An isopentenyl transferase transgenic wheat isoline exhibits less seminal root growth impairment and a differential metabolite profile under Cd stress

Cadmium is one of the most important contaminants and it induces severe plant growth restriction. In this study, we analyzed the metabolic changes associated with root growth restriction caused by cadmium in the early seminal root apex of wheat. Our study included two genotypes: the commercial variety ProINTA Federal (WT) and the P SARK ::IPT (IPT) line which exhibit high-grade yield performance under water deficit. Root tips of seedlings grown for 72 h without or with 10 μ M CdCl 2 (Cd-WT and Cd-IPT) were compared. Root length reduction was more severe in Cd-WT than Cd-IPT. Cd decreased superoxide dismutase activity in both lines and increased catalase activity only in the WT. In Cd-IPT, ascorbate and guaiacol peroxidase activities raised compared to Cd-WT. The hormonal homeostasis was altered by the metal, with significant decreases in abscisic acid, jasmonic acid, 12-oxophytodienoic acid, gibberellins GA20, and GA7 levels. Increases in flavonoids and phenylamides were also found. Root growth impairment was not associated with a decrease in expansin (EXP) transcripts. On the contrary, TaEXPB8 expression increased in the WT treated by Cd. Our findings suggest that the line expressing the P SARK ::IPT construction increased the homeostatic range to cope with Cd stress, which is visible by a lesser reduction of the root elongation compared to WT plants. The decline of root growth produced by Cd was associated with hormonal imbalance at the root apex level. We hypothesize that activation of phenolic secondary metabolism could enhance antioxidant defenses and contribute to cell wall reinforcement to deal with Cd toxicity.

phase.Nearest the quiescent center (QC) is the proximal meristematic zone (MZ), where cells divide several times.Then, cells move through the transition zone (TZ) to the elongation zone (EZ), where they lose the capacity to proliferate but increase their length.Finally, cells reach the differentiation zone (DZ), acquiring their specific characteristics and functions (Baluska et al., 2010).Expansins are cell wall proteins implicated in acid-induced wall extension (Cosgrove, 2015).The members of the expansins superfamily are classified into four subfamilies: α-EXPANSIN (EXPA), β-EXPANSIN (EXPB), EXPANSIN-like A (EXLA), and EXPANSIN-like B (EXLB) (Kende et al., 2004).Together with their function during normal growth, expansins also play a critical role in plant adaptation to environmental stress via relaxation and extension of plant cell walls (Tenhaken, 2015).
However, adjustments in the redox and hormonal homeostasis are necessary for plants to cope with abiotic stress (Bartoli et al., 2013;Raja et al., 2017).Exogenous application of hormones usually alleviates metal toxicity, the modulation of redox balance being one of the mechanisms associated with cell protection (Sytar et al., 2019).The modification in the endogenous cytokinin levels (CKs) during abiotic stress suggests the involvement of this hormone in plant stress response (O'Brien & Benkova, 2013).In this context, engineering CKs metabolism was considered a promising way to improve crop plants to cope with adverse growth conditions (Wani et al., 2016;Zalabák et al., 2013).
The enzyme isopentenyl transferase (IPT) catalyzes the first and rate-limiting step in the biosynthesis of CKs (Sakakibara, 2006).Plants overexpressing IPT under the control of different promoters have been developed the last years to improve the level of CKs.One of the promoter used is the maturation-and stress-inducible SENESCENCE ASSOCIATED RECEPTOR PROTEIN KINASE promoter (P SARK ) isolated from Phaseolus vulgaris.Consequently, transgenic plants expressing the IPT gene under the control of P SARK have been regarded as useful to maintain higher CKs concentration during stress, but not high enough to impinge on developmental processes (Delatorre et al., 2012;Peleg et al., 2011).
The root system of wheat comprises two root types: (1) the seminal roots, which emerge when seeds germinate, and (2) the nodal roots (adventitious or crown roots), which arise from the lower nodes of the shoot.The early seminal roots support the plant growth until the nodal roots emerge and are the primary organ that eventually comes in contact with pollutants present in soils.Thus, in wheat, as in other cereals, control and reprogramming early seminal root growth during abiotic stress is essential for plant emergence and survival.
Previous research conducted by our group indicated that wheat root apex was particularly affected by metal stress, which resulted in increased ROS levels and impaired cell cycle and RAM development, leading to severe root growth decrease (Pena et al., 2012(Pena et al., , 2015)).
However, information regarding primary metabolic responses in the RAM due to toxic Cd levels is still scanty.Herein, we studied the metabolic response induced by cadmium in the root apex and analyzed the antioxidant enzyme activities, the hormonal balance, and root apex's metabolite profile.Since RAM development depends on auxin and cytokinin crosstalk (Su et al., 2011), and considering that Cd stress affects water relations and water limitation restricts plant growth, we decided to use a wheat line expressing the P SARK ::IPT construction.This line was selected because of its high-grade yield performance under water deficit and is a good tool to investigate possible metabolic and biochemical differences in response to Cd stress.

| Plant material and growing conditions
Seeds of Triticum aestivum L. cultivar ProINTA Federal (WT) and a transgenic isoline expressing P SARK ::IPT (IPT) were used for the experiments.The P SARK ::IPT isoline contains the cassette SARK:IPT:NOS, which carries the IPT gene from Agrobacterium tumefaciens under the regulation of the SARK promoter from bean and the nopaline synthase (NOS) terminator (Rivero et al., 2007).Based on the fact that Cd induces dehydration in plants (Ruci nska-Sobkowiak, 2016), the IPT line used in this work-among the six events obtained-was selected because of its better yield performance under water deficit in greenhouse experiments (Beznec, 2016).Seeds were placed in flasks containing 30 mL of demineralized water (WT or IPT) or 10 μM CdCl 2 (Cd-WT or Cd-IPT), 20 seeds per flask.Flasks were randomly distributed in a rotary shaker (100 rpm) and incubated at 24 ± 2 C in darkness.At least eight flasks per treatment were prepared for each experiment and three independent experiments were performed.The Cd concentration used was selected according to our previous experience (Pena et al., 2012) and corresponds to the lowest dose that caused growth differences between lines.It is a realistic concentration that can be found in soils used to cultivate edible crops (Mahmood et al., 2019) and it is also within the range commonly used to study the effect of this metal in wheat plants.To let aside cadmium effects on photosynthesis, we evaluated Cd effect on root apex before the time to which, theoretically, wheat coleoptiles sprout through the soil surface and phototrophic lifestyle begins.After 72 h of incubation, the seedlings contained in each flask were gently washed with distilled water and used for the assays.Germination percentage was always above 80%, and it was unaffected by the experimental conditions.As plant growth parameters, the longest root's length and root fresh (FW) and dry weight (DW) (oven-dried at 75 C until constant weight) were recorded.For analytical determinations, apical root segments (first 10 mm from the root tips) obtained from all seedlings in different flasks were pooled and used to prepare one extract.

| Root meristem-size measure
Roots were fixed overnight in FAA (paraformaldehyde:ethanol:glacial acetic acid 1:4:1) dehydrated in an ethanol series, transferred to xylene, and processed by the inclusion in paraffin.Longitudinal apex sections of 10 μm were cut following standard botanical microtechniques.The slides were stained with a safranin-fast green combination and mounted on a glass slide with dibutylphthalate polystyrene xylene (DPX) and then observed using a Zeiss optic light microscope equipped with a digital photo camera.The distance between the QC and the first cortical elongated cell in the TZ was measured using the software analysis package ImageJ (http://rsb.info.nih.gov/ij).For three independent experiments, at least 5 roots were analyzed, and the mean and SEM were calculated (n = 15).

| Expansin transcripts profile
Transcript profiles of expansins were determined by reverse transcriptase polymerase chain reaction (RT-PCR) in root tip tissues, according to the method described by Marone et al. (2001).Total RNA was extracted from root apical segments using a TRIzol (Quick-ZOL, Kalium Technologies) procedure as described by the manufacturer.
RNA was treated with DNase Turbo (Life technologies) and converted to cDNA with oligo (dT)18 using the RevertAid Reverse Transcriptase (Thermo Scientific).Primers' sequences and PCR conditions are described in Table S1.PCR reactions were performed using a Termocycler T 18 (Ivema).PCR products were separated by electrophoresis on 1% (w/v) agarose gels and visualized with DSView Nucleic Acid Stain (Dongssheng biotech).Wheat TUBULIN was used as the housekeeping gene.Gels were photographed and analyzed with Image J software.Data are expressed as arbitrary units (a.u.) based on the ratio of the absolute integrated optical density of each EXPANSIN band and the corresponding TUBULIN band.Each expression profile shown is representative of three independent experiments with two replicates.

| Hydrogen peroxide determination
Hydrogen peroxide content was determined by homogenizing 200 mg of root apex tissue in 0.5 mL 0.1% (w/v) trichloroacetic acid (TCA).
After centrifuging at 12,000g for 15 min, 0.2 mL of supernatant was mixed with 0.2 mL of 10 mM phosphate buffer (pH 7.0) and 0.4 mL of 1 M potassium iodide (KI), and the absorbance at 390 nm was recorded (Singh et al., 2006).H 2 O 2 content was calculated from a standard curve of H 2 O 2 .

| Antioxidant enzyme activity assays and peroxidase zymogram
Protein extracts were prepared by homogenizing apical root segments (from at least 15 seedlings of each flask) in 1 mL of extraction buffer containing 50 mM potassium phosphate buffer (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v) polyvinylpyrrolidone (PVP) and 0.5% (v/v) Triton X-100 at 4 C. Homogenates were centrifuged at 26,000g for 30 min, and the supernatant fraction was used for assays.Measurements represent the maximal extractable activities of antioxidant enzymes (Noctor et al., 2016).Superoxide dismutase and guaiacol peroxidase activities were quantified in a final volume of 0.2 mL in 96-well UV-microplates using a FlexStation 3 microplate reader, equipped with an internal shaker and temperature incubator (Pena et al., 2020).Superoxide dismutase activity was assayed by the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Giannopolitis & Ries, 1977), and SOD-specific activity was expressed as units per mg of protein.Catalase activity was determined in apex homogenates, according to Aebi (1984).The pseudo-first-order was determined, and catalase content (pmol mg −1 ) protein was calculated using k = 4.7 × 10 −7 M −1 s −1 .Ascorbate peroxidase activity was measured as described by Nakano and Asada (1981).One unit of APX was defined as the number of nmoles of oxidized ascorbate formed per min and mg of protein.Guaiacol peroxidase activity was determined in the homogenates by measuring the increase in absorption at 470 nm due to the formation of tetraguaiacol (ε = 26.6 mM −1 cm −1 ), in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7), 0.1 mM EDTA, 10 mM guaiacol, and 10 mM H 2 O 2 .One unit of GPOX was defined as the amount of tetraguaiacol formed (μmol) per minute.Data are representative of three independent experiments with three replicates.At least three technical replicates of each protein extract were used for each enzyme activity determination.
Protein concentration was determined according to Bradford (1976), using bovine serum albumin as standard.

| Endogenous phytohormone determination
Hormone extraction from the root apex was carried out as described in Durgbanshi et al. (2005) with modifications (Matayoshi et al., 2020).
For hormone analysis, samples were injected into an ultraperformance liquid chromatography system (Acquity SDS or Waters Alliance 2695, Waters Corp.).Chromatographic separations were carried out on a reversed-phase C18 column (Gravity, 50 × 2.1 mm 1.8-μm particle size, Macherey-Nagel GmbH), using a methanol:water (both supplemented with 0.1% acetic acid) gradient at a flow rate of 300 μL min −1 .Gibberellins (GA 3 , GA 4 , GA 7 , GA 20 ), abscisic acid (ABA), jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), JA-isoleucine conjugate (JA-Ile), indole-3-acetic acid (IAA), and salicylic acid (SA) were quantified with a triple quadrupole mass spectrometer (Micromass) connected online to the output of the column through an orthogonal Z-spray electrospray ion source.The spectrometer was operated in negative ionization electrospray mode, and plant hormones were detected according to their specific transitions using a multi-residue mass spectrometric method.
Sample preparation was carried out as described by Zandalinas et al. (2017).Chromatographic separations were performed on a 100 mm × 2.1 mm internal diameter, 1.6 μm, Luna Omega Polar C18 (Phemomenex) column, using acetonitrile and ultrapure water, both supplemented with formic acid at a concentration of 0.1% (v/v), as solvents with an Acquity SDS system (Waters Corp. Ltd.) interfaced to a QTOF Premier from Micromass Ltd. through an ESI source (Zandalinas et al., 2017).Samples were analyzed in both negative and positive ionization modes in the 50-1000 amu scan range using a cap-

| Statistics
Values are expressed as means ± SEM.Differences among treatments were analyzed using InfoStat software (Di Rienzo et al., 2019) by two-way ANOVA, taking P < 0.05 as significant according to Tukey-Kramer's multiple range test.
To investigate any underlying data structure in metabolomics data, raw xcms output matrices were normalized as above and subjected to principal component analysis (PCA).Moreover, following ANOVA and annotation of mass chromatographic features as individual metabolites, results were row-wise standardized and represented as heatmap for a better visualization after row-wise standardization.
Annotated metabolites in rows were subjected to hierarchical cluster analysis (HCA) to investigate potential co-regulations, setting 'Euclidean distance' as the distance metrics and 'complete' as the clustering method.
3 | RESULTS AND DISCUSSION

| Cadmium decreased root elongation and affected root apical meristem size
The decrease in root growth induced by heavy metal has been reported to be related to a decrease in cytokinins production (De Smet et al., 2015).Also, it is well known that heavy metal stress affects plant water relations, reducing plant growth and affecting various biochemical and metabolic processes (Ruci nska-Sobkowiak, 2016).
Thus, to add information regarding root metabolic responses to Cd stress, a comparative analysis was performed using a transgenic wheat line expressing P SARK ::IPT.
Under no stress germination conditions, the IPT line showed similar root length but higher root biomass (20% FW and 14% of DW) compared to the WT line (Table 1).This slight increment in root biomass could represent a better carbon (C) and nitrogen (N) remobilization from seed reserves to the root in the IPT line.
Cadmium treatment negatively affected root biomass in the same proportion in both lines.However, root length reduction was higher in WT than in IPT plants (30 and 20%, respectively, compared to the respective control; Table 1).Li et al. (2011a) reported that root length was the most sensitive to cadmium toxicity among various wheat growth parameters assessed.To evaluate if the cadmium-induced reduction of root elongation could be attributed, at least in part, to differences in cell expansion, the distance from the QC to the TZ was determined in longitudinal root apex cross-sections.Interestingly, the distance in IPT lines was diminished compared to that in the WT under no stress condition (Figure 1).It is well known that an antagonistic interaction between cytokinins and auxins in the TZ regulates RAM development (Perilli et al., 2012;Schaller et al., 2015;Su et al., 2011).
On the one hand, auxins support meristem activity by promoting cell division.On the other hand, cytokinins promote cell differentiation by repressing auxin signaling and transport.Thus, the RAM size decrease is consistent with cytokinins' effect in IPT lines (Dello Ioio et al., 2007).Cadmium decreased RAM size in WT wheat seedlings but increased QC-to-TZ distance in the IPT line (Figure 1).This data reinforces the idea proposed by Bruno et al. (2017), who suggested that Cd differentially affected cell expansion by altering auxin/cytokinin signaling homeostasis.
In this regard, the expression of four wheat EXPANSIN genes with higher expression in the roots than other organs (Han et al., 2019;Zhang et al., 2018) was evaluated in this study.As a general trend, the expression level of the expansin genes analyzed was unaffected by Cd addition in the IPT line but augmented in the WT.The TaEXPA6 expression was similar in both lines and unaffected by the metal, whereas TaEXPB8 expression was higher in the IPT line and rose upon Cd treatment only in the WT line.Among the four analyzed expansins, three of them were upregulated in the IPT line, suggesting that they were CKs-dependent, while Cd increased their expression only in the WT line (Figure 2).Consequently, an enhanced expression of EXPs in the root growth zone could be contributing to the maintenance of primary growth during stress (Cosgrove, 2015).It is important to highlight that 241 expansin genes were recently identified in the wheat genome (Han et al., 2019).However, the specific physiological role of each expansin under normal or stressful conditions is still unknown.In  (Feng et al., 2019).Furthermore, the ectopic expression of TaEXPB23 was associated with phosphorus availability (Han et al., 2014) and drought (Li, Xing, et al., 2011b), as well as in oxidative stress tolerance (Han et al., 2015;Li et al., 2015).Also, the overexpression of the TaEXPA2 gene in tobacco was involved in drought stress tolerance and improved salt tolerance by regulating Na + /K + interchange (Chen et al., 2016(Chen et al., , 2017)).In tobacco, TaEXA2 induced oxidative stress tolerance by upregulating the expression and activity of cell wall peroxidases (Chen et al., 2018); besides, this expansin modulated the absorption and transportation of the metal to cope with Cd stress (Ren et al., 2018).Interestingly, in these studies, it was determined that expansins overexpression was related to hormone response and improved antioxidant capacity during stress.

| Cadmium altered redox status in the root apex
In various wheat cultivars, cadmium tolerance has been related to the antioxidant capacity (Afzal et al., 2019;Guo et al., 2019;Wu et al., 2003).Our results showed that Cd decreased SOD activity by 33 and 24% in Cd-WT and Cd-IPT, respectively, compared to untreated controls (Figure 3).SOD is considered the first line of antioxidant defense to control the relative amounts of O 2 − and H 2 O 2 .
The SOD activity decline could generate a disruption in the balance between O 2 − and H 2 O 2 , and thus affect the normal RAM development (Tsukagoshi et al., 2010).
A slight increment in CAT activity was measured in the IPT line compared to the WT, but a significant increase was observed in Cd-WT (63% over WT control) (Figure 3).Catalase is a peroxisomal antioxidant enzyme involved in regulating H 2 O 2 levels not only under physiological but also during stress conditions (Sandalio & Romero-Puertas, 2015).The role of CAT in overcoming Cd-induced toxicity has been well documented in different plant species, developmental stages, and plant tissues, but its involvement in the root apex is reported here for the first time.Guo et al. (2019) reported CAT activity increases in the leaves of two wheat varieties exposed to Cd with different tolerance to this metal, but no information related to the root apex was reported before.
APX and GPOX significantly increased in Cd-IPT lines compared to Cd-WT (Figure 3), indicating an adjustment of the cell redox balance through the induction or activation of peroxidases.Also, the decrease in peroxidase activities in Cd-WT correlated with the rise in H 2 O 2 (Figure 4), consistently with the ROS accumulation generated by Cd in wheat root tips already communicated (Pena et al., 2012).
The variable responses of peroxidase activity to Cd can be partly associated with the subcellular distribution of the metal, but also to Cd-susceptibility of the antioxidant enzymes located in subcellular compartments (Li, Zhou, et al., 2011a).An elevated activity of phenolic-oxidizing enzymes, such as GPOX, in root tips was associated with increased phenolics contents and lignification in response to Cd (Schützendübel et al., 2001).Likewise, an improvement in antioxidant defenses due to the increased peroxidase activity was described in P SARK ::IPT tobacco plants under nitrogen deficiency (Rubio-Wilhelmi et al., 2011).This response was consistent with the F I G U R E 2 Expansin genes transcript accumulation in root apex.
Semi-quantitative RT-PCR analysis of TaEXPA6, TaEXPA8, TaEXPB8, and TaEXPB10 genes was preformed using specific primers for wheat expansins.TUBULIN4 transcript was used as housekeeping gene.
Values are mean ± SEM of three independent experiments with two replicates (n = 6).Different letters indicate significant differences (P < 0.05) according to Tukey-Kramer's multiple range test Effect of cadmium on superoxide dismutase and peroxidase activities.Enzymatic activities were assayed in root tip extracts, as indicated in materials and methods, and expressed as a percentage of the WT activity.Values of specific activities in control plants: SOD 0.024 ± 0.009 U mg −1 protein, CAT 2.1 ± 0.2 pmol mg −1 protein, APX 23.4 ± 2.1 U mg −1 protein, GPOX 187 ± 20 U mg −1 protein.
Values are mean ± SEM of three independent experiments with three replicates (n = 9).Different letters indicate significant differences (P < 0.05) according to Tukey-Kramer's multiple range test already documented CKs' protective role and the antioxidant system adjustment under stress (Ahanger et al., 2018;Hönig et al., 2018;Synkova et al., 2006).
Despite GPOX activity was differentially affected by Cd in IPT and WT plants, a similar profile in POX isoenzymes was observed in the zymogram (Figure 5).The presence of at least eight bands (POX1 to POX8) was visualized in both genotypes.In particular, the diversity of isoforms, distribution, and the variety of substrates used by these enzymes place peroxidases as essential proteins regarding plant plasticity and response to abiotic stress (Jovanovi c et al., 2018).In the root apex, peroxidases are involved in the control of RAM size by modulating ROS balance in the TZ (Tsukagoshi et al., 2010).Also, in the root apex of wheat genotypes, peroxidases have been associated with different tolerance degrees to stress (Csiszár et al., 2012).

| Cadmium-induced endogenous phytohormone imbalance in the root apex
Plant responses to Cd involve multiple signaling pathways that regulate many aspects of plant growth and development (Gallego et al., 2012).Plant hormones integrate a complex network of interactive pathways involved in plant growth and development and efficient stress responses (Verma et al., 2016).Herein, plant hormone profiles in response to cadmium were analyzed in the root tip.Two-way ANOVA showed that Cd influenced ABA, OPDA, JA, and GA20 contents (P < 0.05).Indole acetic acid was the only hormone that was slightly incremented by Cd exposure in both lines (Table 2), in association with shorter RAM (Figure 1).In line with this finding, the accumulation of IAA and the root growth reduction were previously described in barley root apex after Cd treatment (Tamás et al., 2015).Our results using an IPT line support the idea that not only the amount but also the IAA and CKs homeostasis would determine the size of the RAM during Cd stress.Early studies suggested that perturbation of auxin distribution, more than its content, is the most relevant effect of Cd in the root tip (Bruno et al., 2017).
The presence of Cd decreased the ABA content in the root tip (Table 2).This could be attributed to metal-triggered water imbalance in the root tip (Kuromori et al., 2018).Previous findings demonstrated that root length and seedling fresh weight were comparable in the wild type and ABA-deficient and ABA-insensitive Arabidopsis thaliana mutants during Cd exposure (Sharma & Kumar, 2002), suggesting the lack of involvement of this hormone in Cd-induced growth restrictions.
Cd caused decreases in JA and OPDA (JA-precursor) levels in both lines (Table 2), indicating metal interference with the JA biosynthetic pathway.Other researchers documented JA and ABA reductions in citrus roots subjected to Cd 2+ (López-Climent et al., 2011).
Previous reports correlated the deficiency in endogenous JA level with the enhanced sensitivity of tomato seedlings to Cd stress (Zhao et al., 2016).Conversely, exogenous JA application minimized Cd accumulation in roots and mitigated the negative impact of Cd stress (Ahmad et al., 2017).Among the jasmonates derivatives, JA conjugated with isoleucine (JA-Ile) represents the most active form of this plant growth regulator and triggers defense responses (Fonseca et al., 2009).Interestingly, the IPT line showed increased JA-Ile con- Cd treatment resulted in a decreased level of GA20, the precursor of the bioactive 13-hydroxylated forms GA1 and GA3 (Table 2).Since GA intermediates are involved in the long-distance transport of active GAs (Binenbaum et al., 2018), it is possible to assume that Cd interfered with GA20 movement toward the root tip.Nevertheless, the GA20 transformation into the bioactive forms GA1 and GA3 was unaffected by the metal.The most abundant bioactive GA in the root tip was the non-13-hydroxylated GA7.Cd produced an opposite effect in the non-13-hydroxylated GAs by decreasing GA7 and increasing GA4 amount (Table 2).Interestingly, since bioactive GAs synthesis occurs at the same site of action, Cd could have differentially impacted each cell line at the RAM level.Thus, although RAM morphology changes may have been caused mainly by a hormonal imbalance governed by the auxin/cytokinin ratio, gibberellins involvement cannot be excluded.of the total variation, 54.5% for PC1 and 21.9% for PC2 for the negative ion mode.Thus, from the PCA score plots of both ion modes, it may be deduced that differentiation among groups occurred mainly along PC1, and the largest source of variation was Cd treatment.This result suggests that metal addition is the main driver of the changes observed.After multivariate and univariate analyses, the selected parent ions were retrieved using online databases to obtain the corresponding candidate compounds (Table S2).
An unsupervised multivariate HCA was applied to the hydrophilic metabolites that showed statistical differences among the samples to visualize general grouping information (Figure 6).In general, the dendrograms grouped compounds into two clusters: metabolites that were reduced (up cluster) or increased (down cluster) by Cd in both lines.
Fatty acid, phospholipids, and sphingolipids are among the metabolites decreased by the metal (Figure 6).Taken into account that these compounds comprise a metabolic group that is not long-distance transported in the plant, this result could indicate that Cd induced modification or redistribution of lipid metabolism at the apex level.
Remodeling membrane lipid composition has been described as a potential mechanism to enhance plant abiotic stress tolerance (Liu et al., 2019).Also, the decrease in JA content would be in agreement with the observed alterations in the metabolism of unsaturated fatty acids (Table 2).
Metabolic adjustment included the increment of some phenolic secondary metabolites such as flavonoids and phenylamides.Flavonoids are mainly biosynthesized by the phenylpropanoid pathway from shikimate (Nabavi et al., 2020) and are recognized for their key role in plant defense against both biotic and abiotic stress and also for their involvement in plant-microorganism interactions (Falcone Ferreyra et al., 2012).Among their various molecular properties, antioxidant and ion-chelating capacities were described (Cherrak et al., 2016).Phenylamides (PhA)-conjugates of biogenic aliphatic polyamines or arylmonoamines and hydroxycinnamic acids are a widespread class of secondary metabolites distributed in plants (Bassard et al., 2010).Cadmium increased the PhA level, particularly those formed from putrescine and agmatine with hydroxycinnamoyl, coumaroyl, or feruloyl substitutions.During abiotic stress, PhA may act as antioxidant agents and ROS scavengers (Edreva et al., 2007).As substrates for peroxidases, they may have a role in eliminating H 2 O 2 and participating in the cell-wall reinforcement by cross-linking (Bassard et al., 2010).The participation of PhA in the crosstalk between phenolic and nitrogen metabolism has been suggested previously (Bassard et al., 2010).Further studies specifically designed to gain a deeper knowledge about PhA metabolism in wheat root apex under cadmium stress are certainly needed.

| CONCLUSION
In the present work, we added valuable information regarding redox, hormonal, and metabolite adjustments occurring in the wheat root apex in response to Cd stress, using transgenic wheat plants expressing P SARK ::IPT construction, taking advantage of the water deficit tolerance of this line to study its behavior under Cd stress.
Our results suggest that the P SARK ::IPT construction could add an adaptive advantage to the normal cell homeostasis, enabling better performance of young seedlings to cope with Cd stress.Among these features, hormonal and metabolic adjustments in the root apex and upregulation of several expansin genes at the root apex may be illary and cone voltages of 3.5 KV and 30 V, respectively.Mass chromatograms were centroided using the accurate mass of the molecular ion of leucine enkephalin(ESI+ 556.2771 and ESI− 554.2625).After acquisition, files were converted to NetCDF for subsequent xcms processing.Chromatographic peak detection was performed using the matchedFilter algorithm, and retention time correction was achieved in three consecutive iterations reducing bandwidth.Ion type identification, adduct annotation, and grouping of related mass chromatographic features were achieved with the CAMERA package(Kuhl et al., 2012).Normalization of peak areas was achieved by taking into account the internal standard biochanin area and the actual sample weight.Significantly-altered mass chromatographic features were identified after ANOVA using genotype and treatment as factors.Annotation of metabolites was achieved by comparison of mass spectra and retention time with an in-house-built database.Additionally, unknown metabolites were identified by manual curation of mass pseudospectra (generated by CAMERA) and comparison of actual pseudomolecular ions and fragmentation patterns with those available in public databases (Metlin, MassBank, and HMDB).
Wheat wild type (WT) and transgenic plants expressing P SARK ::IPT (IPT) seeds were germinated and grown in flasks containing 30 mL of water or 10 μM of CdCl 2 for 72 h.Fresh and dry weight (FW and DW, respectively) are expressed in mg per 25 seedlings.Values represent means ± SEM.Different letters indicate significant differences at P < 0.05 according to Tukey-Kramer's multiple range test.F I G U R E 1 (A) Schematic longitudinal view of the root apical meristem (RAM) structure: Quiescent center (QC), meristem zone (MZ), transition zone (TZ) and elongation zone (EZ).(B) Representative images of longitudinal cross-sections of the wheat root apex.Wheat seeds from WT and IPT line (20 per flask) were germinated in 30 mL of water or 10 μM CdCl 2 for 72 h.empty and filled black arrowheads indicate the QC and the cortex TZ in the RAM, respectively.(C) Distance from the QC to TZ.Values are mean ± SEM of three independent experiments with five roots analyzed (n = 15).Different letters indicate significant differences (P < 0.05) according to Tukey-Kramer's multiple range test this regard, it has been demonstrated that overexpression of TaEXPB7-B in Arabidopsis enhanced plant tolerance to lowtemperature stress tent compared with the WT, but Cd treatment reduced JA-Ile content in the root apex.Given that JASMONOYL-ISOLEUCINE SYNTHASE (JAR1) is involved in JA-Ile synthesis, further studies are necessary to decipher the factors that regulate this enzyme activity in the RAM.F I G U R E 4 Hydrogen peroxide content in the root apex.Wheat seeds from WT and IPT line (20 per flask) were germinated in 30 mL of water or 10 μM CdCl 2 for 72 h.values are mean ± SEM of three independent experiments with three replicates (n = 9).Different letters indicate significant differences (P < 0.05) according to Tukey-Kramer's multiple range test F I G U R E 5 Peroxidase (POX) isoform profile.Proteins (50 μg protein per well) were separated by native-PAGE and stained for peroxidase activity, as described in materials and methods

3. 4 |
FiguresS1 and S2.The first principal components (PC) explained almost 69% of the total variation, 51.5% for PC1 and 17.1% for PC2 for the positive ion mode.Similarly, the first PC explained almost 77%

F
I G U R E 6 Metabolites heatmaps combined with hierarchical clustering analysis.Wheat seeds from WT and IPT line (20 per flask) were germinated in 30 mL of water without or containing 10 μM CdCl 2 for 72 h. the column represents the samples and the row displays metabolite.The blue color represents a decreasing trend and red a rising trend mentioned.Changes in phenolic secondary metabolites could lead to an improved antioxidant ability and cell wall reinforcement, resulting in less Cd toxicity inside root apex cells.
Phytohormone contents in root apex segmentsNote: Wheat wild type (WT) and transgenic plants expressing P SARK ::IPT (IPT) seeds were germinated and grown in flasks containing 30 mL of water or 10 μM of CdCl 2 for 72 h.Mean values ± SEM are shown.Different letters indicate significant differences (P < 0.05) according to Tukey-Kramer's multiple range test.
T A B L E 2