4.1. Biomass Production and Vanadium Accumulation in Lettuce Plants
In the present study, the application of vanadium into the nutrient solution had no effect on the biomass of lettuce roots and leaves. Even the application of the highest dose of vanadium (0.4 µM V) did not cause any visible symptoms of its toxicity to plants. This clearly suggests that the applied doses were on the safe level for lettuce plants. Welch and Huffman [17
] did not note any significant effect of 1 µM V (as NH4
) on the yield of lettuce and tomato as compared to the control combination (characterized by a trace amount of vanadium, i.e., <0.07 µM V).
The studies by Vachirapatama et al. [40
] revealed the possibility of increasing root-to-leaf transfer of vanadium when very high, potentially lethal doses of that element are applied to plants. In the plants of Chinese green mustard, the high content of vanadium in the nutrient solution between 0.39 and 1.57 mM V caused a proportional increase in vanadium content in respective plant organs according to the order: roots > steams > leaves. Importantly, roots contained a few hundred times more V than leaves and stems [40
]. Similar relations were found in the plants of sweet basil fertilized with NH4
in doses of 0.1, 0.1, 0.39, and 0.79 mM V [41
]. It was also revealed that fertilization with 1 µM V (as NH4
) proportionally increased the content of vanadium in the leaves and roots of lettuce and tomato [17
In the present studies, increasing vanadium doses caused a gradual increase in its content only in the roots (Figure 2
). The obtained results are in agreement with observations made on various crops, including tomato and Chinese green mustard [40
], soybeans [19
], rice [42
] and lettuce [43
]. In these studies, vanadium accumulated in roots rather than in above-ground parts of the plant. It needs to be mentioned that the applied doses between 0.05 and 0.40 µM V, apart from being safe for plants, may have been too low to observe an efficient root-to-leaf distribution of that element. Vanadium doses exceeding 0.79 mM V impaired the growth and development of tomato and Chinese green mustard plants [40
]. A toxic dose of vanadium (as VOSO4
) for soybean plants was 1.2 mM V[19
]. In the case of rice plants, vanadium toxicity was revealed for the 0.39 mM V dose [42
]. When excessive vanadium concentrations were applied, plastid degradation occurred in maize and horse bean plants [43
]. Gil et al. [44
] observed a 15% decrease in lettuce biomass, even for the lowest dose of 0.002 mM V (as NH4
) and the application of 0.02 mM V reduced plant biomass by approximately 64%. Moreover, root darkening, a decrease in the number of secondary roots, and a loss of leaf turgidity was observed.
4.2. Iodine Accumulation vs. Vanadium Application and vHPO Activity
The enzyme vanadium-dependent haloperoxidase (vHPO) that is present in marine alga Laminaria digitata
participates in the process of iodine uptake/release from cells with hypoiodous acid (HIO) as an intermediate [27
]. An indirect role of vHPO in the volatilization of elemental iodine I2
by marine algae is related, among others, to plant response to oxidative stress. Furthermore, the release of volatile methyl iodide also occurs in marine algae [4
] and higher plants such asArabidopsis thaliana
, rice [45
], or lettuce [47
In the analyzed literature, no information can be found on the interaction between vanadium application and the level of iodine accumulation in crop plants. The effect of vanadium on the activity of vHPO in crop plants also has not been studied. Basically, the description of the structure, activity, and role of vHPO rely on the results obtained for enzymes isolated from various species of marine algae. Considering the structure and functioning of vHPO, it is classified into the group of histidine phosphatase/peroxidase super family [48
]. Studies conducted by Colin et al. [49
] revealed that in vitro activity of vHPO isolated from Laminaria digitata
increased when KI was applied in the range of 0–10 mM I and dropped for KI applied in doses >20 mM I.
The trial was undertaken to determine the enzymatic activity of vHPO in lettuce with no previous analytical protocol and based on the assumption that lettuce extracts may exhibit enzymatic activity typical for vHPO. The assayed activity of vHPO is a total activity of vanadium-dependent peroxidases in plant extract and is a derivative of interaction between iodine and vanadium or other halogens. The results of the study indirectly indicate the functioning of various mechanisms regulating the activity of vHPO in lettuce, depending on the application of vanadium and different iodine compounds (KIO3, 5-ISA and 3,5-diSA).
A significant increase in vHPO activity after the application of vanadium was noted only in the leaves of plants non-fertilized with iodine(Experiment No. 1) and was positively correlated with iodine content in roots (r2 = 0.78*) and leaves (r2 = 0.79*).These results suggest that vanadium fertilization may improve via increasing vHPO activity, the process of iodine uptake, and accumulation in lettuce roots and leaves only when plants are cultivated in the presence of a trace amount of that element in the nutrient solution (Experiment No. 1). Application of 3,5-diISA reduced vHPO activity in lettuce roots to levels lower than those noted for 5-ISA. At the same time, application of the highest dose of V together with 3,5-diISA decreased the activity of vHPO in leaves the most. That observation was accompanied by a slight increase in T3 content in leaves, but no effect on leaf accumulation of SA, BeA, 3,5-diSA, 5-ISA, 2-IBeA, 4-IBeA and 2,3,5-triIBeA was observed.
The reduction in the vHPO activity in roots of plants treated with 5-ISA and 3,5-diISA suggests that these iodosalicylates are taken up by the roots by different mechanisms than those related to iodide uptake with the participation of vHPO as described in marine alga. It is worth mentioning that iodates (IO3−
) undergo reduction to I−
in the root zone most probably by a specific reductase [50
] or, alternatively, nitrate reductase [4
4.3. SA and Iodosalicylate Metabolism
In the plant organisms, benzoic acid (BeA) is a precursor of SA, the latter being considered as a signaling molecule and plant growth regulator [51
]. On the other hand, 2,3,5-triiodobenzoic acid (2,3,5-triIBeA) plays a role of auxin inhibitor [52
]. Van de Wouwer et al. [53
] revealed that p
-iodobenzoic acid (synonym: 4-IBeA) and its derivatives inhibit the process of lignification by reducing the activity of cinnamate 4-hydroxylase—a key enzyme in the phenylopropanoid pathway leading to the synthesis of lignin polymers. Crisan [54
] revealed that exogenous 3-iodobenzoic acid (3-IBeA—its content was not determined in our study) stimulated root elongation and the formation of adventitious roots.
Previous studies on tomato plants showed that plant preference towards the uptake and accumulation of 5-ISA and 3,5-diISA in leaves and roots depended on the growth stage of plant: stage of 5–6 true leaves [26
], intensive vegetative growth, and fruiting [39
]. In the present studies, iodine applied as 5-ISA was taken up and distributed more easily than 3,5-diISA.
It was revealed that 3,5-diSA, 5-ISA, 2-IBeA, 4-IBeA, and T3 are present and synthesized in lettuce plants and, to our knowledge, this is a first report of that matter. In the case of PDTHA
(Plant Derived Thyroid Hormone Analogs), its synthesis in plant tissues has been previously hypothesized by Lima et al. [55
]. The exact pathway of PDTHA synthesis is yet to be described. It is worth mentioning that the first report of PDTHA was presented by Fowden [56
], who revealed that, after fertilization with iodine, the plants of astra and Salicornia
sp. contained (and therefore synthesized): 3,5-diiodothyrosyne, 3,5-diiodothyronine and 3,5,3′-triiodothyronine. Bean and barley plants grown in the presence of iodine contained only 3,5-diiodothyrosyne. A specific enzymatic system is required for the production of PDTHA in plants. Fenical [57
] informed that the enzymatic process of halogenation, i.e., the incorporation of iodine (or other halogens), was described for mushrooms. Furthermore, the presence of 3-iodothyrosyne, 2,5-diiodothyrosyne, and 3,5,3′-triiodothyronine has been confirmed in Rhodophyta
]. To our knowledge, the process of synthesis and metabolism of iodosalicylates and T3 in higher plants has not yet been described. Based on quantitative relations between analyzed iodosalicylates and iodobenzoates in the roots and leaves of lettuce from all four experiments, a hypothetical overview of that process can be proposed (Figure 2
and Figure 3
It needs to be underlined that the synthesis and metabolism of the analyzed compounds in lettuce plants varied depending on the form of applied iodine rather than the dose of vanadium. In all four experiments, the highest content of 2,3,5-triIBeA, 5-ISA, 2-IBeA and 4-IBeA was measured in the leaves of control plants, i.e., not fertilized with iodine (Experiment No. 1). On the other hand, the content of 3,5-diSA in the leaves of the control plants was slightly lower than after the application of 3,5-diSA (Experiment No. 4). It seems that the basic iodine metabolites in the plants grown in the presence of trace amounts of iodine were iodosalicylates, iodobenzoates, and T3. However, the obtained results do not provide the background for the possible physiological function of these organoiodine compounds in lettuce plants.
The application of KIO3 into the nutrient solution decreased the level of iodosalicylates and iodobenzoates in lettuce leaves. Most likely, in the case of increased accumulation of inorganic iodine, different pathways of its metabolism were activated (i.e., including iodine volatilization through methylation) than those engaged in the synthesis of iodosalicylates and iodobenzoates. Based on these results, it can be concluded that, after the application of iodosalicylates, endogenous iodobenzoates (2-IBeA, 4-IBeA and 2,3,5-triIBeA) as well as 3,5-diISA and 5-ISA are degraded, conversed into other compounds or volatilized in methyl forms. One of the formed compounds includes T3 as its level was higher in lettuce leaves fertilized with 5-ISA and 3,5-diISA than in the control and KIO3 plants.
Taking the above into consideration, this supports the proposed description of possible synthesis of T3 and the metabolism of exogenous iodosalicylates in lettuce plants (Figure 3
). Biosynthesis of T3 is independent of applied vanadium dose as well as of vHPO activity and most likely occurs in roots. The transport of T3 from roots to leaves is strongly limited or alternatively; T3 is conversed in leaves into other compounds from the PDTHA group.
Plant fertilization with KIO3
did not modify the level of T3 in leaves and roots of lettuce, which suggests that the content of T3 in lettuce leaves remains stable for a trace and increased concentration of inorganic iodine in the root zone and plant tissues. In the conditions of increased concentrations of 5-ISA and 3,5-diSA in plants, an efficient distribution of both iodosalicylates into the leaves was observed. This was followed by a substantial increase in the T3 level in leaves and a decrease of T3 content in roots as compared to plants from the control and KIO3
experiments.Therefore, it can be concluded that 5-ISA was converted into 3,5-diISA in both leaves and roots and the latter compound may have been utilized for the synthesis of T3 through, for instance, its joining with 3-iodo-L-tyrosine or other metabolic pathways that are presented in Figure 3
A slight decrease in the biosynthesis of BeA was also observed in the roots of plants grown in the presence of KIO3 as compared to the control plants (Experiment No. 2 versus Experiment No. 1). Plant fertilization with KIO3 also contributed to a simultaneous decrease of the content of (a) SA in roots and leaves; and(b) 5-ISA, 3,5-diSA, 2-IBeA, 4-IBeA, and 2,3,5-triIBeA in leaves. These results indicate that different pathways of iodine metabolism were activated by KIO3applicationthat was directed, among others, on iodine methylation. In the case of plant fertilization with 5-ISA and 3,5-diISA, iodine metabolism was directed to the synthesis of T3 or other organic iodine compounds including those classified as PDTHA. This may have been a direct cause of obtaining lower biomass after plant treatment with iodosalicylates. In addition, the content of 5-ISA, 3,5-diSA, and T3 in the leaves of plants treated with iodosalicylates was higher than the physiological level noted in the control plants. This could have been another factor that modified the functioning of phytohormones related to the growth and development of lettuce plants fertilized with iodosalicylates.
Furthermore, the obtained results indirectly indicate that the exogenous 5-ISA and 3,5-diSA weakened the synthesis of 2-IBeA, 4-IBeA, and 2,3,5-triIBeA in lettuce plants. A significantly higher content of SA as well as a decreased content of BeA in the roots of plants fertilized with 5-ISA and 3,5-diISA suggest that some share of iodosalicylates, apart from T3/PTDHA synthesis, could have undergone the process of de-iodination into SA (Figure 3