Analysis of the role of MTP1, NRAMP4 and ZNT1 metal transporters in Ni hypertolerance and hyperaccumulation in Noccaea caerulescens and Identification of miRNAs involved in response to Zn excess in Arabidopsis species

Among hyperaccumulators, i.e. plants able to accumulate extremely high concentrations of heavy metals (HMs) in shoots, the Brassicaceae Arabidopsis halleri and Noccaea caerulescens, zinc (Zn)/cadmium (Cd) and Zn/Cd/nickel (Ni) hyperaccumulators respectively, represent two of the most interesting species, due to the huge variability between different ecotypes and populations in metal tolerance and accumulation. In this PhD work, two independent approaches were used aiming for a deeper understanding of metal hypertolerance and hyperaccumulation. The first approach exploits N. caerulescens ecotype Monte Prinzera (MP, Italy), native of a serpentine soil, that is able to hypertolerate and hyperaccumulate Ni in addition to Zn. Molecular mechanisms responsible for Ni tolerance and accumulation are still mostly unknown, although several studies suggested that metal transporters, also essential for metal homeostasis, have a fundamental role in HM tolerance and accumulation. Therefore, the vacuolar transporters MTP1 and NRAMP4 and of plasma membrane transporter ZNT1 were taken in consideration, thanks to their proposed role in Ni tolerance and accumulation in N. caerulescens MP (Visioli et al., 2014). At first, the expression of all three genes in N. caerulescens MP exposed to different Ni concentrations was analysed and compared with those of T. arvense (non-accumulator) and N. caerulescens ecotype Ganges (GA, Zn/Cd hyperaccumulator). Higher expression levels of all genes were found in N. caerulescens MP under normal and Ni excess conditions, confirming the different regulation of metal transporter proteins in hyperaccumulator plants and suggesting their role in Ni tolerance. The Ni transport properties of both NcNRAMP4 and NcZNT1 was tested by yeast complementation assays, using a WT strain of S. cerevisiae; according to the results previously found for the other Ni hyperaccumulator plant N. japonica (Mizuno et al., 2005), NcNRAMP4 reduced recombinant yeast survival under Ni treatment and the opposite result was found for NcZNT1, indicating a possible direct involvement of both proteins in Ni transport. To better understand the role of NcNRAMP4 and NcZNT1 in Ni tolerance and accumulation, plants of Arabidopsis thaliana were transformed with constructs carrying CaMV35S::NRAMP4 and CaMV35S::ZNT1 and the transgenic lines were crossed to obtain plants overexpressing both genes. Single 35S::NRAMP4 and double 35S::NRAMP4/35S::ZNT1 lines displayed bigger shoot compared to WT plants and the same results were also found for two of the three lines overexpressing ZNT1, although with a minor impact compared to the other gene. The impact of both genes on Ni tolerance and accumulation in planta was also elucidated by in vivo and in vitro analysis. Compared to WT plants, transgenic lines of A. thaliana expressing NcNRAMP4, NcZNT1 or NcNRAMP4/NcZNT1 in combination showed higher tolerance to Ni excess in vivo, thanks to a reduction of Ni accumulation in shoots. In addition, single 35S::NcNRAMP4 and double 35S::NcNRAMP4/35S::ZNT1 transgenic lines displayed longer roots in vitro, under standard and Ni excess condition compared to the WT, although no significative differences were found regarding 35S::ZNT1 overexpressing lines. These data may suggest that NRAMP4 and ZNT1 could participate in the transport and accumulation of Ni in N. caerulescens MP, although probably these proteins are more associated with the transport of other micronutrients than Ni itself. The possible involvement of the vacuolar transporter MTP1 in Ni hyperaccumulation and hypertolerance in N. caerulescens MP was also investigated. Gene expression analysis have initially confirmed the high constitutive levels of MTP1 expression previously found in different hyperaccumulator-hypertolerant plants belonging to Noccaea genus (Assunção et al., 2001; van de Mortel et al., 2006) under normal and stressful conditions, although in N. caerulescens MP this gene seem to be downregulate upon Ni treatment. In both N. caerulescens GA and MP two different CDSs of MTP1 gene, characterized by different length, were found in cDNA and genomic DNA; these proteins, called MTP1-long and MTP1-short, based on the presence or absence of the conserved His-loop region, are localized in the vacuolar compartment, as confirmed by subcellular localization experiments, suggesting their role in metal detoxification. The metal binding properties of both NcMTP1-long and NcMTP1-short were tested by yeast complementation assays using zrc1cot1 double mutant yeast of S. cerevisiae, which lacks these two vacuolar proteins, and WT strain of S. cerevisiae considering AtMTP1 as control for Zn transport ability (Desbrosses-Fonrouge et al., 2005). The two MTP1 forms displayed different metal specificity regarding Zn, Ni and Co, suggesting their possible different involvement in detoxification of metal excess in N. caerulescens MP. Therefore, A. thaliana mtp1+/+ homozigous mutants were transformed with constructs carrying CaMV35S::MTP1-long and CaMV35S::MTP1-short and also CaMV35S::AtMTP1 as control to elucidate their different metal binding specificity in planta. The transgenic lines were selected and the biological effect of MTP1 in planta will be investigated. With the second approach the attention is focused on the identification of miRNAs involved in the response to Zn excess. In eukaryotes, these small non-coding RNAs are essential for the regulation of gene expression during development and stress response, as well as nutrient homeostasis. miRNAs are small regulatory RNAs (20-24 nt length), with important role in plant development and response to many abiotic and biotic stress in Arabidopsis and other plant species. Moreover, several works have led the attention on metal -responsive miRNAs, which are responsible for the morphological and metabolical adaptation of environmental cues in plants, representing an interesting starting point for further analysis. To identify miRNAs putatively involved in response to Zn excess in Arabidopsis, miRNA-seq analysis was performed on miRNAs extracted from shoots of A. thaliana, treated and not treated with an excess of Zn for one week, and from untreated A. halleri. This hyperaccumulator plants was used in order to evaluate the existence of a possible different regulation mechanisms which regulates miRNA-target interaction compared to the non-hyperaccumulator A. thaliana. 100 miRNAs resulted to be differentially modulated in A. halleri compared to A. thaliana, including metal responsive miRNAs. Some of them were experimentally validated by Northern Blot Analysis and Real Time RT-PCR, in particular miRNAs with a role in plant development (miR157, miR159, miR390) and in nutrient homeostasis regulation (miR395, miR398, miR408), confirming the data obtained by miRNA-seq analysis and suggesting that some miRNAs could have an important role in metal hypertolerance and hyperaccumulation. Then, the attention was focuses on miR398b and miR408, two conserved miRNAs principally involved in the regulation of copper homeostasis in A. thaliana and other plant species; several works have elucidated their role in response to many abiotic stresses, including oxidative stress and heavy metals (Yamasaki et al., 2009; Ma et al. 2015; Pilon et al., 2017; Jalmi et al., 2018); moreover, the coordinate action of both miRNAs in planta have been suggested to be required for Cd basal tolerance in Arabidopsis (Gayomba et al., 2013; Gielen et al., 2016), representing an interesting starting point for further analysis. In order to investigate the possible involvement of both miR398b and miR408 in response to Zn excess, the promoter sequence of both miRNAs was amplified from genomic DNA of A. thaliana and A. halleri and fused to GUS reporter gene. A bioinformatic analysis was initially done comparing the entire sequences obtained by A. thaliana and A. halleri, and several DNA motifs involved in response to abiotic stresses were identified. GUS assay on A. thaliana transgenic lines expressing pAtMIR398::GUS, pAhMIR398b::GUS, pAtMIR408::GUS, pAhMIR408::GUS have shown that both miRNAs are expressed in roots and shoots of Arabidopsis, particularly in the vasculature tissues, suggesting their mobility into the whole plant. In addition, GUS expression was also evaluated on transgenic plants grown under Zn and Cu treatment, to better elucidate a possible competition between Cu and Zn, as previously proposed (Remans et al., 2012), and study the response of these two miRNAs under metal exposure. Both miRNAs resulted to be modulated by high concentration of these two micronutrients, suggesting a competition between Zn and Cu as previously proposed (Remans et a., 2012). Finally, the effect of the overexpression of pre-miR408 of A. halleri upon Zn treatment was investigated, since it is known its fundamental role for the adaptation to environmental cues in plants as demonstrated for the homologous of A. thaliana (Ma et al., 2015; Zhang and Li, 2013; Song et al., 2018). Compared to WT plants, transgenic lines overexpressing the precursor sequence of miR408 of A. halleri displayed higher shoot biomass and a significative reduction of root length, suggesting that high expression of miR408 could modify Zn tolerance in planta. Further investigations will be also required to better elucidate the possible role of miRNAs in response to Zn excess in Arabidopsis.