can destroy the chloroplast structure, reduce the ability of
   plants to capture light energy, and inhibit photosynthesis. This may be
   because the heavy metal cadmium replaced the plasma of iron and
   magnesium, which was bound with the sulfhydryl group on the chloroplast
   protein, reduced the activity of the chlorophyll enzyme, inhibited the
   synthesis of chlorophyll, and led to a decrease in the photosynthetic
   rate, thus decreasing the content of carbohydrates and organic acids
   and inhibiting plant growth [[164]75]. Therefore, the changes in
   carbohydrates and organic acids showed positive relationships between
   the length and fresh weight and negative relationships with the MDA
   content. There were two differential nucleotide VIPs > 1 in the leaves,
   which were negatively correlated with leaf length and leaf fresh weight
   but positively correlated with MDA ([165]Figure 4A–C, [166]Table S1).
   In comparison, three differential nucleotides’ VIPs > 1 in the roots,
   which were positively correlated with root length and fresh weight,
   were also negatively correlated with MDA ([167]Figure 4D–F, [168]Table
   S2). Nucleotides are closely related to protein synthesis. The two
   nucleotides in the leaves were increased, while the three nucleotides
   in the roots were decreased, which partially confirms that Cd hindered
   protein synthesis in the roots.

Figure 4.

   [169]Figure 4
   [170]Open in a new tab

   Relationships between the differential metabolite changes and
   biological endpoints (BEs) of fountain grass induced by Cd stress (n =
   3). (A) Leaf length as the BE. (B) Root length as the BE. (C) Leaf
   fresh weight as the BE. (D) Root fresh weight as the BE. (E) Leaf MDA
   content as the BE. (F) Root MDA content as the BE. The red and blue
   circles represent active and passive coefficients, respectively. The
   sizes of the circles represent the variable importance projection (VIP)
   values.

2.6. The Metabolic Pathway Changes under Cd Stress

   The metabolic pathways significantly changed under Cd stress. The Cd
   ions not only significantly impacted more metabolites and metabolic
   pathways in the roots than in the leaves but also indicated the larger
   impact of Cd stress on the roots than on the leaves. The metabolic
   pathways analysis showed that the Cd stress significantly altered seven
   pathways in the leaves, including glyoxylate and dicarboxylate
   metabolism (0.06, 2.03), purine metabolism (0.15, 1.83), pentose and
   glucuronate interconversions (0.1, 1.56), the citrate cycle (TCA cycle;
   0.15, 1.42), amino sugar and nucleotide sugar metabolism (0.14, 1.39),
   zeatin biosynthesis (0.03, 1.39), and pyruvate metabolism (0.15, 1.35)
   ([171]Figure 5A). In contrast, Cd stress significantly altered eight
   pathways in the roots, including the purine metabolism (0.16, 3.68),
   glycine, serine, and threonine metabolism (0.02, 3.48), arginine and
   proline metabolism (0.25, 2.25), aminoacyl-tRNA biosynthesis (0, 1.93),
   phenylalanine metabolism (0.19, 1.91), nicotinate and nicotinamide
   metabolism (0.21, 1.81), arginine biosynthesis (0.17, 1.42), and
   cysteine and methionine metabolism (0.09, 1.37) ([172]Figure 5B).

Figure 5.

   [173]Figure 5
   [174]Open in a new tab

   Top 10 metabolic pathways in the leaf or root of fountain grass
   affected by cadmium stress via pathway analysis from differential
   metabolites (n = 3). The pathway impact was computed from the pathway
   topological analysis and represented via the circle size. The p values
   obtained from pathway enrichment analysis were shown both by the x-axis
   (−log[10]) and color. (A) Leaf. (B) Root.

   Among the top 10 metabolism pathways in the roots or leaves, three
   shared metabolic pathways existed in both the roots and leaves,
   including purine metabolism, aminoacyl-tRNA biosynthesis, cysteine
   metabolism, and methionine metabolism. Purine metabolism is the only
   metabolic pathway that was shared in both the leaves and roots that was
   significantly affected by Cd stress, which was also closely correlated
   with nucleotide metabolism and was found to be significantly altered
   under Cd stress and other stresses ([175]Figure 6). It has been
   reported that purine metabolism plays a pivotal role in the
   accumulation, tolerance, and detoxification of Cd in Amaranthus
   hypochondriacus ‘K472’. In addition to the purine metabolism,
   aminoacyl-tRNA biosynthesis was found to be significantly changed under
   Cd stress [[176]6], salt stress [[177]76], chilling stress [[178]77],
   and drought stress [[179]40]. The aminoacyl-tRNA biosynthesis was
   strongly associated with significant changes in the protein metabolism
   and the large accumulation of amino acids ([180]Figure 6), leading to
   phenotypic damnification in fountain grass. According to a
   transcriptomic analysis, the cysteine and methionine metabolism
   pathways also significantly changed under Cd stress [[181]78]. Plants
   could produce cysteine-rich peptides and chelate Cd ions to produce
   non-toxic complexes that are transferred into the vacuoles to obviate
   high levels of free cytotoxic Cd in the cytosol [[182]79].

Figure 6.

   [183]Figure 6
   [184]Open in a new tab

   Effects of cadmium stress on the metabolic map of fountain grass (n =
   3). The metabolites in blue text were the differential ones. Green or
   yellow arrows denote metabolites in the leaf or root, respectively. The
   up or down arrow direction indicates if the metabolite was up-regulated
   or down-regulated under cadmium stress. The linking arrows among
   metabolites with full or dotted lines denote the direct or indirect
   reactions, respectively. (For an interpretation of these references