SPAD readings

Figure 1 shows the SPAD readings of the interaction of line and Fe in the solution. When Fe was not added to the solution, line 496 had higher SPAD readings than lines 33 and T₂. The highest SPAD reading without addition of Fe corresponded to line T₂, statistically similar to line 496 but higher than line 33. This shows that line 496 has a better ability to adapt to iron chlorosis, while line T₂ responds favorably only when Fe is present in the nutrient solution, but negatively when it is absent. Line 33 has the highest degree of susceptibility of all lines studied, both in presence and absence of Fe.

Figure 1 Leaf SPAD readings of bean lines and Fe in the solution. 

The symptom of Fe deficiency in the foliage is a yellowing (chlorosis) of young leaves. Authors such as ^{Dell’Orto et al. (2003)} found in Parietaria diffusa plants grown in the absence of Fe that they showed chlorosis and a reduction in growth. Iron chlorosis causes a decrease in photosynthetic capacity, affecting pigments in different magnitudes (^{Abadía and Abadía, 1993}). Chlorophyll reduction may be due to inhibition of the synthesis or degradation of pigments or their precursors (^{Bertamini and Nedunchezhian, 2005}).

In beet plants susceptible to this physiopathy, the concentration of chlorophyll in the leaf decreases to a greater degree with respect to tolerant ones (^{Campbell and Nishio, 2000}). Yellowing is due to an enrichment in carotenoids (xanthophylls). Xanthophyll synthesis works in Fe-deficient plants, but not in other yellowings, it also occurs in carotenoid-enriched materials; etiolated plants or senescent leaves (^{Abadía et al., 2000}). In field conditions, the intensity of the symptoms does not always correlate with low concentration of Fe in the leaf and in some cases, this is even higher in chlorotic leaves than in green ones, due to an inactivation of Fe in the apoplast (^{Morales et al., 1998}; ^{Römheld, 2000}; ^{López-Millán et al., 2013}).

Nutritional concentration of young leaves

Table 1 shows the results of the mineral composition of young leaves exposed to 0 and 1 mg L^-1 of Fe and pH 6 and 8.5. With a Fe concentration of 1 mg L^-1 in the nutrient solution, the concentration of Fe increased, but when it was 0 mg L^-1 the leaf K, Ca, Mg, Mn, Zn, Cu and B increased. Fe deficiency stimulates the accumulation of K (^{Marschner, 1995}; ^{Maldonado-Torres et al., 2006}), Mg (^{Belkhodja et al., 1998}), Mn, Zn and Cu (Jolley et al., 2004) in young leaves.

The increase in K in the leaves is a consequence of an increased activity of the ATPase of the plasma membrane of the root involved in the extrusion of protons (Alcántara et al., 1991; ^{Marschner, 1995}; Blekhodja et al., 1998). ^{Welkie et al. (1993)} have associated the high concentration of K with the accumulation of organic acids in Fe-deficient plants. ^{Thomas et al. (1998)} found a higher concentration of K in chlorotic oak trees compared to non-chlorotic ones. For their part, in non-grass dicotyledons and monocotyledons with Fe deficiency that use strategy I, Alam et al. (2001); ^{Marschner (1988)} have determined a greater mobilization and absorption of Mn.

In the nutrient solution with pH 6, the leaf concentration of Mn, Zn and Cu increased, while at 8.5, P, K, Ca and B increased. The concentrations of N, Mg and Fe were not affected. At a low pH in the rhizosphere, roots absorb more cations than anions (^{Schaller, 1987}; ^{Schmidt et al., 2020}). When cations are absorbed in excess with respect to anions, acidification of the rhizosphere occurs (^{Curtin and Wen, 2004}). The pH of a nutrient solution affects dissociation, complexation and precipitation reactions, which significantly impacts the speciation and availability of essential elements, especially metal cations (^{Rijck and Schrevens, 1997}).

In Table 2, the results show that where Fe was not added, bean line 33 had the lowest concentration of P, K and B, while line 493 presented a higher concentration of P and K. In the lines where Fe was added, there were no significant differences in leaf nutrient concentration. Although lines 496 and 33 had the same concentration of Fe, line 33 had a higher degree of chlorosis (Figure 1), indicating that it is inefficient in the use of Fe.

The leaves with chlorosis show physiological alterations, such as a decrease in the concentration of chlorophylls, radiation capture and photosynthetic capacity. Light absorbed and not used in photosynthesis can photo-inhibit processes and photo-oxidize compounds (^{Abadía et al., 2000}; ^{Rout and Sahoo, 2015}). In chlorotic leaves, Fe accumulates in unavailable forms (^{Marschner, 1991}).

Cells of chlorotic leaves have a limited capacity for the absorption of Fe, and this could be accumulated in the apoplast, which can be amplified under field conditions. Thus, this accumulation in chlorotic leaves would be the consequence of the enrichment of Fe in leaves when the plant no longer has the capacity to use it (^{Bohórquez et al., 2001}).

Some chlorotic species green when dilute acid is applied to the petiole and when the leaves are treated with compounds that promote the functioning of plasma membrane ATPases (^{Kosegarten, 1999}). In Fe-deficient plants, high pH in the cytoplasm has been reported and this is associated with immobilization (^{Abadía, 1992}).

The concentration of Mn in stem and root follows an inverse relationship with Fe, the mobilization and absorption of Mn increases during Fe deficiency in plants that use mechanism I (Alam et al., 2001). In Figure 2, the plants with nutrient solution without Fe show a generally higher Nutrient Imbalance Index (NII), but where Fe was added, lines 496 and 33 had the lowest indices, while in T₂ it was higher.

Figure 2 Interaction of bean lines and Fe in the nutritional imbalance index (NII). 

This was contrary to SPAD readings, where T₂ values were higher. Although Fe was not applied, line T₂ had the same degree of chlorosis (Figure 1) as line 33 but had a higher NII. Conversely, it occurred when Fe was applied, since T₂ had higher NII and SPAD readings, while in line 33, NII and SPAD readings were lower, so T₂ adapted better to Fe deficiency than line 33.

Regarding nutritional ratios, it was found that where Fe was not added, the ratios N/P, B/P and Ca/P were higher in line 33 (Table 3), which indicated that in this there was a higher amount of P in the young leaves, while line 496 was lower. High concentrations of P in the medium or plant increase iron chlorosis (^{Clark, 1991}). ^{Keshirad et al. (1978)} suggest that high doses of P interfere with the absorption and translocation of Fe, inactivating and precipitating it in the apoplast of chlorotic plants, preventing its translocation to mesophyll cells.

In chlorotic plants, the ratios P/Fe, K/Ca, K/Fe, Mn/Fe, Cu/Fe and Zn/Fe were higher (Table 4). This indicates a lower amount of Fe compared to other cations (K, Mn, Cu and Zn) and a higher amount of K, P and Mn, which coincides at least for the P/Fe ratio, since ^{Köseoglu (1995)}) and ^{Maldonado-Torres et al. (2006)} found that this ratio is higher in chlorotic leaves than in green leaves. It has been found that the K/Ca ratio is high in pear tree leaves with iron chlorosis (^{Abadía et al., 1989}), peach (^{Belkhodja et al., 1998}) and Mexican lime (^{Maldonado-Torres et al., 2006}).

It has often been verified that the chlorotic organs have high concentrations of K (^{Loué, 1988}), this effect is attributed to the fact that excess K in the leaves can displace Fe from the metabolic surfaces of enzymes (El-^{Kassas, 1984}), but K deficiency is considered a susceptibility to iron chlorosis. K favors the absorption of Fe as a result of acidification of the rhizosphere soil and an increase in the plant in the citric acid that transports Fe within the plant (^{Marschner, 1995}).

Mineral composition in the root

The addition of 1 mg L^-1 of Fe to the nutrient solution influenced the increase in the concentration of N, P, K and Fe (Table 5). In the roots of plants that did not receive Fe, Mn, Zn and Cu increased, while the concentrations of Ca, Mg and B were not influenced. ^{Alhendawi et al. (1997)} examined that the concentration of K⁺ and NO₃ ^- in the roots is decreased by HCO₃ ^-, due to an increase in the flow of these ions into the medium. High concentrations of CO₃ ^2- and HCO₃ ^- (alkaline pH) induce low availability of nutrients such as Fe and sometimes P (^{Dell’Orto et al., 2003}; ^{Parrilla and Schmidt, 2019}).

^{Alhendawi et al. (1997)} mention that increasing the concentration of HCO₃ ^- decreases the content and concentration of Fe in the roots and a similar percentage in the stem. This indicates that the reduction of Fe induced by HCO₃ ^- in the stem is not a specific effect of translocation of Fe from the root to the stem, but in the absorption of Fe by the root. Alam et al. (2001) found that the concentration of Mn is higher than in the root in plants with and without Fe. The pH of six in the solution increased the concentration of P and at a pH 8.5, that of Ca increased. The concentration of other nutrients was affected by the pH.

The line T₂ reached a higher concentration of N and B in the roots of plants without application of Fe (Table 6). Regarding K, lines 496, 493 and 33 were the ones that obtained the highest concentration and T₂ the lowest. The concentration of Fe and Mg was higher in line 496. Line 142 had a higher concentration of Mn and Zn, but was the lowest in Fe, which agrees with different authors who mention that the concentrations of Mn and Fe follow an inverse relationship (Roomizadeh and Karimian, 1996; ^{Foy et al., 1998}; Ghesemi-Fasaei et al., 2003). Line 493 had the highest concentration of Fe in the root when they received Fe. However, the degree of chlorosis was the same as that of the lines with smaller magnitude (Figure 1).

The transfer coefficient of all nutrients increased in chlorotic leaves (Table 7), so, Fe deficiency increases their translocation to young leaves. The root and stem interact by transferring nutrients between vessels of xylem that nourish the leaves and those to the stem (Jeschke and Hartung, 2000).

The content and concentration of Fe in barley, sorghum and corn in all parts of the plant are significantly reduced by elevated levels of bicarbonate (^{Alhendawi et al., 1997}). ^{Alhendawi et al. (1997)} observed that the supply of bicarbonates results in an accumulation of organic acids in roots and a decrease in the absorption and translocation of Fe. Bicarbonate has been found to avoid the translocation of Fe to young organs and the transport of Fe from the apoplast within the cytoplasm to the apex level (Mengel, 1994), although it is not a general thesis (^{Römheld, 2000}). ^{Toulon et al. (1992)} and ^{Susin et al. (1996)} have shown that the reduction of Fe³⁺ in the apoplast of intact roots occurs at low pHs.

Root volume

In plants without Fe, there was no statistically significant difference (Figure 3), but where Fe was present, lines 496 and 33 had greater root volume, while in line 101 it was lower. Fe deficiency produces physiological responses in the root (^{Abadía et al., 2000}; ^{Tripathi et al., 2018}), which include transformation of epidermal roots into transfer cells (^{Schmidt and Bartels, 1996}), acidification of the rhizosphere (Alcántar et al., 1991) and increased iron reduction capacity of roots (^{Robinson et al., 1999}).

Figure 3 Effect of bean line and iron concentration on root volume. 

The development of morphological and physiological changes in the root is another response found in Fe-deficient dicotyledons. The proliferation of root hairs is a modification induced by Fe deficiency, in plants that carry out strategy I (^{Landsberg, 1996}). In Casuarina glauca, Fe deficiency is one of the main factors, in addition to P, in the induction of cluster root (proteoids) formation, even reversing cluster root formation due to P deficiency due to the addition of Fe to the nutrient medium. In addition, the formation of this type of roots appears to be an efficient morphological response to Fe stress (^{Arahou and Diem, 1997}; ^{Connorton et al., 2017}).

^{Campbell and Nishio (2000)} found that plants under Fe deficiency decrease the growth of the root tip, retardation of root tissue growth and production of lateral roots, in addition to the formation of numerous root hairs, which correlates with a significant decrease in chlorophyll. These roots produce and excrete phosphatases and organic acids that acidify the soil and chelate minerals, mobilizing P and other micronutrients. The detriment of the acquisition of Fe may be a consequence of the inhibition of root growth (^{Alhendawi et al., 1997}).

Bicarbonates absorbed through the roots become used in the synthesis of organic acids, being fixed in situ by phosphoenolpyruvate carboxylase (PEPase) to form oxaloacetate, which is reduced to produce malate (^{Cramer et al., 1993}). In rice, inhibition of root growth due to the high bicarbonate concentration is linked to high concentrations of organic acids in the roots (^{Wairich et al., 2019}).

Dry matter

There were no significant differences in dry matter in the plants that grew without Fe supply (Figure 4). When Fe was added, lines 496, 33 and T₂ produced more dry matter and the lowest value corresponded to line 101. This agrees with the root volume, as they have the same tendency, thus a good root system causes a higher production of biomass. However, only in T₂ were SPDAD readings higher. (^{Belkhodja et al., 1998}; ^{Connorton et al., 2017}) mention that the decrease in chlorophyll concentration is not always due to Fe deficiency, but to a dilution effect; and that it can be the result of two processes; a dilution of chlorophyll caused by growth and subsequent inability to produce or stabilize new molecules in thylakoid membranes.

Figure 4 Dry biomass production of bean lines exposed to deficiency and supply of Fe. 

In barley, sorghum and corn, it was reported that increases in the concentration of bicarbonate ions in the nutrient medium promote iron chlorosis, significantly decrease the dry biomass of root and stem (^{Alhendawi et al., 1997}; ^{Jia et al., 2018}; ^{Li et al., 2021}). (^{Bertamini and Nedunchzhian, 2005}; ^{Riaz et al., 2021}) found a 37% reduction of dry biomass in Fe-deficient grape leaves.