Ammonium Uptake, Mediated by Ammonium Transporters, Mitigates Manganese Toxicity in Duckweed, Spirodela polyrhiza

Nitrogen is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants. Here we investigated the influence of the two major sources of inorganic nitrogen, nitrate and ammonium, on the toxicity caused by excess of Mn in great duckweed, Spirodela polyrhiza. The revealed alleviating effect of ammonium on Mn-mediated toxicity, was complemented by detailed molecular, biochemical and evolutionary characterization of the species ammonium transporters (AMTs). Four genes encoding AMTs in S. polyrhiza, were classified as SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. Functional testing of the expressed proteins in yeast and Xenopus oocytes clearly demonstrated activity of SpAMT1;1 and SpAMT1;3 in transporting ammonium. Transcripts of all SpAMT genes were detected in duckweed fronds grown in cultivation medium, containing a physiological or 50-fold elevated concentration of Mn at the background of nitrogen or a mixture of nitrate and ammonium. Each gene demonstrated an individual expression pattern, revealed by RT-qPCR. Revealing the mitigating effect of ammonium uptake on manganese toxicity in aquatic duckweed S. polyrhiza, the study presents a comprehensive analysis of the transporters involved in the uptake of ammonium, shedding a new light on the interactions between the mechanisms of heavy metal toxicity and the regulation of the plant nitrogen metabolism.


Introduction
Together with carbon, hydrogen and oxygen, nitrogen (N) is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants [1][2][3]. Nitrate (NO − 3 ) and ammonium (NH + 4 ) are the two major forms of N supply for plants [4]. With different plant species demonstrating preferences for either nitrate or ammonium as a preferential source of nitrogen [5][6][7][8][9], many studies revealed the dependence between nitrogen resource and the plants' responses to stresses [10,11]. A number of studies also demonstrated that the form of supplied nitrogen affects the response of different plant species to stress caused by heavy metals such as aluminum, cadmium, copper, iron or mercury [12][13][14][15][16].
For example, the alleviating effect of NH + 4 compared to NO − 3 related to Al [15,17] and Cd toxicity [16] has been demonstrated in rice and wheat [18]. Similarly, increasing the between duckweed grown on a mixture of nitrate and ammonium supplied with high (M-Mn50) of normal (M-Mn1) amounts of Mn ( Figure 1). and high Mn (N-Mn50) compared to nitrate medium with usual concentration of Mn (N-Mn1). Simultaneously, almost no difference in biomass accumulation was registered between duckweed grown on a mixture of nitrate and ammonium supplied with high (M-Mn50) of normal (M-Mn1) amounts of Mn ( Figure 1). Suppression of biomass accumulation in the N-Mn50 medium was accompanied by severe chlorosis and death of whole duckweed fronds profoundly manifested at day 12.
To the contrary, after 12 days cultivation, duckweed grown in the medium supplied with NH (M-Mn50), mostly remained green with partial local browning of frond apexes, but no total chlorosis observed. In course of duckweed growth, the relevant changes in media such as consumption of nitrogen and pH have been monitored (Figure 1).
The dynamics of NO 3 consumption by S. polyrhiza growing on N-Mn1 and N-Mn50 media were pretty similar, with the available nitrogen reduced by about a half by day 12. Duckweed cultivated in the media containing both NO 3 and NH 4 (M-Mn1 and M-Mn50) preferentially utilized ammonium, with nitrate almost untouched in the M-Mn50 and only slightly consumed in M-Mn1 by day 12 ( Figure 1C).
During the growth period, we observed contrasting dynamics of medium acidity between medium containing NO 3 , as a soul source of nitrogen and a mixture of NO 3 and NH 4 . The original medium pH of 5.8 climbed up to 7.01-7.18 in the medium supplied Suppression of biomass accumulation in the N-Mn50 medium was accompanied by severe chlorosis and death of whole duckweed fronds profoundly manifested at day 12.
To the contrary, after 12 days cultivation, duckweed grown in the medium supplied with NH + 4 (M-Mn50), mostly remained green with partial local browning of frond apexes, but no total chlorosis observed. In course of duckweed growth, the relevant changes in media such as consumption of nitrogen and pH have been monitored (Figure 1).
The dynamics of NO − 3 consumption by S. polyrhiza growing on N-Mn1 and N-Mn50 media were pretty similar, with the available nitrogen reduced by about a half by day 12. Duckweed cultivated in the media containing both NO − 3 and NH + 4 (M-Mn1 and M-Mn50) preferentially utilized ammonium, with nitrate almost untouched in the M-Mn50 and only slightly consumed in M-Mn1 by day 12 ( Figure 1C).
During the growth period, we observed contrasting dynamics of medium acidity between medium containing NO − 3 , as a soul source of nitrogen and a mixture of NO − 3 and NH + 4 . The original medium pH of 5.8 climbed up to 7.01-7.18 in the medium supplied with nitrate, whereas the pH gradually dropped to 3.94-4.02 on day 12 in the medium containing both nitrate and ammonium. There were no differences in pH dynamics in the pairs of media containing usual or high concentration of Mn, N-Mn1 and N-Mn50 or M-Mn1 and M-Mn50 ( Figure 1D).

Genome of S. polyrhiza Containes Four Genes Coding for Ammonium Transporters
A blast search on the S. polyrhiza genome (ecotype Sp9509 [44]), available on the NCBI website (taxid: 29656, GCA_900492545.1), using the protein sequences of rice ammonium transporters in the queries revealed four genes potentially encoding ammonium transporters SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. The exon/intron structures of the gene sequences were deduced by their similarities with the corresponding rice sequences, showing three intron-free genes belonging to the AMT1 subfamily and only SpAMT2 containing two short introns. A similar arrangement of the AMT genes has been revealed also for the second species in the genus Spirodela, S. intermedia ( Figure S1) by using BLAST against the corresponding genome at the NCBI website (taxid: 51605). To validate the sequences available in the GenBank, we sequenced the amplification products for the four SpAMTs derived from cDNA of ecotype NB5548 used in this study (the corresponding sequence accession numbers are: OP730321, OP730322, OP730323 and OP730324).
Phylogenetic analysis of the deduced protein sequences showed that SpAMT1;1 and SpAMT1;2 are closely related to each other, clustering together with their homologues from S. intermedia and taro, Colocasia esculenta, a representative of Araceae, the sister family to Lemnaceae [46]. The sequences also demonstrate significant similarity to more remote monocotyledonous species (date palm, Phoenix dactylifera; Cocos, rice and wheat), as well as the representative of dicotyledonous, Arabidopsis thaliana ( Figure 2). Similar phylogenetic distances relative to other monocotyledonous and dicotyledonous were revealed also for SpAMT1;3 and SpAMT2.

Functional Activity of the Ammonium Transporters in Yeast and Xenopus Oocytes Systems
To test general transport function of the identified potential SpAMTs we performed a complementation analysis by expressing the four duckweed ammonium transporter proteins from the pDR199 vector in a yeast strain deficient in AMT function (31019b, ∆∆∆mep; [49]). The yeast was grown on medium supplied with NH + 4 as a sole source of Nitrogen at two concentrations, 0.3 and 1 mM and three different pH: 4.5, 5.5 and 6.5 ( Figure 3). The highest complementation activity in all experimental settings, demonstrated the duckweed ammonium transporter SpAMT1;3, followed by the SpAMT1;1. The complementation efficiency of SpAMT1;3 was comparable to that of Arabidopsis AtAMT1;1, with the activity of SpAMT1;1 resembling that of AtAMT2. We were not able to detect any complementing activity of SpAMT1;2. The SpAMT2 showed some activity slightly above the pDR199 control only at 1 mM concentration of NH + 4 and medium pH 6.5. In general, there was no pH effect on any of the transporters. When grown on 60 mM methylammonia (MeA), a toxic analog of NH + 4 , the active transporters conferred transport activity of MeA as well, which led to strongly reduced yeast growth.  To analyse the two functional duckweed AMTs in more detail, the proteins were expressed in Xenopus leavis oocytes and the ammonium induced currents where recorded by two electrode voltage clamp. Ammonium induced significant inward currents in the oocytes expressing SpAMT1;1 and SpAMT1;3 ( Figure 4). As expected, the currents increased with more negative membrane potential ( Figure 4A). Total currents by SpAMT1;3 were in the low nano ampere range and increased with increasing ammonium concentration with a low affinity Km = 5.4 mM. The SpAMT1;1 mediated currents were about three times higher than the currents by SpAMT1;3 ( Figure 4B) and saturated with a high affinity Km = 116 µM tipical for plant AMTs ( Figure 4C). No currect induction was detected in the oocytes transfected with SpAMT1;2 or SpAMT2 (data not shown). Based on the obtained results, we can characterise SpAMT1;1 as a tipical high affinity plant To analyse the two functional duckweed AMTs in more detail, the proteins were expressed in Xenopus leavis oocytes and the ammonium induced currents where recorded by two electrode voltage clamp. Ammonium induced significant inward currents in the oocytes expressing SpAMT1;1 and SpAMT1;3 ( Figure 4). As expected, the currents increased with more negative membrane potential ( Figure 4A). Total currents by SpAMT1;3 were in the low nano ampere range and increased with increasing ammonium concentration with a low affinity K m = 5.4 mM. The SpAMT1;1 mediated currents were about three times higher than the currents by SpAMT1;3 ( Figure 4B) and saturated with a high affinity K m = 116 µM tipical for plant AMTs ( Figure 4C). No currect induction was detected in the oocytes transfected with SpAMT1;2 or SpAMT2 (data not shown). Based on the obtained results, we can characterise SpAMT1;1 as a tipical high affinity plant ammonium transporter while SpAMT1;3 showed uncommon low affinity for ammonium.

Expression Profiling of S. polyrhiza AMT Genes by qRT-PCR
The expression of the SpAMT genes was evaluated at the growth stage follo days of cultivation in the four media formulations mentioned above, using the represented in Table S1 according a previously described RT-qPCR protocol [42]. qPCR reactions were performed in triplicate, normalized against the expression housekeeping genes (β-actin and histone H3), and normalized to the gene expressio at the starting point (SP). The expression of S. polyrhiza genes encoding AMT AMT1;3, which demonstrated clear activity in oocytes and yeast tests, showed a c and distinct regulation by Mn and the source of nitrogen. Adding of nitrogen in NO at the background of physiological concentration of Mn (N-Mn1) led to a slig vation of both SpAMT1;1 and SpAMT1;3, whereas a mixture of NO and NH stim expression of SpAMT1;3 but did not SpAMT1;1 ( Figure 5). The 50-fold elevated con

Expression Profiling of S. polyrhiza AMT Genes by qRT-PCR
The expression of the SpAMT genes was evaluated at the growth stage following 4 days of cultivation in the four media formulations mentioned above, using the primers represented in Table S1 according a previously described RT-qPCR protocol [42]. All RT-qPCR reactions were performed in triplicate, normalized against the expression of two housekeeping genes (β-actin and histone H3), and normalized to the gene expression levels at the starting point (SP). The expression of S. polyrhiza genes encoding AMT1;1 and AMT1;3, which demonstrated clear activity in oocytes and yeast tests, showed a complex and distinct regulation by Mn and the source of nitrogen. Adding of nitrogen in form of NO − 3 at the background of physiological concentration of Mn (N-Mn1) led to a slight activation of both SpAMT1;1 and SpAMT1;3, whereas a mixture of NO − 3 and NH + 4 stimulated expression of SpAMT1;3 but did not SpAMT1;1 ( Figure 5). The 50-fold elevated concentration of Mn affected the expression of SpAMT1;1 and SpAMT1;3 in opposite directions, enhancing SpAMT1;1 and suppressing SpAMT1;3, with these effects manifested much stronger in the presence of nitrate as a sole source of N, N-Mn50, compared to nitrate and ammonium mixture, M-Mn50.  The expression of SpAMT1;2, was downregulated in all experimental sampl pared to SP. On the contrary, SpAMT2, which similar to SpAMT1;2, did not show tion either in oocyte or in the yeast test, demonstrated a 3.3-fold increased level scripts in the N-Mn50 sample, while expression was downregulated both in the and the M-Mn50 samples compared to the starting point.

Regulatory Cis-Elements in the Promoters of Spirodela AMT Genes
To get further insights into the expression regulation of S. polyrhiza AMT ge conducted an in silico analysis of cis-elements in the gene promoters. The 1.5 kb lo moter sequences upstream from the protein translation starts, extracted from the a online genome sequence, version 3 of S. polyrhiza, strain 9509 (GCA_900492545 analyzed using the New Place promoter analysis software (https://www frc.go.jp/PLACE, accessed on 7 October 2022) [50]. In parallel, we conducted a analysis of corresponding gene promoters in a recently published genome of S. in (taxid: 51605) a closely related duckweed species, with the aim to further highl evolutionary conserved regulatory elements ( Figure 6). The expression of SpAMT1;2, was downregulated in all experimental samples compared to SP. On the contrary, SpAMT2, which similar to SpAMT1;2, did not show a function either in oocyte or in the yeast test, demonstrated a 3.3-fold increased level of transcripts in the N-Mn50 sample, while expression was downregulated both in the M-Mn1 and the M-Mn50 samples compared to the starting point.

Regulatory Cis-Elements in the Promoters of Spirodela AMT Genes
To get further insights into the expression regulation of S. polyrhiza AMT genes, we conducted an in silico analysis of cis-elements in the gene promoters. The 1.5 kb long promoter sequences upstream from the protein translation starts, extracted from the available online genome sequence, version 3 of S. polyrhiza, strain 9509 (GCA_900492545.1) were analyzed using the New Place promoter analysis software (https://www.dna.affrc.go.jp/PLACE, accessed on 7 October 2022) [50]. In parallel, we conducted a similar analysis of corresponding gene promoters in a recently published genome of S. intermedia (taxid: 51605) a closely related duckweed species, with the aim to further highlight the evolutionary conserved regulatory elements ( Figure 6). A prominent structural feature revealed in all analyzed promoters, was the abundant presence of GAGA motifs or its complement CTCT, involved in regulation of eukaryotic genomes through chromatin modulations [51][52][53]. Especially intriguing is the presence of a more than 50 bp long GAGA stretch in promoters of AMT1;1 gene of S. polyrhiza and S. intermedia, closely adjacent to the ATG of protein translation start. Within the first 500 bp upstream of the translation start, all analyzed promoters feature one (AMT1;1 and AMT1;3) or multiple (AMT1;2 and AMT2) TATA motifs. The TATA-box is recognized as binding sites for a basic transcription factor TBP (TATA box-binding protein) involved in transcription initiation of many eukaryotic genes [54]. Moreover, inside 500 bp proximity to the ATG, all AMT1 genes promoters contain general eukaryotic cis-element CCAATbox [55] a target for transcription complex NF-Y (Nuclear Factor Y), which plays an essential role in many processes related to plant development, productivity and stress responses [56,57]. The SpAMT2 and SiAMT2 promoters have multiple CCAAT sites located between −1000 and −500 bp relating to the first ATG.
Transcription factors IDD (Indeterminate Domain) and DOF (DNA binding with one finger) were shown to be involved in regulation of the AMT genes in a number of different plants [58]. We located the recognition site for IDD, with the signature ACAAA, within the first 500 bp at the identical positions in the AMT2 promoters of both S. polyrhiza and S. intermedia. In the promoters of the AMT1 genes the site is scattered at various locations more remote from the translation start. As for the DNA motif recognized by DOF, T/AAAAG, multiple copies are found within the first 500 bp of all AMT1 promoters, as well as scattered at more distant locations in promoters of both AMT1 and AMT2 genes.
The members of the NIGT1 (Nitrate-Inducible GARP-Type Transcriptional Repressor-1) subfamily in the superfamily of GARP transcription factors [59], participate in fine-tuning of gene expression in response to the plant nitrogen and phosphorus status [60]. The proteins bind to two types of cis-elements, GAATC and GAATATTC demonstrating dual modes of promoter recognition [61,62]. The GAATATTC site has been A prominent structural feature revealed in all analyzed promoters, was the abundant presence of GAGA motifs or its complement CTCT, involved in regulation of eukaryotic genomes through chromatin modulations [51][52][53]. Especially intriguing is the presence of a more than 50 bp long GAGA stretch in promoters of AMT1;1 gene of S. polyrhiza and S. intermedia, closely adjacent to the ATG of protein translation start. Within the first 500 bp upstream of the translation start, all analyzed promoters feature one (AMT1;1 and AMT1;3) or multiple (AMT1;2 and AMT2) TATA motifs. The TATA-box is recognized as binding sites for a basic transcription factor TBP (TATA box-binding protein) involved in transcription initiation of many eukaryotic genes [54]. Moreover, inside 500 bp proximity to the ATG, all AMT1 genes promoters contain general eukaryotic cis-element CCAAT-box [55] a target for transcription complex NF-Y (Nuclear Factor Y), which plays an essential role in many processes related to plant development, productivity and stress responses [56,57]. The SpAMT2 and SiAMT2 promoters have multiple CCAAT sites located between −1000 and −500 bp relating to the first ATG.
Transcription factors IDD (Indeterminate Domain) and DOF (DNA binding with one finger) were shown to be involved in regulation of the AMT genes in a number of different plants [58]. We located the recognition site for IDD, with the signature ACAAA, within the first 500 bp at the identical positions in the AMT2 promoters of both S. polyrhiza and S. intermedia. In the promoters of the AMT1 genes the site is scattered at various locations more remote from the translation start. As for the DNA motif recognized by DOF, T/AAAAG, multiple copies are found within the first 500 bp of all AMT1 promoters, as well as scattered at more distant locations in promoters of both AMT1 and AMT2 genes.
The members of the NIGT1 (Nitrate-Inducible GARP-Type Transcriptional Repressor-1) subfamily in the superfamily of GARP transcription factors [59], participate in fine-tuning of gene expression in response to the plant nitrogen and phosphorus status [60]. The proteins bind to two types of cis-elements, GAATC and GAATATTC demonstrating dual modes of promoter recognition [61,62]. The GAATATTC site has been identified only in the AMT2 promoters of S. polyrhiza and S. intermedia about 550 bp upstream of the first ATG, while the GAATC recognition site was located along the sequence of all promoters with the highest representation in the promoters of AMT1;2 and AMT2 genes.

Discussion
Our previous study demonstrated that the extend on Mn toxicity in four aquatic species of duckweed (Lemnaceae) depends on the source and concentration of nitrogen in the cultivation medium [43]. In particular, in great duckweed, Spirodela polyrhiza, the first characteristic symptoms of Mn stress appeared in form of characteristic brown spots when concentration of Mn was 40 mg/L (0.73 mM); however, the symptoms were largely reduced by the presence of ammonium in the medium in addition to nitrate. This observation, together with (i) increasing pollution of water resources with heavy metals [34][35][36][37], (ii) multiple previous reports linking the level of toxicity caused by various heavy metals to the nitrogen metabolism [12][13][14][15][16] and (iii) availability of the solid set of data on whole Spirodela genome sequences [45,63,64], encouraged us to take a closer look at Mn toxicity in S. polyrhiza in relation to the source of inorganic nitrogen with a special attention on the species's ammonium transporters (AMTs). , looked perfectly healthy and produced higher biomass compared to the medium with a mixture of a 50% of NO − 3 and 50% of NH + 4 ( Figure 1A). This observation is in accordance with the previously published data showing that growth rate of two duckweed species, S. polyrrhiza and Lemna aequinoctialis was lower in the medium containing ammonia compared to the medium with nitrate [65]. In addition to reduced biomass, selected fronds in the M-Mn1 medium showed some signs of chlorosis. These symptoms can be attributed to general ammonium toxicity [29][30][31], which we also observed in our previous study on six duckweed species, including S. polyrhiza [42].

NH
The picture dramatically changed when the cultivation medium was supplied with elevated Mn. The fronds' appearance and biomass accumulation remained practically the same for duckweed in the medium with mixture of NO − 3 and NH + 4 (M-Mn50) compared to M-Mn1, except of occasional brown spots characteristic for Mn-mediated stress. In contrast, duckweed cultivated in the N-Mn50 medium with NO − 3 as a sole N source, manifests severe senescence, probably even death, and significant reduction in biomass accumulation compared not only to the N-Mn1 medium, but also to duckweed grown in medium containing the mixture of NO − 3 and NH + 4 supplied with either normal (M-Mn1) or high concentration of Mn (M-Mn50). It is worth to notice that high concentration of Mn had no effect on the dynamics of NO − 3 and NH + 4 uptake or the dynamics of pH during the cultivation period ( Figure 1C,D). Confirming our previous study [42], utilization of NO − 3 led to a pH increase in the medium, whereas the uptake of NH + 4 resulted in acidification of the nutrient medium, as has been shown earlier for a number of plants [42,66,67].
The lowering of pH in the media containing NH + 4 might be a part of the mechanism of the observed alleviation of Mn toxicity mediated by NH + 4 in S. polyrhiza. For example, NH + 4 was shown to alleviate Mn stress symptoms in spruce and rice [68,69], and the decrease in Mn uptake by medium acidification was proposed as a mechanism underlying this phenomenon [69]. pH sensitivity has been reported as a general feature of transmembrane solute movement [70] as well as for many kinds of specific transporters in different organisms [71][72][73][74]. The reported data demonstrate that conformation and functionality of the plant transmembrane transporters are dependent upon cellular and intercellular pH. Dependence on pH, is especially well established in relation to the activity of aquaporins [75,76]. Aquaporins represent a large family of plant proteins with members in the model plant Arabidopsis thaliana [77] responsible not only for the plants water homeostasis, but for transporting many other molecules including ammonia and other small solutes [78]. Amounting evidence from across phyla suggests that some aquaporin types act as ion channels, and are modulated by divalent cations such as Ca 2+ and Cd 2+ [79]). Moreover, in Arabidopsis, in addition to pH, manganese was identified as potent inhibitors of aquaporin AtPIP2;1 [80]. In order to check the possibility that aquaporins could contribute to Mn stress in our system by responding to Mn and/or ammonium/pH, we have tested expression of two duckweed homologues of Arabidopsis aquaporins, SpTIP2;1 and SpPIP2;2 by quantitative RT-qPCR ( Figure 7, Table S1). Indeed, both genes showed differential expression in response to the applied experimental conditions. The SpTIP2;1 was significantly suppressed in the N-Mn50 environment but slightly upregulated in the M-Mn1 and M-Mn50, whereas SpPIP2;2 was upregulated in all experimental medium, especially in the medium containing NH + 4 (M-Mn1 and M-Mn50).
Plants 2023, 12, x FOR PEER REVIEW Dependence on pH, is especially well established in relation to the activity of aq [75,76]. Aquaporins represent a large family of plant proteins with members in th plant Arabidopsis thaliana [77] responsible not only for the plants water homeos for transporting many other molecules including ammonia and other small sol Amounting evidence from across phyla suggests that some aquaporin types a channels, and are modulated by divalent cations such as Ca and Cd [79]). M in Arabidopsis, in addition to pH, manganese was identified as potent inhibitors porin AtPIP2;1 [80]. In order to check the possibility that aquaporins could contribute to Mn stre system by responding to Mn and/or ammonium/pH, we have tested expressio duckweed homologues of Arabidopsis aquaporins, SpTIP2;1 and SpPIP2;2 by qua RT-qPCR (Figure 7, Table S1). Indeed, both genes showed differential expressi sponse to the applied experimental conditions. The SpTIP2;1 was significantly su in the N-Mn50 environment but slightly upregulated in the M-Mn1 and M-Mn50, SpPIP2;2 was upregulated in all experimental medium, especially in the medium ing NH (M-Mn1 and M-Mn50). We can assume that the acidification of the cultivation media and probably may inactivate the transporters supporting inward Mn transport into the cytosol to Mn exclusion from plant tissues. The presented data on differential expression porins may pave a way for future research in this area.

Ammonium Transporter (AMT) Genes in the Genome of S. polyrhiza
Ammonium transporters (AMTs) are transmembrane proteins, usually con a N-terminal non-cytoplasmic domain, 11 transmembrane helices and C-termi plasmic domain [81,82], responsible for transporting NH from the outside into t of the cell. Intensive studies on AMTs from different plant species revealed that tein family is divided into two distinct subfamilies, the AMT1 and the AMT2, rep by different number of members in different species [58,83]. Compared to other polyrhiza contains fewer genes coding for the AMTs. For example, Arabidopsis tha five genes coding for AMT1 variants and one gene for AMT2 [83], and rice has thr bers of the AMT1 subfamily [84] and nine representatives of AMT2 [85]. Our an the S. polyrhiza genome revealed three variants of AMT1: SpAMT1;1, Sp SpAMT1;3 and a single SpAMT2, all encoded by single copy genes located on chromosomes. This finding further illustrates the reduced number of protei genes in S. polyrhiza originally observed by the first genome sequencing [63], whe reduction in genes compared to Arabidopsis thaliana and 50% reduction compare was shown. This was later confirmed by Michael et al. (2017) [45] showing that m families in Spirodela have less members as compared with the model dicotyledono We can assume that the acidification of the cultivation media and probably Mn itself may inactivate the transporters supporting inward Mn transport into the cytosol and lead to Mn exclusion from plant tissues. The presented data on differential expression of aquaporins may pave a way for future research in this area.

Ammonium Transporter (AMT) Genes in the Genome of S. polyrhiza
Ammonium transporters (AMTs) are transmembrane proteins, usually consisting of a N-terminal non-cytoplasmic domain, 11 transmembrane helices and C-terminal cytoplasmic domain [81,82], responsible for transporting NH + 4 from the outside into the inside of the cell. Intensive studies on AMTs from different plant species revealed that this protein family is divided into two distinct subfamilies, the AMT1 and the AMT2, represented by different number of members in different species [58,83]. Compared to other plants, S. polyrhiza contains fewer genes coding for the AMTs. For example, Arabidopsis thaliana has five genes coding for AMT1 variants and one gene for AMT2 [83], and rice has three members of the AMT1 subfamily [84] and nine representatives of AMT2 [85]. Our analysis of the S. polyrhiza genome revealed three variants of AMT1: SpAMT1;1, SpAMT1;2, SpAMT1;3 and a single SpAMT2, all encoded by single copy genes located on different chromosomes. This finding further illustrates the reduced number of protein-coding genes in S. polyrhiza originally observed by the first genome sequencing [63], where a 28% reduction in genes compared to Arabidopsis thaliana and 50% reduction compared to rice was shown. This was later confirmed by Michael et al. (2017) [45] showing that most gene families in Spirodela have less members as compared with the model dicotyledonous plant Arabidopsis and monocotyledonous Brachypodium. Sequence analysis of the cloned cDNA from the Chinese ecotype NB5548 shows very high homology to the corresponding sequences of European ecotype 9509 (BlastN GCA_900492545.1) at the level of 99.6-99.8% similarity, with just a few observed C↔T or G↔A substitutes in the 1500 bp long sequences of the three SpAMT1 genes, and 100% similarity for SpAMT2. This level of sequence conservation between ecotypes of S. polyrhiza is in a good agreement with the previously postulated extremely low rate of mutations revealed by comparing genomes of 68 worldwide geographic ecotypes of this species [86]. The SpAMT gene sequences also show similar arrangement, no introns in all three AMT1 gene sequences, two short introns within the AMT2 gene, and 95-97% sequences similarities with their homologues in the genome of the closely related S. intermedia [64], as represented in Figure S1.
The deduced SpAMT protein sequences demonstrate most close phylogenetic relationship to their counterparts from S. intermedia, followed by taro, Colocasia esculenta [87], a plant species which belongs to the group of early-branching monocotyledonous family Araceae [88], a sister family to Lemnaceae [46]. The SpAMT proteins also demonstrate significant levels of similarity to the homologues from other monocotyledonous banana, cocos, oil palm, rice and wheat, as well as dicotyledonous Arabidopsis (Figure 2), stressing strong evolutionary conservation of this important protein family along the plants [83,89]. Among S. polyrhiza AMTs, SpAMT1;1 and SpAMT1;2 share the highest homology, whereas the two proteins have certain differences in their C-termini ( Figure S2). This offers possibilities for differential post-translational regulation since the proteins' C-terminal domain was reported as a target for environmental regulation through phosphorylation by protein kinases [82,[90][91][92].

SpAMT1;1 and SpAMT1;3 Demonstrate Clear Activity in Yeast and Oocytes
From the four SpAMT proteins expressed in yeast and Xenopus oocytes, SpAMT1;1 and SpAMT1;3 demonstrated highly specific activity in parallel with the Arabidopsis controls ( Figure 3). Interestingly, the homologues of these transporters in Arabidopsis, AtAMT1;1 and AtAMT1;3, have been reported for conferring 60% of total ammonium uptake combined [93,94]. To the contrary, SpAMT1;2 showed no activity in all of the experimental conditions and SpAMT2 was slightly activated at elevated pH in yeast complementation test. One might ponder about the silence of the SpAMT1;2, considering its phylogenetic closeness with the SpAMT1;1 (Figure 2) which shows a quite good activity in transporting NH + 4 . However, when looking on the structure of both proteins in more details, one can see that despite a high homology of the transmembrane part in the middle of the proteins, the non-cytoplasmic N-termini domains and the cytoplasmic C-termini domains show significant differences ( Figure S2). For example, specific mutations of a conserved Gly in this domain was shown to inactivate AMTs from yeast and plants [49,95]. The mentioned mutation, however, cannot explain the functional differences between SpAMT1;1 and SpAMT1;2. The Gly is intact within the conserved first half of the C-terminal domain in both proteins. Another well-documented option of AMT regulation, is by post-translational phosphorylation of the Thr, located 4 residues downstream of the above mentioned Gly [96]. This position also does not differ between SpAMT1;1 and SpAMT1;2, as evident from the protein alignment in Figure S2. However, additional Thr residues uniquely positioned within both N-terminal and C-terminal domains of SpAMT1;2 ( Figure S2), offer an opportunity of differential regulation of this protein compared to SpAMT1;1, through phosphorylation. These suggestions are purely speculative at the moment, and require an experimental verification.

The Four S. polyrhiza AMT Genes Show Different Expression Patterns in Response to Nitrogen Source and Mn
Our data show that both nitrogen source and elevated concentration of Mn affect expression of each of the SpAMT genes in a unique and in some instances opposite direction under the applied experimental conditions (Figure 3). Although NO − 3 slightly stimulated expression of SpAMT1;1 and SpAMT1;3, it downregulated SpAMT1;2 and SpAMT2. NH + to nitrogen is not too surprising. It is well in line with multiple previous reports showing diversity of AMT responses in other species such as Arabidopsis [97], rice [98], maize [99], poplar [14,100], apple [101] or tobacco [102]. The availability of multiple AMT genes with individual expression patterns, and multiple capacity of the expressed transporters for NH + 4 uptake is considered to provide plants with great flexibility to respond differentially to varying environmental and nutritional conditions by fine tuning gene expression.
Simultaneous, presence of high concentrations of Mn in the cultivation media containing nitrate, N-Mn50, or a mixture of nitrate and ammonium, M-Mn50, dramatically changes the expression of SpAMT genes compared to the media with physiological concentration of Mn, N-Mn1 and M-Mn1. Thus, we observed drastic increase in expression of SpAMT1;1 and SpAMT2 in the medium containing NO − 3 as a sole source of nitrogen, N-Mn50, whereas the increase in the M-Mn50 medium was rather mild ( Figure 5). Based on this specific expression dynamics, we are inclined to explain the observed expression boost not by direct influence of Mn, but rather by the process of senescence started by Mn toxicity and manifested in fronds cultivated in the N-Mn50 to a significantly greater extend, compared to the duckweed grown in the M-Mn50 (Figure 1). Observations in other plants, showing upregulation of the SpAMT1;1 and SpAMT2 homologues in old tissues, suggesting the role of these proteins in nitrogen remobilization [103], might be used in support of this notion. For example, van der Graaff et al., (2006) [104] revealed enhanced expression of AtAMT1.1 in senescing leaves of Arabidopsis, and more recently, Liu et al., (2018) [102] showed that of nine AMT transporter genes identified in tobacco, only two, NtAMT1.1 and NtAMT2.1, were strongly expressed in the old leaves, implying their dominant roles in N remobilization from senescent tissues.
Contrary to SpAMT1;1 and SpAMT2, expression of the SpAMT1;2 and SpAMT1.3 seems to be influenced by Mn in a more direct manner. High Mn concentration suppressed expression of both genes independent of the: (i) supplied nitrogen formulation, N-Mn50 or M-Mn50 as, respectively, compared with N-Mn1 or M-Mn1 ( Figure 5) and (ii) the differences in the acidity of the cultivation medium, correspondingly pH~6.5 for N-Mn1 and N-Mn50 and pH~4.0 for M-Mn1 and M-Mn50 (Figure 1). The question whether expression of SpAMT1;2 and SpAMT1.3 is directly controlled by Mn or through some Mn-mediated physiological changes such as hormonal status, generation of reactive oxygen species (ROS), or some other signaling pathway remains to be investigated.

Distinctive Promoter Elements in the AMT Genes in S. polyrhiza
Many environmental and physiological factors, nitrogen status, soil pH, photoperiod, developmental stage [58], have been recognized to influence ammonium transporter expression in plants. These factors regulate expression of AMT genes primarily via interaction of various transcription factors with specific cis-elements in the promoter of the genes [55]. Our analysis of AMT gene promoters in Spirodela, revealed characteristic ciselement patterns which might affect transcriptional regulation of ammonium uptake, and are distinctive from the genes encoding major nitrogen assimilation genes responsible to both NO − 3 and NH + 4 [42]. The promoters of AMT genes are populated by (GA/CT) n repeats (Figure 6), targets for GAGA/TCTC-binding transcription factors (GAFs). Abundance of the GAGA in the promoters is not very surprising, considering that genomes of both S. polyrhiza and S. intermedia, are un-proportionally enriched in these motifs [45,64]. However, the contrast in distribution of the sequences in AMTs and nitrogen assimilation genes promoters is noticeable. In the analyzed AMT promoters, relatively short GAGA-stretches are more or less evenly distributed along the 1.5 kb length of the promoter, with a unique~50 bp GAGAstretch located just upstream of the ATG in the AMT1;1 promoter ( Figure 6), whereas in most of the promoters of nitrogen assimilation genes the (GA/CT) n repeats are represented by long stretches located outside of the 500 bp region adjacent to the genes' translation start.
Among prominent conservative cis-elements present within the 500 bp region upstream of translation start in all analyzed Spirodela AMTs are the TATA-box, a binding site for the general eukaryotic transcription factor TBP [54]. All AMT promoters also contain another general eukaryotic cis-element CCAAT-box [55], a binding site for NF-Y (nuclear factor Y) [105], involved in many important growth and developmental processes [106], including stress and hormone responses [107,108].
Overall, the range of cis-elements revealed in the Sprirodela AMT genes promoters in combination with the wide selection of corresponding transcription factors provide huge flexibility to regulate the AMT transcripts in response to various developmental, environmental and nutritional challenges faced by a plant during the life span.

Summary
Here we showed that the toxicity of manganese in duckweed plants could be mitigated by addition of ammonium as a nitrogen source. This mitigation effect might be pH related since the preferential uptake of ammonium by S. polyrhiza resulted in a pH decrease in the media which in turn might lead to a decreased activity of Mn uptake transporters e.g., aquaporins. The uptake of ammonium from the media could be mediated by four SpAMT proteins. The expression of the AMT genes was uniquely regulated by Mn and the available nitrogen source and two of the AMT proteins showed strong activity in heterologous systems. Their interesting unique transcriptional regulation is orchestrated by multiple transcription factor binding sites in the AMT promoters. The exact mechanism of Mn resistance by ammonium as well as the identification of transcription factors involved in SpAMT regulation are interesting questions which should be addressed in future studies.

Plant Material
The S. polyrhiza ecotype (collection ID: NB5548) used in this study was selected from the duckweed live in vitro collection recently established in the School of Life Sciences at Huaiyin Normal University, Huai'an, China. The NB5548 ecotype was originally collected in fall 2017 (N 33"17 40; E 118"49 45), surface sterilized and propagate on solid agar medium supplemented with SH salts [109] under sterile conditions.

Duckweed Cultivation Parameters
To accumulate biomass, duckweed plants grown on solid agar medium were initially transferred into 200 mL sterile liquid SH medium supplemented with 5 g/l sucrose contained in 500-mL flasks and cultivated at 23 ± 1 • C with a photon flux density of 50-60 µmol·m −2 s −1 provided by cool white fluorescent bulbs in a 16-h light/8-h dark cycle. After four weeks of growth, the accumulated fronds were washed 3 times with autoclaved water, and 200 mL sterile N-and sugar-free basal salt SH medium was added. After 3 days cultivation, 0.5-g portions were blotted and weighed in aseptic condition and then inoculated into 100-mL flasks containing 60 mL of autoclaved basic SH medium (no sugar) supplemented with two different formulations of N (N and M) and two concentrations of MnSO 4 SO 4 ). The medium pH was adjusted to 5.5 and autoclaved. In total, 18 flasks with growing duckweed have been prepared for every variant of tested media. Three flasks (as 3 independent biological repeats) were randomly taken after 4 and 12 days for each of the analyses (RNA isolation for RT-qPCR, biomass measurement, determination of the total concentration of N). The duckweed biomass was also collected at the starting point (designated as SP), which corresponds to the moment before washing and 3-days cultivation on a medium containing no nitrogen (starvation).

Determination of N Concentration
The total nitrogen concentration was determined using standard alkaline potassium persulfate digestion followed by UV spectrophotometry as previously described [110]. The NO − 3 concentration in the growth media was measured spectrophotometrically as the difference in absorption between 220 and 275 nm [111]. The NH + 4 concentration in the growth media was measured calorimetrically using the Nessler method [112].

Cloning and Analysis of S. polyrhiza Genes Encoding Ammonium Transporters
The AMT1;1, AMT1;2, AMT1;3 and AMT2 genes were obtained by sequencing and cloning the PCR-amplified gene regions using cDNA prepared from local S. polyrhiza, ecotype NB5548, mRNA as a template. The PCR fragments were amplified with genespecific primers designed according to the in silico sequence information available at NCBI (taxid: 29656, GCA_900492545.1) for S. polyrhiza, ecotype 9509. The generated DNA fragments, cloned into the vector pMD19 (Takara, Dalian, China) following the manufacturer's instructions were custom sequenced (Sangon Biotech, Shanghai, China), and the obtained nucleotide sequences were analyzed using the CLC Main Workbench (Version 6.9.2, Qiagen) software. The specific primers used for gene amplification are listed in Table S2.

Phylogenetic Analysis
Protein sequences of AMT proteins were compared with a selection sequence available in public databases. The corresponding sequences available for monocotyledonous and dicotyledonous were extracted from the GenBank (National Center for Biotechnology Information (NCBI). Available online: https://www.ncbi.nlm.nih.gov (accessed on 15 August 2022) by blasting with the S. polyrhiza sequences as a query. The maximumlikelihood phylogenetic trees were constructed using NGPhylogeny webservice accessible through the https://ngphylogeny.fr [47] using MAFFT Multiple Sequence Alignment [113] and PhyML algorithm [114] with the Jones-Taylor-Thornton (JTT) model. Cleaning aligned sequences was made by utilizing BMGE tools [115]. Bootstrap support (BS) was estimated with 100 bootstrap replicates. iTOL (https://itol.embl.de) was used for displaying and annotating the generated phylogenetic trees [48].

Construction of Expression Vectors and Testing AMTs Functionality in Yeast and Xenopus oocytes
For testing in yeast and oocytes the coding sequences of SpAMP1;1, SpAMP1;2, SpAMP1;3 and SpAMP2 were cut out from the original plasmid pMD19 and ligated into the vector pDR199 for expression in yeast [82] or the oocyte expression vector pOO2 [116]. Following verification by sequencing, the resulting vectors pDR199-SpAMP1;1, pDR199-SpAMP1;2, pDR199-SpAMP1;3 and pDR199-SpAMP2 were introduced into the AMTdeficient yeast strain 31019b [49]. The complementation analysis was performed according to the earlier described procedure [81], using the pDR199 vector as negative control and the pDR199 featuring Arabidopsis thaliana genes AtAMT1;2 and AtAMT2 as positive controls. Correspondingly, the four S. polyrhiza AMT genes were fitted into the pOO2 vector, and the resulting expression cassettes were used to test the proteins' abilities for transporting NH + 4 in the oocyte according to an earlier established protocol [92]. Oocytes were ordered at EcoCyte Bioscience (Castrop-Rauxel, Germany), selected, washed and injected with 50 nl of linearized cRNA (0.4 µg/µL) of SpAMTs. Oocytes were kept in ND96 for 3 days at 18 • C and then placed in a recording chamber containing the recording solution (in mM): 110 CholineCl, 2 CaCl 2 , MgCl 2 , 5 N-morpholinoethane sulfonate (MES), pH adjusted to 5.5 with Tris(hydroxymethyl) aminomethane (TRIS). Ammonium was added as Cl salts. Oocytes were impaled with 3 M KCl-filled glass capillaries of around 0.8 Mohm resistance connected to a two-electrode voltage clamp amplifier (Dagan CA-1). Transport was measured at an array of NH + 4 concentrations (10, 30, 100, 300, 1000, 3000 and 10,000 µM). Currents without ammonium were subtracted from currents with ammonium to give ammonium induced current.

Analysis of AMT Genes Expression in S. polyrhiza by RT-qPCR
After DNAase treatment, 600 ng of total RNA was reverse transcribed using Reverse Transcriptase cDNA synthesis kit (Takara, Beijing, China) following the manufacturer's manual. The qPCR reactions were performed using CFX Connect Real-Time detection system (Bio-Rad, Hercules, CA, USA) using the UltraSybr Mixture (High Rox) supplied by CWBio (Taizhou, China). The cycling conditions were as follows: initial denaturation at 95 • C for 10 min followed by 40 cycles of 30 s at 94 • C, and 20 s at the annealing temperature of the respective primers (Table S1). The SYBR Green I fluorescence was monitored consecutively after the annealing step. The quality of products was checked by a thermal denaturation cycle. Only results providing a single peak were considered. The coefficient amplification efficiency for each pair of primers was determined by 10-fold serial dilutions. The level of relative expression was calculated by the 2 −∆∆Ct method [117]. Expression data for the target genes were normalized using the average expression of two S. polyrhiza ecotype NB5548 housekeeping genes, histone H3 (GenBank ID: MZ605911) and β-actin (GenBank ID: MZ605912), according to the geNorm protocol [118]. Three replicates were performed for all samples. Normalized expression was estimated using the ∆∆Cq algorithm All data were analyzed using the program BIO-RAD CFX Manager 3.1 (Bio-Rad, USA) and Microsoft Excel 2016 software.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12010208/s1, Figure S1: Schematic structure of AMT coding regions in genomes of S. polyrhiza and S. intermedia; Figure S2: Schematic structure of AMTs of S. polyrhiza; Figure S3: Sequence alignment of AMT1 proteins from S. polyrhiza and A. thaliana. Table S1: List of RT-qPCR primers used for evaluating expression of AMTs and aquaporin genes in S. polyrhiza line NB5548; Table S2: List of primer used for cloning AMT genes of S.polyrhiza ecotype NB5548.