Zn supply-dependent modification of Zn root/shoot distribution due to expression of AtHMA4 in tomato is accompanied in roots by tissue-specific differential expression of metal-homeostasis genes
Our previous study showed that expression of 35S::AtHMA4 and AhHMA4
p
::AhHMA4 in tobacco and tomato led to changes in Zn and Cd accumulation and root/shoot distribution. The pattern was different, however, at a range of metal concentrations in the medium, indicating involvement of endogenous processes specifically induced in transgenic plants [10–13]. Here it was shown that the Zn supply-dependent modifications of Zn accumulation due to AtHMA4 expression (increased Zn root-to-shoot translocation at 5 μM Zn, no change at 0.5 μM Zn) were accompanied in the roots by tissue-specific expression patterns of metal-homeostasis genes that were different than in the wild type. Importantly, the pattern of modifications detected in transgenic plants (as compared with the wild type) was not the same upon exposure to 0.5 and 5 μM Zn in the medium. According to the presented model (Fig. 10), changes in the expression of endogenous genes in transgenic plants are induced in response to the mineral imbalance resulting from the export activity of AtHMA4 in transgenic tomato. These alterations contribute to the generation of the phenotype of transgenic plants, including changes in the accumulation and distribution of metal(s) between roots and shoots. It has already been shown [22] that plants grown under different conditions of exposure to metals (metal deficiency, sufficiency, and excess) differ greatly in their expression profiles. Therefore, it was assumed that these very different molecular backgrounds of tomato (used for transformation) grown at varying Zn levels in the medium will interact with the changes in the mineral status at the cellular level (resulting from AtHMA4 export activity), the extent of which is different at lower and higher Zn exposure. As a result, in specific cells/tissues/organs endogenous genes known to respond to the availability of a metal(s) are up- or downregulated in transgenic plants in a tissue-dependent fashion. The activation of endogenous metal cross-homeostasis mechanisms in transgenic plants is considered a key factor that contributes to the generation of their characteristics.
Changes specific for the EC (epidermis + cortex)
In numerous plant species, Zn excess decreases the Fe level in aerial parts and induces an Fe-deficiency response due to a shoot-born systemic signal [22–24]. In the wild-type tomato, the shoot Fe content was lower at elevated Zn (10 and 20 μM) compared with 1 μM Zn [13]. In line with these observations, here it was shown that in the EC of roots from wild-type tomato grown at 5 μM Zn, the expression of transcription factor LeFER, and LeFER-dependent LeIRT1 that mediates Fe, Zn and Cd uptake [20, 25, 26] was higher compared with 0.5 μM Zn exposure (Fig. 4a, b; Fig. 5a, b). However, expression of AtHMA4 changed the intracellular Fe status, which was different at the lower and higher Zn levels in the medium.
Thus, expression of the Fe deficiency-inducible Strategy I Fe acquisition genes (LeIRT1 and LeFER), ethylene genes (LeNR, LeACO4, and LeACO5), and Chln found to be higher in transgenic plants than in the wild type grown at 0.5 μM Zn, indicates generation of Fe deficiency status due to AtHMA4 expression. Conversely, the lower expression of these genes in AtHMA4-plants grown at 5 μM Zn indicated that their Fe status had changed to Fe sufficiency. These contrasting changes in the expression of endogenous genes show how deeply the Zn export activity of HMA4 alters endogenous metal cross-homeostasis mechanisms (Fig. 10). In transgenic plants they did not lead, however, to marked changes in total root and leaf Fe concentrations, except in transgenic line 4 grown at 5 μM Zn (Fig. 1g-i), suggesting that in most experimental variants, there occurred the induction of pathways counteracting mineral imbalance generated by the export activity of AtHMA4 in tomato.
Ethylene is necessary for upregulation of Fe-uptake genes [27]. The examined genes included LeNR (an ethylene receptor that responds to environmental stresses), LeACO4, and LeACO5. Ethylene is the product of the reaction catalyzed by 1-aminocyclopropane-1-carboxylate oxidase (ACO), therefore, the site of ACO expression is considered the best indicator of ethylene production [28, 29]. Ethylene affects Fe deficiency-inducible genes, including IRT1, by regulating the FER transcription factor level [30]. Expression of LeIRT1 also depends on the expression of LeChln, the only NAS gene in tomato involved in nicotianamine (NA) synthesis that is known to regulate cross-homeostasis of Fe, Zn, Mn, Cd, and Ni, among others [26, 31, 32]. Upon exposure to 0.5 or 5 μM Zn, in the EC the expression of LeIRT1 in AtHMA4-tomato was higher or lower than in the wild type, respectively, and was accompanied by respectively higher or lower expression of LeChln (Fig. 4a).
Moreover, in the EC of transgenic plants grown at 0.5 μM Zn, the expression of NRAMP1 and NRAMP3 was lower than in the wild type in both lines, whereas at 5 μM Zn—only in line 4 (Fig. 4a; Fig. 10). It cannot be excluded that the detected modified expression of LeNRAMPs could contribute to regulation of the amount of Zn available for radial transport and root-to-shoot translocation, thus, to the difference in Zn and Fe root and shoot concentrations detected between lines 4 and 15 (Fig. 1b-c, h-i). The NRAMPs consist of a group of membrane importer proteins that exhibit functional divergence and broad substrate specificity, including Fe, Mn, Ni, Cd, Zn, Pb [33]. However, up to now the tomato LeNRAMP1 and LeNRAMP 3 localized to the internal membranes have only been shown to mediate Mn transport [20]. Other metals, including Zn, were not tested, hence it was not determined if Zn is a substrate. Further research aimed at elucidation of LeNRAMP1 and LeNRAMP3 function is needed to understand the contribution of these proteins to Zn homeostasis and partitioning in organs.
Changes specific for the S (stele)
In the S (where Zn is loaded into xylem vessels) the Zn supply-dependent contribution of AtHMA4 expression to Zn root-to-shoot translocation efficiency (increase at 5 μM Zn, no change at 0.5 μM Zn; Fig. 1c) was accompanied primarily by a distinct expression pattern of LeNRAMP2. In plants exposed to 0.5 μM Zn, LeNRAMP2 was downregulated, and upregulated at 5 μM Zn (Fig. 4d, e). Thus, LeNRAMP2 was found to respond in the S of both transgenic lines in a unique way to the changes in mineral status resulting from AtHMA4 export activity (see model in Fig. 10). This suggests that LeNRAMP2 could be considered a candidate gene for involvement in the Zn supply-dependent efficiency of Zn translocation to shoots, which is distinct in transgenic vs. wild-type plants (Fig. 1c). To corroborate this supposition, it is necessary to characterize LeNRAMP2. Currently only its sequence is known. The highest level of sequence identity was found between LeNRAMP2 and the Arabidopsis NRAMPs from the second sub-family, which include AtNRAMP2-5 (Additional file 14). It shares 74.2 % amino acid identity with AtNRAMP2 which does not complement the fet3fet4 yeast mutation, indicating the lack of ability to transport Fe [21]. LeNRAMP2 also shows high identity with AtNRAMP3 and TcNRAMP3 (72.1 % and 71.5 %, respectively), and with AtNRAMP4 and TcNRAMP4 (69.6 % and 68.2 %, respectively). They encode tonoplast-localized proteins implicated in the release of Fe, Mn, and Cd from vacuoles [21, 34–36]. It cannot be excluded that LeNRAMP2, as an import protein, is localized to the internal membranes and participates in metal redistribution from intracellular stores. Detailed molecular and functional characteristics are needed, however, to conclude about its specific role in the S relating to modifications of Zn supply-dependent alteration of Zn translocation to shoots in the AtHMA4-tomato.
Cd-dependent modifications of Zn/Cd root/shoot distribution due to expression of AtHMA4 in tomato involves root tissue-specific alteration of LeNRAMP1-3 and LeChln
Expression of AtHMA4 also contributed to enhanced Cd root-to-shoot translocation. Moreover, in the presence of Cd, the efficiency of Zn translocation was significantly higher, however, the difference was noted primarily in transgenic line 4 (Fig. 1). These changes were accompanied at the molecular level by differences in the abundance of LeNRAMP1-3 mRNA, especially in the EC (Fig. 4c, f; Fig. 10) indicating that they were regulated in a tissue-specific manner directly or indirectly by Zn/Cd. It is noteworthy that there is a growing amount of data suggesting the involvement of NRAMP transporters in mediating Cd uptake and in intracellular distribution in plants [34, 36, 37]. The changes in Cd and Zn accumulation distinct for line 4 were accompanied by the reduction to a barely detectable level of the expression of LeNRAMP2, LeChln (LeNAS), and yet uncharacterized transcription factors LebZIP44 in the S of transgenics’ roots. The role of NAS in Zn and Cd translocation to shoots has been shown for AhNAS2 from A. halleri [32, 38]. However, the tissue-specific regulation of LeChln upon exposure to a range of Zn and to Cd had not been investigated thus far. The expression of LeFER and LeChln in the S of transgenics’ roots was lower than in the wild type and corresponded with decreased expression of ethylene-related genes (Fig. 10). In the EC this correlation was not as obvious, however. The role for ethylene in a plant’s response to Cd was indicated in experiments showing higher tolerance to Cd in the Nr tomato mutant [39], as well as in the etr1-1 and ein2-1 Arabidopsis mutants [40].
In leaves, ectopic expression of AtHMA4 modifies the ethylene-dependent pathway in a tissue-dependent fashion
Expression of HMA4 in tobacco and tomato led to the appearance of necrosis within leaf blades when plants were exposed to elevated Zn in the medium [11,12]. It was shown that loading of Zn into “Zn-storage cells” was initiated upon a high Zn concentration in the apoplast, however, the nature of the signal was not proposed. In this study, in the leaves of AtHMA4-tomato plants grown at moderately toxic 5 μM Zn its accumulation was also restricted to groups of mesophyll Zn-storage cells, whereas it remained low in neighboring non-accumulating ones. Upon exposure to a higher (10 μM) Zn concentration, necrotic regions developed [13], likely originating from the groups of Zn-accumulating cells identified in this study. In contrast, in wild-type tomato Zn was distributed uniformly across mesophyll cells (Fig. 9).
Here we demonstrate that formation of clusters of Zn-accumulating cells in leaves of AtHMA4-tomato plants (Fig. 9) is accompanied by dramatically lower, relative to the wild type, expression of two ethylene receptors LeNR, both in the EPP (upper epidermis + palisade parenchyma) and in the ESP (lower epidermis + spongy parenchyma), and LeETR1 in the ESP (Fig. 7c, g). Lower expression of ethylene receptors in transgenic tomato might indicate fewer receptors within the ER of mesophyll tissues, leading to higher sensitivity to ethylene than in the wild type [41].
These results were the basis for formulating the hypothesis linking the appearance of groups of Zn-accumulating mesophyll cells (Fig. 9) with modifications of the expression profiles of genes from the ethylene biosynthesis pathway detected in transgenic tomato plants exposed to 5 μM Zn (Fig. 7c, g). According to this hypothesis, the combination of higher sensitivity to ethylene than in the wild type with the probably higher production of ethylene (resulting from higher expression of certain ACO genes in the mesophyll) could be involved in the pathway signaling Zn excess in the apoplast, leading to induction of Zn accumulation in groups of mesophyll cells. Our previous research on tobacco expressing AtHMA4 led to the conclusion that until the Zn concentration is sensed as too high, Zn is accumulated uniformly in mesophyll cells, but when the apoplastic Zn reaches a threshold, a signal is generated to redirect Zn to groups of cells (“Zn-storage cells”). The nature of the signal is not known; however, the results of this study suggest that ethylene could be a part of it. These results open a new direction in the search for the mechanisms behind formation of necrotic regions, which, according to our recent data, could be considered a mechanism of tolerance to Zn, protecting neighboring non-accumulating cells from the toxic effects of Zn rather than only being a symptom of toxicity [15]. Future research should clarify this issue. Ethylene is involved in the regulation of numerous physiological processes, including the response to biotic and abiotic stresses [42]. Interestingly, in tomato leaves ethylene accounts for formation of ozone-dependent lesions from specific mesophyll cells, which were considered groups of cells disposed to die upon a certain signal resulting from increased ethylene [43]. Moreover, a recent study on ethylene insensitive mutants etr1-1 and ein2-1 indicated that ethylene signaling is involved in the early Cd stress response in A. thaliana leaves [40].
Expression of AtHMA4 in tomato modifies expression of cell-wall remodeling genes
Plant cell wall composition and structure play a variety of functions in a plant’s response to metals, including regulation of the capacity for metal accumulation and signaling of metal status [44]. These functions might be distinctly affected in different root and leaf tissues of tomato plants due to overloading of the apoplast with Zn as a result of AtHMA4 export activity.
Roots
Expression of AtHMA4 induced processes leading to strengthening of the cell wall structure by enhanced expression of cell wall structural proteins, extensins, and downregulation of several cell wall-modifying enzymes (Fig. 3d, h). Upregulation of the cell wall structural protein, extensin uJ-2, was noted in both root sectors. Stress-induced expression of extensin genes is usually related to the requirement for a fortified cell wall [45]. Significantly higher expression of uJ-2 in both examined sectors of transgenic roots, with a much higher increase of the transcript level in the EC, indicate hitherto unknown regulation resulting from the high Zn status in the apoplast due to the export activity of the AtHMA4 protein. Increased expression of the cell wall structural gene was accompanied by strong downregulation of expansin-encoding LeEXP8 and LeEXP18 genes that facilitate cell wall extension and contribute to cell wall disassembly [46, 47]. Studies have shown that the mRNA of LeEXP8 accumulates in germinating seeds in the cortical tissue of the root elongation zone [48]. In agreement with this, expression of LeEXP8 in the wild-type tomato was detected at a high level in the EC only, whereas in the S, the transcript level remained almost undetectable (Fig. 3d). In turn, the expression of LeEXP18 was detected in aerial parts of young soil-grown seedlings and not in roots [49]. In our experiments, expression of LeEXP18 decreased to almost zero in transgenics in both sectors, however, in the wild type it was very low in the S, and high in the EC. Thus, this study demonstrates that LeEXP18 in wild-type tomato is expressed specifically in the EC of the roots, and that a high-Zn apoplastic status in transgenic plants contributes to downregulation of its transcript level. Other downregulated genes in transgenics were plant cell wall-remodeling genes, THT7-1 and THT7-8, that encode an enzyme responsible for the synthesis of hydroxycinnamoyl tyramines. It has been proposed that they promote accumulation of cell wall-bound phenolic amines, and respond to wounding and ozone [50]. It seems that this is an example of common pathways for biotic and abiotic stresses, including the action of heavy metals. Finally, the XTHs and XET genes involved in modification of the cellulose/xyloglucan network [46, 51] were differentially expressed in transgenic tomato. XTHs encodes xyloglucan endotransglucosylase/hydroxylases with two distinct activities. Most act as transglucosylase referred to as XET, while some XTHs, preferentially as hydrolases. The encoded enzymes participate in a range of physiological processes including wall loosening, wall strengthening, cell-wall remodeling during abiotic stress [46, 51]. Interestingly, the accumulation profiles of these genes were different in AtHMA4-tomato, with moderate downregulation of tXET-B1 in both EC and S root sectors, and very strong upregulation of XTH3, specifically in the S. Compared with the wild type, the lower accumulation of tXET-B1 mRNA in transgenics is in agreement with the overall expression pattern of cell-wall remodeling genes, indicating strengthening of this structure in AtHMA-tomato roots. Transglycosylation by XET can increase or reduce the length of polysaccharides, which could result in either cell wall expansion or disassembly [51].
Expression of the cell wall-remodeling genes was not further analyzed upon longer exposure to the lower (0.5 μM) Zn concentration and in the presence of Cd, as cell wall modification in response to metals was not the major aim of this study. However, these data constitute a basis for further research with a focus on the specific role of the apoplast in a plant’s response to Zn.
Leaves
The genes found to be expressed differently in the leaves of transgenic tomato vs. wild type were, except for LeXTH3, not the same as those found in roots (Figs. 3 and 7). LeXTH3 showed higher transcript levels in leaves, specifically in the EPP. In transgenic tomato, in addition to three XTHs (LeXTH1, LeXTH3, LeXTH7), differential expression of three Cel genes encoding endo-1,4-beta-D-glucanases (LeCel2, LeCel5, LeCel7) and LePMEU1 encoding pectin-methyl-esterase (PME) were identified. Endoglucanases are hydrolytic enzymes related to hemicellulose degradation involved in cell wall disassembly during vegetative growth and fruit ripening [46], but also involved in cell wall signaling. Both identified XTHs and Cel genes are ethylene-inducible [52, 53], and their tissue-specific expression in transgenic plants is accompanied by downregulation of LeETR1 and LeNR receptor genes and upregulation of ACO genes, which is very much specific for the EPS and EPP (Fig. 7). In this context, the detected higher expression of LePMEU1 in transgenic leaves in both EPS and EPP, known from its role in cell wall loosening and cell wall signaling by generation of active oligogalacturonides (OGAs) [54, 55], could be related to signaling the modification of the cell wall structure due to the apoplastic Zn excess. Higher expression of LePMEU1 was also detected in tomato exposed to 10 μM Zn with enhanced Zn concentration in the apoplast due to expression of AhHMA4::AhHMA4 [11]. Not much is known about the role of the identified classes of cell wall proteins in plant responses to metals, nonetheless, our study points to their significance in this process. More detailed analysis is needed to demonstrate the nature and physiological significance of these relationships, and to indicate whether they likely contribute to signaling and/or accommodation of Zn excess in the cell wall.