Root morphology and kinetics of Zn absorption by roots of common bean influenced by Zn status of the root environment¹

ABSTRACT

Understanding the kinetics of Zn absorption by roots and its effect on morphology of this organ is relevant for improving crop management, but still poorly studied for common beans. Therefore, an experiment was conducted in a hydroponic system with five initial concentrations of Zn (CZnI): 0.0; 1.0; 4.0; 16.0 and 48.0 µmol L^-1. The experiment was installed with plants at V3 stage of development and aliquots of the solution collected over 24 h. The maximum absorption rate (Vmax), Michaelis-Menten constant (Km) and the absorption power (α) increased as a function of CZnI. The minimum concentration of Zn estimated for its absorption (Cmin) was at 0.0028 mg L^-1. The influx of Zn (Imax) was higher in higher CZnI, 16,0 µmol L^-1. Root length, root volume, root Zn content and Zn absorption efficiency increased with the increase of CZnI. Therefore, the increase of CZnI positively influenced kinetic parameters of root Zn absorption and common bean root morphology, characteristics that favor Zn absorption by roots and improves overall plant nutrition, favoring agronomical biofortification practices for Zn and other nutrients.

Keywords:
root Zn inflow; absorption efficiency; kinetic parameters; Vmax; Cmin; Km

INTRODUTION

Zinc (Zn) is an essential element for plant growth and development, which is absorbed primarily by the roots (Longnecker & Robson, 1993; Cakmak, 2008). Due to the metabolic functions of Zn, this nutrient is a plant growth promoter, regulating different morphological, physicochemical, molecular and metabolic processes in cultivated plants (Patel et al., 2018).

The absorption of Zn²⁺ from the soil solution by the roots is a dynamic, complex and still poorly understood process. The accumulation of Zn in the roots over time is known to be biphasic, comprising the initial phase of rapid intake due to its binding on the negative charges present on the root cell walls, followed by the slower phase when the nutrient is transported through the plasmalemma (Hacisalihoglu et al., 2001; Meng et al., 2014). The absorption kinetics of Zn by wheat follows the Michaelis-Menten model and shows the rapid phase in the first 6 h and the slowest phase during the subsequent period (Hacisalihoglu et al., 2001). In bean plants, Zn absorption occurs predominantly fast, irreversibly and without metabolic energy expenditure (Joseph et al., 1971; Broughton et al., 2003).

Common beans, despite being one of the most consumed foods in underdeveloped countries in Latin America and Africa, few studies have been conducted on the absorption of Zn by the roots of the plant, especially when the interest is mineral nutrition for the biofortification of grains with Zn. Blair (2013) reviewed the advantages and needs of bean biofortification, however, his focus was on improving the development of new biofortified varieties, with a lesser approach to agronomic biofortification strategies. Cambraia et al. (2019), investigating the agronomic biofortification of beans with Zn, found that combined Zn in the soil and foliar application increases its concentration in beans. However, the processes related to the enrichment of the grains with Zn is still a gap, which deserves to be deepened so that it can understand how agronomic biofortification can be related to plant characteristics such as ion absorption kinetics and root system morphology.

The Zn concentration in the soil solution is one of the main factors related to the rate of Zn absorption by the roots. The ion concentration in the soil solutioninfluences ion absorption kinetics, including: Vmax (maximum absorption rate), Km (external ion concentration providing half of Vmax), Cmin (minimum ion concentration in the solution required for the nutrient to be absorbed) and Imax (inflow or ion absorption rate in a solution with low ion concentration); and in the morphology of plant root tissues (Epstein & Hagen, 1952; Nie et al., 2017).

Plants can change root morphology depending on the available Zn concentration in the medium. Coffee plants grown without Zn showed changes in root morphology caused by the deficiency of this nutrient, with bigger root stamen diameter, thicker epidermis, and bigger cross-sectional area of the cortex and stele (Rosolem et al., 2005). The increase in root cortex and stele diameter increased the surface area for nutrient absorption, which led to a lower Cmin (from 13.8 to 3.4 μmol L^-1) and higher Vmax (from 0.50 to 2.1 μmol cm^-2 h^-1) (Rosolem et al., 2005).

Identifying plant characteristics that increase plant's ability to accumulate Zn is important for plants and human nutrition. Even though common bean has not been the focus of biofortification studies yet, it is a staple food highly consumed by the low-income population of Latin America and certain African countries, which could definitely help to deliver Zn and keep people healthier. It is already a good source of Fe and, as other leguminous plants, it has a great potential for Zn and other metals accumulation in the grains, giving its high protein content.

Thus, it is important to understand Zn uptake to improve agronomic biofortification management practices and to guide plant breeding studies for genotypes that are more efficient in Zn absorption and accumulation. Therefore, the objective of this work was to evaluate the absorption kinetics of Zn by common bean roots and the influence of Zn status on the morphology of the root system at the V3 phenological stage, aiming to improve the agronomic biofortification of common bean grains.

MATERIAL AND METHODS

Application of Zn concentrations

For the experiment we used the common bean (Phaseolus vulgaris L.) cultivar BRSMG. The whole experiment was carried out in a greenhouse with temperature control (24 ^oC) and photoperiod of 13 h of light. Common bean seeds were germinated in washed sand, and after 10 d seedlings with fully expanded cotyledonary leaves were transplanted to a vessel covered with aluminum foil and with capacity for 150 mL of solution. Clark's solution with 1/8 ionic strength (Clark, 1977) was used and plants were kept in this condition until they reach V3 stage of development, which corresponds to the first pair of fully expanded leaves. Then, plants were transferred to 1/2 ionic strength Clark's solution, where they remained for 5 d. The solution received continuous aeration and the pH was adjusted daily to 6.0 (± 0.5).

After 3 d the nutrient solution was replaced by the solution with 0.2 mmol L^-1 of CaSO₄ and 12.5 µmol L^-1 of H₃BO₃ by 48 h, to ensure integrity of cell membranes and increase Zn absorption capacity by roots (Lee & Kathryn, 1985). After that plants were transferred to the solutions containing the treatments, which consisted of five initial concentrations of Zn in solution (CZnI) - 0.0; 1.0; 4.0; 16.0 and 48.0 µmol L^-1 of Zn as ZnSO₄.7H₂O, plus 0.2 mmol L^-1 of CaSO₄ and 12.5 µmol L^-1 of H₃BO₃.

Zn absorption kinetics

Once the treatments were applied, samples of 2 mL of the solution were collected manually 11 times throughout the day (0, 1, 2, 3, 4, 5, 6, 7, 8, 12, 24 h after treatments application), on which pH and Zn concentration were measured. At the end of 24 h, the remaining solution volume was quantified and stored. The roots were separated from the shoot, washed with 10% (v/v) alcohol and placed in 25% (v/v) alcohol and stored at 5 ºC for later evaluation of morphological characteristics in a professional Epson XL 10000 scanner using the software WinRHIZO Pro 2009.

Morphological evaluations and Zn content in roots and shoot

The plants morphological evaluations were: root mean volume (Vroot), root mean length (Lroot), root mean area (Aroot), root mean diameter (Droot), root dry matter mass (DDNroot), Zn content in roots (ZCnroot), length of shoot (LS), leaf area (LA), dry matter mass of shoot (DMMS) and Zn content in shoot (ZCS). For root and shoot Zn quantification, samples were oven dried with forced air circulation at 65 ºC until reaching constant weight. Then the material was milled to a size smaller than 20 mesh in a Wiley sieve mill, the samples were opened using nitro perchloric acid digestion (4:1, v/v) and Zn subsequently quantified by atomic absorption spectrometry.

Zn Absorption Kinetic Parameters

Data on the volume of the remaining solution, DDMroot and Zn concentration in the aliquots over time were used to obtain the values ​​of the kinetic parameters: maximum absorption rate of Zn (Vmax); Michaelis-Menten constant (Km), and estimated minimum concentration of Zn for absorption (Cmin) to occur. In addition, we calculated: i) root Zn absorption power (α) - root capacity to absorb Zn²⁺ from the solution, which is the relationship between Vmax and Km; ii) Zn influx (Imax) - value representing the influx equal to the nutrient efflux (Eq. 1); iii) efficiency of Zn absorption (EAZn), relationship between plant Zn content by root dry matter mass; iv) efficiency of translocation n (ETZn), relationship between the Zn content in the shoot by the Zn content in the plant; and v) Zn utilization efficiency in the production of shoot dry matter mass (EUZn), ratio of shoot dry matter mass to shoot Zn content.

$Imax=\frac{ENT\#091;Vmax\left(ZnI-Cmin\right)ENT\#093;}{ENT\#091;Km+\left(ZnI-Cmin\right)ENT\#093;}$ Eq. 1

Mathematical graph method and statistical evaluation

The estimated Vmax, Km and Cmin values ​​were obtained by the mathematical graphical method using the "Kinetic" program (Ruiz, 1985; Ruiz & Fernandes Filho, 1992). The experimental design was randomized with three replications. The analysis of variance and regression for all variables were performed using the SAEG 9.0 (SAEG,2005) program, the graphs and tables were made in excel.

RESULTS

The Zn depletion curve in the solution indicates that there was a decrease on Zn concentration in all CZnI (Figure 1). However, evidence that there was no Zn efflux by bean roots when the solution had no Zn. The pH of the solution, measured in every sample, did not change for all CZnI, kept on average at 6.1.

Figure 1:
Zn depletion curve in the solution over 24 h as a function of the application of the initial concentrations of Zn in solution (CZnI) at the V3 stage of common bean development.

For CZnI of 1.0; 4.0 and 16.0 µmol L^-1; concentrations at the end of the 24 h reached 0.03; 0.04 and 0.24 mg L^-1 (Figure 1). While for CZnI of 48 µmol L^-1 the final Zn concentration after 24 h was close to 1.0 µmol L^-1 (Figure 1). The remaining volume of the solution at the end of the experiment was similar for all CZnI, averaging 104.5 mL.

The kinetic parameters, Vmax, Km and α, increased linearly as a function of CZnI (Figure 2). By the adjusted regression equations, the highest values of Vmax, Km and α were 2.27 µmol/ g h; 1.58 µmol L^-1 and 0.69 L/ g h; obtained at the highest CZnI of 48 µmol L^-1 (Figure 2). Cmin values did not differ between CZnI. Cmin were very low, close to zero, with an average of 0.0028 mg L^-1 (Figure 2).

Figure 2:
Regression equations adjusted for the kinetic parameters, Vmax, Km and α, as a function of the initial concentrations of Zn in solution (CZnI). Vmax (µmol/g h) - maximum ion absorption velocity, Km (µmol L^-1) - Michaelis-Menten constant, and (L/g h) - ion absorption power. **, ***, significant at 1 and 10% probability.

The estimated Imax of Zn by common bean roots differed between CZnI (Figure 3). For the two smallest CZnI; 1.0 and 4.0 µmol L^-1, the inflow did not reach the maximum, characterizing crescent line. For the two highest concentrations; 16.0 and 48.0 µmol L^-1; had a hyperbolic response. At a concentration of 48.0 µmol L^-1, Zn absorption saturation was reached at a concentration well below 48 µmol L^-1. While at a concentration of 16 µmol L^-1 Imax was reached close to µmol L^-1 (Figure 3). Imax curves overlapped in the lowest CZnI concentration ranges (up to 16 µmol L^-1), for all concentrations studied (Figure 3).

Figure 3:
Estimated Zn Influx - Imax (mg/gh), as a function of the initial concentrations of Zn in solution (CZnI).

Morphological variables of root and shoot of common bean plants; Aroot, Droot, LS, ZCS, ETZn and EUZn did not respond to CZnI, averaged 1415.88 mm²; 3.31 mm; 11.12 cm; 26.87 mg kg^-1; 0.59 and 0.02 (Table 1). Already Vroot, Lroot, DMMroot, ZCroot, LA, DMMS and EAZn were positively influenced by the increase of CZnI, the highest values of these variables were obtained in CZnI of 48.0 µmol L^-1 (Table 1).

Table 1:
Regression equations adjusted for the variables; Vroot, Lroot, MMSroot, TZnroot, Aroot, Droot, LAP, FA, MMSAP; TZnAP, EAZn, ETZn, EUZn; as a function of the initial concentrations of Zn in solution (CZnI), with the coefficients of determination

Variables	Ajusted equations	R2
Vroot (mm3)	ŷ = 13.10 + 974.74** CZnI	0.72
Lroot (mm)	ŷ = 246.56 + 89.97* CZnI	0.53
MMSroot (g)	ŷ = 0.047 + 0.0027 CZnI	0.90
TZnroot (mg kg-1)	ŷ = 33.01 + 99.93* CZnI	0.91
Aroot (mm2)	ŷ = ȳ = 1415.88	-
Droot (mm)	ŷ = ȳ = 3.31	-
LAP (cm)	ŷ = ȳ = 11.12	-
FA (cm2)	ŷ = 15.97 + 10.33** CZnI	0.95
MMSAP (g)	ŷ = 0.076 + 0.079* CZnI	0.91
TZnAP (mg kg-1)	ŷ = ȳ = 26.87	-
EAZn	ŷ = 136.78 + 35.038* CZnI	0.80
ETZn	ŷ = ȳ = 0.59	-
EUZn	ŷ = ȳ = 0.02	-

• Vroot - root mean volume, Lroot - root mean length, Aroot - root mean area, Droot - root mean diameter, DMMroot - dry matter mass of root, ZCnroot - Zn content in roots, LS - length of shoot, LA - leaf area, DMMS - dry matter mass of shoot, ZCS - Zn content in roots, EAZn - Zn absorption efficiency, ETZn - Zn transport efficiency, EUZn - Zn utilization efficiency. ** and *significant at 1 and 5% probability.

DISCUSSION

Studies of ion absorption kinetics and morphological data of bean roots influenced by the concentration of Zn available in the solution have not been reported in the literature. In this work, the kinetic parameters Vmax and Km have increased as a function of CZnI in solution, corroborated with Joseph, et al. (1971). As Cmin was low for all CZnI, it indicates that common bean has a high ability to absorb Zn from their roots (Nie et al., 2017).

In addition, high Vmax ​​also favors root absorption as it is associated with low selectivity of Zn²⁺ by the ion-transporting proteins (Epstein & Hagen, 1952; Fageria & Baligar, 1997; Hafeez et al., 2013). High Vmax and low Cmin are very interesting for Zn absorption. When common beans are grown in dilute solutions, when the soil has low levels of available Zn, as occurs naturally in most agricultural areas, including those in Brazil plants are still able to absorb Zn due to the low Cmin (Barber, 1995; Malavolta et al., 1997). On the other side, when Zn is applied as fertilizers, high Vmax also helps plants absorbing high amounts of the nutrient, favoring their accumulation in edible parts of the plant.

Zn absorption by bean roots was possibly performed by low and high affinity systems, as was observed for crops such as rice and wheat (Hacisalihoglu et al., 2001; Meng et al., 2014). The evidence that high affinity systems were active is that Cmin was very low. Besides that, Km values ​​between 0.6 and 2 µmol L^-1 activate these transporters active (Hacisalihoglu et al., 2001) and in this work, the highest Km value was 1.58 µmol L^-1.

Absorption of Zn by common bean roots by low affinity transporters may have been mediated by ion channels and facilitated by the physical or physicochemical connection of Zn²⁺ to the cell wall or free space components (Rathore, 1970; Joseph et al., 1971). In fertilizer management aimed at agronomic biofortification of the common bean with Zn, ion absorption by ion channels is a highly favorable feature. This is because ion channels allow a high influx of ions in a short period of time (Robson, 1993).

EAZn, α and Imax are characteristics of plants that measure the ability of roots to absorb ions. The higher the α value, the higher the rate of ion absorption per root unit and consequently the higher the nutrient acquisition by the plant (Nye & Tinker, 2000; Smith & Mullins, 2001; Sanes et al., 2013). The common bean presented higher EAZn and α when it increased CZnI, which may be related to the efficiency of the common bean in responding to Zn fertilization in the soil and enriching the grains when agronomic biofortification techniques are applied in the crop (Cambraia, 2019; Ram et al., 2016; Figueiredo et al., 2017).

Imax is obtained when all ion transport sites are loaded and ion absorption reaches a plateau. In the lowest CZnI, 1.0 and 4.0 µmol L^-1, inflow did not peak, a condition in which high affinity carriers can establish (Hacisalihoglu et al., 2001; Glass et al., 2002; Pedas et al., 2005). On the other hand, in CZnI 16.0 and 48.0 µmol L^-1 the curve was characteristic of this variable, a hyperbole. There was saturation of Zn absorption at the concentration from 16.0 µmol L^-1, so it is likely that at the concentration of 48.0 µmol L^-1 deluxe accumulation of Zn occurred, and that the CZnI of 16.0 µmol L^-1 s the interesting concentration to study the absorption of Zn by bean roots.

The concentration of Zn in the medium may alter root morphology, as well as root system morphological characteristics may explain the difference between genetic materials in nutrient absorption, translocation and accumulation (Sanes et al., 2013; Pinto & Nazareth, 2016). Because Zn is one of the components responsible for indole acetic acid (IAA) biosynthesis and activity in plants, it is expected to find differences in root systems of Zn deficient plants compared to non-deficient plants in Zn (Schäfer et al., 2016).

The root morphology variables, Aroot and Droot, did not respond to CZnI, this may have occurred because the minimum tissue Zn concentration achieved in this experiment was not low enough to decrease the auxin level to interfere with the cell elongation (Riseman & Craig; 2000). While Vroot, Lroot and DMMroot increased with CZnI. Nie et al., 2017 observed that Zn concentrations in solution interfered with Lroot, Aroot, Vroot and Droot when the mineral wassupplied to the plants of. More Lroot and Aroot indicate that the plants can exploit better the soil, increasing its capacity to absorb nutrients (Batista et al., 2016). This is interesting especially for less mobile nutrients in the soil, which are transported primarily by diffusion such as P, K, Zn and Mn (Barber, 1995; Zonta et al., 2006). In addition, as the root system represents the biggest carbon (C) input for soil organic matter formation, Zn must be included in the fertilization practices in order to increase root system development and C input to the soils.

ETZn and therefore EUZn were not replied to the CZnI because ZCS did not change. Thus, the absorbed Zn was more concentrated in the root than in the shoot, possibly due to the short experiment period and root supply of Zn, only 24 h. Thus, the experiment conduction time was not sufficient for activation of compounds related to the requirement, mobility and activation of fundamental Zn transporters for Zn transport and utilization, such as carbonic anhydrases, alcoholic dehydrogenases, nicotianamine, metallothionein, glutathione, among others (Wilcox & Fageria, 1976; Sadeghzadeh & Rengel, 2011; Gupta et al., 2016; Patel et al., 2018).

The information generated in this study shows that common beans responded to the increase of Zn concentration in the solution by adjusting ion absorption kinetic parameters and altering root morphology, favoring Zn absorption. These common bean characteristics are fundamental to assist studies of agronomic and genetic biofortification with Zn, considered a worldwide challenge and important alternative to improve the nutritional quality of food and ensure a balanced diet (Pedraza, 2017; Cakmak & Kutman, 2018; Balk et al., 2019).

CONCLUSIONS

The common bean responded to the increase in the concentration of Zn in the solution, adjusting kinetic parameters of ion absorption and changing the root morphology to favor the absorption of Zn.

Common beans have an efficient form of Zn absorption, with low Cmin and high Vmax and Km. Low Cmin ensures the absorption of Zn even at very low concentrations of Zn in the solution, which is important for plant nutrition. On the other hand, high Km and Vmax guarantee a high Zn intake when the concentration of Zn in the soil solution is high, as when Zn is added in fertilization practices, for example. This high Zn intake is highly appreciated for biofortification purposes, since translocation of Zn from the roots to the edible parts of the plants is a very difficult process.

Interestingly, there were also the rapid morphological responses of the bean roots to zinc in the external solution. Although the experiment lasted only 24 h, the volume, length and biomass of the root increase with the availability of Zn, showing the importance of this nutrient for the development of the bean root, influencing the absorption of water and nutrients and the C cycle itself.

ACKNOWLEDGMENTS

We thank CAPES, CNPq, the Department of Soils of the Federal University of Viçosa and the technicians of the Stable Isotope Laboratory.

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Publication Dates

• Publication in this collection
  06 Aug 2021
• Date of issue
  Jul-Aug 2021

History

• Received
  06 Feb 2020
• Accepted
  09 Jan 2021