Metabolic And Biochemical Changes Associated With Root Growth Restriction Under Cd Stress During Maize Pre-Emergence

Cadmium (Cd) pollution of agricultural soils is a growing global problem. Plant growth restriction is the main visible symptom of Cd toxicity, and this metal may be particularly harmful to the preformed, seminal root during the pre-emergence stage. In the present study, we focused on Cd phytotoxicity on seminal root growth, nutrient composition, redox status, and hormone homeostasis during the pre-emergence stage, distinguishing between the root apex and the remaining root tissue. After 72 h of metal exposure (50 and 100 µM CdCl 2 ), root length and biomass was diminished, as well as Ca, Fe, Mg, and Mn contents. A redox imbalance was evidenced by changes in peroxidase activities and decreased ascorbate-dehydroascorbate ratio in both root parts. There was less accumulation of carbonylated proteins in both root fractions upon exposition to 50 µM Cd, compared to 100 µM Cd, and this was related to increased 20S proteasome activities. Cd incremented ABA, IAA, and SA contents, but drastically reduced the biologically active gibberellin GA4 and the conjugate jasmonoyl isoleucine (JA-Ile). We demonstrated that the whole root tissue is involved in maize response to Cd stress, which entails redox and hormonal rearrangements, probably directed to widen plant defense lines at the expense of root growth.


Introduction
Cadmium is a transition metal ion released to the environment by industrial activities and urbanization. In cultivated soils, Cd derives mainly from P fertilizers (Sterckeman et al. 2018). Due to its relatively high mobility and high toxicity for living organisms-even at very low doses-, cadmium is considered a While each plant hormone has its speci c pathway that acts in a non-redundant way, their activities are interconnected by a complex network, and it is the interaction and cooperation between hormones that dynamically regulate plants' development and physiology (Vanstraelen y Benková 2012). It has been described that the exogenous application of phytohormones reduces the toxic effects of metals, in part through the improvement of the cell antioxidant potential (Singh et al. 2016).
On the other hand, it is known that plant cell redox homeostasis is controlled by a complex system known as the Foyer-Halliwell-Asada pathway, responsible for reactive oxygen species (ROS) scavenging (Foyer y Noctor 2011). This antioxidant defense machinery includes enzymes and low molecular weight compounds, like glutathione (GSH) and ascorbic acid (ASC) (Foyer y Noctor 2016). Nevertheless, an excessive ROS production that overwhelms the protective antioxidant mechanism can occur when plants are subjected to adverse environmental conditions (Gill y Tuteja 2010). A consequence of cell redox imbalance is protein oxidative damage, commonly expressed by carbonyl group increases (Møller et al. 2007). Carbonylated proteins can form high-molecular-weight aggregates that compromise several cellular functions (Nyström 2005). Because protein carbonylation is a covalent, non-reversible modi cation, oxidatively damaged proteins have to be rapidly degraded, mainly by the 20S proteasome The emergence of maize coleoptile from the soil surface delimits the onset of plant phototrophic lifestyle and takes place at 5 to 7 days after planting under favorable, natural conditions (Abendroth et al. 2011). The embryonically preformed root type dominates during this stage of development (Hochholdinger 2009). Apart from being vital for the vigor of young maize plants during the rst weeks after germination, the embryonic root system is the rst plant organ expected to interact with the underground environment and eventually suffer the toxic effects caused by Cd present in soils (Tai et al. 2016). Several reports indicate a higher Cd 2+ in ux in the root tip region, even when cadmium acquisition could be achieved through the entire root utilizing metal transporters. Direct xylem loading due to the lack of Caspary band and higher expression of transport systems associated with Cd uptake located close to the root tip have been related to this phenomenon (Piñeros et al. 1998;Laporte et al. 2013;Chen et al. 2018). Thus, the root apex could be the main site prone to suffer the toxic effects of metal ions.
In this work, the impact of Cd on the nutrient composition, redox balance, and phytohormone pro le of embryonic maize roots was analyzed, distinguishing between the rst 5 mm from the root tip, considered the root apex (Ap), and the remaining root tissue (Rt). Because plants are still under a chemoheterotrophic lifestyle at the pre-emergence stage, our analysis lets aside the well-known effects of cadmium on photosynthesis.
Materials And Methods

Plant material and growing conditions
Seeds of maize (Zea mays L. cv 2741MGRR2 kindly provided by DON MARIO Semillas, Buenos Aires, Argentina) were imbibed and germinated on lter paper in a plastic box containing deionized water for 72 h. Then, uniformly developed seedlings with primary roots of approximately 1.5 cm length were carefully transferred to a hydroponic system containing 250 mL of diluted (1/10) Hoagland's nutrient solution (Hoagland y Aron 1950) without (control, C) or with 50 or 100 µM CdCl 2 ; 30 seedlings were distributed in each container. Cd speciation was calculated with Visual MINTEQ ver. 3.1 (J P Gustafssons, KTH, Sweden). In the nutrient solution containing 100 µM of Cd, about 88% was in Cd 2+ form, the most phytoavailable. Plants were grown in a controlled climate room at 24 ± 2°C. All the experiments were carried out in the darkness to mimic soil conditions during germination and post-germinative growth. After 72 h of treatment, roots were gently washed with distilled water. The tissue collected from each container was considered a biological replicate. Root length, fresh weight (FW), and dry weight (DW, determined after drying the roots at 80 ºC until constant weight) were measured. Also, dried root powder was used to determine Cd and nutrient content. Determinations were performed in parallel using root apical segments obtained from the rst 5 mm from the tip (Ap) or all the remaining root tissue (Rt).

Nutrient composition of maize roots
Elemental analysis was performed at the Spectrometry Core Facility INQUISAL, Universidad Nacional de San Luis (UNSL-CONICET). Brie y, dried root powder (50 mg) was homogenized in 1 mL of 65% (v/v) HNO 3 in an ultrasonic bath during 30 s. Then, 0.5 mL of H 2 O 2 was added, and the mixture was incubated for 1 h at 60°C in a thermostatic bath. After diluting the samples with ultrapure water, inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer Elan DRC) was applied to estimate Cd, Cu, Ca, Fe, K, Mg, Mn, P, S, and Zn content.

Enzymatic and non-enzymatic antioxidants
Protein extracts were prepared from 0.1 g of fresh tissue homogenized in 1 mL of 50 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.5% (v/v) Triton X-100, at 4°C. The homogenates obtained centrifuged at 13,000×g for 30 min at 4°C, and the supernatants were used for the assays. Protein content was estimated according to Bradford (1976).
Extracts were obtained by homogenizing 0.1 g of root tissue in 1 mL of 0.1 N HCl. After centrifugation (13,000×g, 30 min, at 4°C), the supernatants were used for the assays. A standard curve of commercial ASC was used for calibration.

Quantitative dot blot analysis of carbonylated proteins
Protein extracts were prepared by homogenizing 0.1 g of root tissue in 0.5 mL of loading buffer (60 mM Tris-HCl (pH 6.8), 5% (v/v) β-mercaptoethanol). After centrifugation at 26,000xg for 15 min at 4 ºC and protein derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) dot blot analysis was performed as described Weher and Levine (2012). Membranes were photographed and then analyzed with Gel-Pro software, and the amount of oxidized proteins was expressed as arbitrary units (assuming control value equal to 100 units), based on the absolute integrated optical density of each dot.

Proteasome activities
Proteasome activity in root tissue was determined as described by Kim et al. (2003). Protein extracts were prepared in 135 mM Tris-acetate buffer (pH 7.5) containing 12.5 mM KCl, 80 µM EGTA, 6.25 mM 2mercaptoethanol, and 0.17% (w/v) octyl-β-D-glucopyranoside. After homogenizing 100 mg of root tissue (Ap or Rd) in 0.5 mL buffer, the extracts were centrifuged at 6,400 g for 30 min at 4°C, and the supernatants were further used to determine chymotrypsin-like (Q), trypsin-like (T), and peptidylglutamylpeptide hydrolase (PGPH) activities (Matayoshi et al. 2020). Due to the extraction buffer interference with Bradford reaction, protein content was determined by the method of Lowry et al. (1951).

Plant hormone analysis
Hormone extraction and analysis were carried out as described in Durgbanshi et al. (2005), with few modi cations (Matayoshi et al. 2020). Absolute levels of hormones (indole-3-acetic acid, IAA; abscisic acid, ABA; salicylic acid, SA; jasmonic acid, JA; jasmonoyl isoleucine, JA-Ile, and gibberellins, GA3, 4, 7, 20) were determined using an ultra-performance liquid chromatography system (Acquity SDS, Waters Corp., Milford, MA, USA or Waters Alliance 2695, Waters Corp.) coupled to a triple quadrupole mass spectrometer (TQS, Micromass Ltd., UK). All data were acquired in negative electrospray mode and processed using MassLynx v4.1 software. Quantitation was achieved after external calibration with standards of known amount and referenced to actual sample weight.

Statistical analysis
Each container had 30 seeds from which 0.1 g of tissue was collected and considered as a biological replicate. Tables and gures show means ± SEM of three or ve independent experiments, with three biological replicates per treatment. Differences among treatments were analyzed by 1-way ANOVA, taking P < 0.05 as signi cant, followed by Tukey's multiple comparisons test Results And Discussion 3.1. Cadmium accumulation reduced maize root growth and modi ed root nutrient composition The presence of Cd in the hydroponic solution reduced maize root growth by about 70% in length and 45% in biomass (Table 1)  Plants have not developed any specialized uptake system for cadmium because this element has no biological function. Nevertheless, this metal can be easily taken up by plant roots through membrane transporters of essential nutrients (Sterckeman y Thomine 2020). Current evidence indicates that Cd root symplastic in ux in maize is controlled by high-and low-a nity transport systems (Redjala et al. 2009(Redjala et al. , 2010. Furthermore, cadmium can be strongly adsorbed on the maize cell wall, resulting in a large amount of Cd 2+ being retained in the root apoplast (Redjala et al. 2009). As

Cadmium differentially affected peroxidase activities along the root and disrupted ascorbate homeostasis
In maize seminal root, CAT and APX activities were mostly localized in the root tip (Ap), while GPX activity was predominantly in the remaining tissue (Rt) (Fig. 2 To counteract an excessive H 2 O 2 formation in plant tissues, non-speci c peroxidases acting on one-or two-electron donors (including phenolic compounds such as guaiacol) are usually induced. In plants, GPX activity is mainly located in vacuoles and cell walls but not in organelles (Asada 1992 (Bocova et al. 2012), but the activity of this enzyme was particularly impaired in the Rt, dropping to near to half under both Cd concentrations (Fig. 2). Because of a higher APX a nity for H 2 O 2 than CAT and GPX, it has been suggested that this enzyme has a more crucial role in the scavenging of ROS during abiotic stress (Sofo et al. 2015;Anjum et al. 2016a).
In both root portions, total ASC (ASC plus DHAs) levels augmented under Cd treatment due to a pronounced rise in DHAs content, resulting, at the same time, in the reduction of ASC/DHAs ratio (Table  3). This nding suggests that Cd altered the adequate functioning of the ASC-GSH cycle. 3.3 Cadmium-induced accumulation of oxidatively damaged proteins was prevented by 20S proteasome increased activity Protein carbonylation is considered a reliable parameter of oxidative stress (Shulaev y Oliver 2006). Also, the accumulation of oxidized proteins re ects the balance between their production and degradation, mainly by the 20S proteasome activity. Under our experimental conditions, only 100 µM Cd incremented protein carbonyl group content along the whole root (Fig. 3).
A time-dependent analysis of three peptidase activities was assayed for 50 µM Cd treatment. As it is shown in Fig. 4, the metal incremented 20S peptide hydrolyzing activities. At 72 h, all of them were signi cantly increased in the Ap, and also T and Q in the Rt. Thus, the lack of carbonylated protein accumulation in maize roots subjected to 50 µM treatment could be attributed to the increase in the activity of the 20S proteasome, in a similar way to that previously described (Pena et al. 2007).

Cadmium altered hormonal root homeostasis
Cadmium enhanced IAA and ABA levels in the entire root tissue, whereas SA content increased only in the Rt portion (Table 4). IAA increments by Cd in rice roots were related to the overexpression of the biosynthetic genes OsASA2 and OsYUCCA1 (Ronzan et al. 2019). Also, it has been described that Cd affects not only IAA content but also its distribution, metabolism, and transport (Chmielowska-Bak et al. 2014), suggesting an eventual switch to an alternative morphogenic root program to counteract metal stress (Hu et al. 2013;Fattorini et al. 2017;Piacentini et al. 2020  Cadmium increased the root concentration of GA20, the precursor of the active form 13-hydroxylated GA3. Interestingly, the total root content of GA3 remained similar to the control at 100 µM Cd but decreased in the Ap and increased in the Rt at 50 µM Cd. On the other hand, the contents of non-13hydroxylated GA7 and GA4 were reduced under both Cd treatments. A similar drastic decrease in GA4 content was reported during copper stress (Matayoshi et al. 2020).
The mechanism by which metals affect GA4 homeostasis could involve interference with hormone biosynthesis but also with subsequent gibberellin transformations. Conclusion Maize seedlings exposed to Cd arrested root growth, and the entire primary root was found to be involved in redox and hormonal adjustments to trigger and/or to support defense mechanisms to cope with Cd stress. The integrated analysis of our experimental data shows that Cd addition decreases the root content of several essential nutrients, disrupts ASC homeostasis, and causes a strong decline in GA4 and JA-Ile levels, along with root growth inhibition. Faced with the incapacity of maintaining ASC homeostasis, CAT and GPX would be alternative enzymatic defense lines for seminal roots to remove ROS excess during Cd stress. Finally, the 20S proteasome seems to be a relevant defense component to cope with the oxidative damage generated by cadmium during this early stage of plant development.

Declarations
Acknowledgments We thank Dr. Myriam S. Zawoznik for her helpful criticism and for improving the English. CLM is a Research Fellow at the UBA (Argentina). LBP and SMG are Career Investigators from CONICET (Argentina). Hormone measurements were performed at Servei Central d'Instrumentació Cientí ca (SCIC) of Universitat Jaume I (Spain).

Funding
This work was supported by the Universidad de Buenos Aires (20020130100132BA UBACYT), and from Consejo Nacional de Investigaciones Cientí cas y Técnicas (PIP 0266).

Con icts of interest
The authors have no con icts of interest to declare that are relevant to the content of this article. Figure 1 Root cadmium content. Maize seedlings were subjected to hydroponic culture without (control, C) or containing 50 and 100 µM of CdCl2. After 72 h of treatment, roots were harvested and used for Cd determination. Data are means ± SEM of ve independent experiments, with three biological replicates per treatment. Different letters indicate signi cant differences (P < 0.05), according to Tukey's multiple range test.

Figure 2
Effect of Cd on peroxidase activities. Maize seedlings were subjected to hydroponic culture without (control, C) or containing 50 and 100 μM of CdCl2 for 72 h. Determinations were performed on extracts