THEIR POTENTIAL USE IN MODERN AGRICULTURE
Juan D. Lira-Morales1, Marino Valenzuela-López2, María A. Islas-Osuna3, Tomas Osuna-Enciso1, José A. López-Valenzuela4, J. Adriana Sañudo-Barajas1*
1Centro de Investigación en Alimentación y Desarrollo A. C., Culiacán, Sinaloa, México.
2Facultad de Agronomía, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México.
3Centro de Investigación en Alimentación y Desarrollo A. C., Hermosillo, Sonora, México
4Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México
* Correspondencia:
J. Adriana Sañudo-Barajas [email protected]
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58 EXPRESSION PATTERNS OF ZIP PROTEINS UNDER ZINC DEFICIENCY IN TOMATO LEAVES (Solanum lycopersicum L.)
Juan Lira-Morales1, Tomás Osuna-Enciso1, José López-Valenzuela3, Rosabel Velez de la Rocha1, Carmen A. Contreras-Vergara2, J. Adriana Sañudo-Barajas1 and María A. Islas- Osuna2*.
1Centro de Investigación en Alimentación y Desarrollo A. C., Culiacán, Sinaloa, México.
2Centro de Investigación en Alimentación y Desarrollo A. C., Hermosillo, Sonora, México.
3Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, México
ABSTRACT
Plants require zinc (Zn) for optimal growth and they have several mechanisms to achieve homeostasis under mineral stress, including the activation of several families of proteins that control the influx and efflux of Zn such as the ZIP family (ZRT, IRT-like Protein). Several ZIP- like sequences were found in the Micro-Tom tomato database and in silico analyses were conducted with those sequences to find similarities with ZIP members reported in other species.
Tomato (Solanum lycopersicum cv. Micro-Tom) plants were treated with 0 µM (-Zn) or 0.57 µM (Zn), and evaluated for expression changes of three genes from the ZIP family of proteins (ZIP4, ZIP5 and ZIP8) after 0, 6 and 48 h. Zn deficient plants showed more internodes and less distance between internodes, a characteristic symptom of Zn deficiency. The in silico analyses showed the presence of the Zip superfamily (cl00437) conserved domain, 6 to 9 putative transmembrane domains (TM), a length between 355-490 amino acids, and a “variable region” rich in histidine.
The relative expression of the gen ZIP4 remained constant under Zn deficiency over all the evaluated times, whereas ZIP5 showed a peak of expression after 6 h and then decreased after 48 h. On the other hand, ZIP8 showed its maximum expression levels at 48 h. These results suggest a coordinated system of ZIP proteins in tomato dependent of the time of exposition to stress (-Zn),
59 which could be an evolutionary response to avoid functional redundancy. Additional studies are required to elucidate the roles of individual proteins, uptake kinetics, and specific localization on tissues that could be useful for crop improvement.
Keywords: gene expression, homeostasis, tomato, transcription zinc, ZIP
INTRODUCTION
Tomato (Solanum lycopersicum L.) is a crop with high demand in the global market (FAOSTAT, 2015), due to its nutrimental composition and versatility (Acosta-Quezada et al., 2015). The optimal growth of this crop requires an adequate mineral supply and Zinc (Zn) is one of the most important micronutrients since it is essential in processes like CO2 fixation, photosynthesis (Sasaki et al., 1998) and protein biosynthesis (Prask and Plocke, 1971). Plants acquire Zn bivalent cation (Zn2+) from the rhizosphere via the apoplast of the radicular tissue then follow a symplastic pathway using auxiliary membrane Zn transporters that facilitate its diffusion into the cytoplasm of epidermal root cells and thus cross the caspari band (Eide et al., 1996; Lee et al., 2010; Tiong et al, 2014; Olsen and Palmgren, 2014; Broadley et al., 2007). Zn deficiency has a negative effect on the enzymatic activities of carbonic anhydrase, Cu-Zn superoxide dismutase, ribonucleases, acid phosphatase (Pandey et al., 2002), as well as peroxidase (Kosesakal and Unal, 2009). Also, Zn deficiency has been associated with the accumulation of pigments like anthocyanins and carotenoids (Kosesakal and Unal, 2009), the synthesis of auxins, indole-3-acetic acid (Tsui, 1948), gibberellins and tryptophan (Sekimoto et al., 1997) and the folding of proteins through zinc finger domains (Perales-Calvo et al., 2015). In eukaryotes, it is estimated that around 9% of the proteome is dependent of Zn in different ways, where most of these proteins have enzymatic or transcriptional functions (Andreini et al., 2009). The high relevance of Zn in plant development
60 requires its steady supply during the whole life cycle of plants. However, the absence or insufficiency of Zn in about 50% of worldwide soil has motivated practices of agronomical management oriented to minimize yield affectation (White and Zasoski, 1999). There are other conditions of Zn absorption impedance that force plants to challenge its normal growth, but in all cases the limiting conditions stimulate an adaptative response at the molecular level to gather the surrounding Zn available below the hostile environments. Several protein families take part in the regulation and transport of Zn in the plant such as metal tolerance proteins (MTPs; Desbrosses- Fonrouge et al., 2005); heavy-metal ATPases (HMAs; Morel et al., 2009); NRAMP4 (Natural Resistance Associated Macrophage Protein 4; Oomen et al., 2009; Verret et al., 2004); the YSL (yellow stripe-like) family (Waters et al., 2006); and PCR2 (Plant Cadmium Resistance2; Song et al., 2010), nicotinamide (Deinlein et al., 2012), MTPs and ZIF1 Like (ZIFL; Zinc-Induced Facilitator; Sinclair and Kramer, 2012). Some genes that encode for members of the ZIP family of proteins (those required for the influx of zinc) have been characterized in several species; they have showed up-regulation at the transcriptional level under Zn deficiency, some with ~200-fold change, to make more efficient the transport of this element into the cytoplasm from outside the cell (Grotz et al., 1998; Jain et al., 2013). Zinc transporters have from 6 to 9 putative transmembrane domains (TMDs), 355-490 amino acids, and contain a “variable region” between TMD-3 and TMD-4, which is rich in histidine and it is suggested as the site with the Zn affinity.
TMD-4 has a highly conserved region that is predicted to form an amphipathic helix with a fully conserved histidine residue. Additionally, zinc proteins contain predicted histidine residues in TMD domains II, IV y V, and some studies indicated that these residues are implicated in metal transport (Gainza-Cortes et al., 2012; Li et al., 2013). These proteins have been identified in several species; there are twelve ZIPs in A. thaliana (Grotz et al., 1998; Jain et al., 2013), and six proteins (HvZIP5, HvZIP7, HvZIP8, HvZIP10, HvZIP13 and HvZIP3) in Hordeum vulgare. All
61 these proteins were characterized as Zn transporters, although not all of them were responsive to Zn deficiency (Pedas et al., 2009; Tiong et al., 2015). Similar members present in other species include MtZIP1-7in Medicago truncatula like (Burleigh et al 2003.; López-Millán et al., 2004), OsZIP1-10 and OsIRT1-2 in Oryza sativa (Bughio et al., 2002; Ramesh, 2003; Ishimaru et al., 2005; Ishimaru et al., 2006; Chen et al., 2008; Lee et al., 2010; Miyadate et al., 2011), PvZIP1-19 in Phaseolus vulgaris (Astudillo et al., 2013), VvZIP1.1, 2-3; 5.1; 6.1; 8; 11.1, and 13 in Vitis vinifera (Gainza-Cortes et al., 2012); GmZIP1 in Glycine max (Moreau et al., 2002) and TdZIP1 in Triticum turgidum (Durmaz et al., 2010). A more recent report showed the identification of ZIP proteins in the leaves of Solanum lycopersicum L. (Pavithra et al., 2016). However, the information obtained of the studies mentioned above indicate that the expression of these genes is different depending on the tissue, time of exposition to metal stress, and nutritional level of this element. In this work, tomato (Solanum lycopersicum L. cv. Micro-Tom) was chosen to evaluate the differential expression of ZIP genes in the leaves of plants grown under low Zn levels to evaluate their possible role in plant homeostasis under Zn deficiency.
MATERIALS AND METHODS
Putative Zn transporters from the tomato genome. Nucleotide sequences of confirmed members of the ZIP family of proteins from different plants (Arabidopsis thaliana, Populus trichocarpa, Medicago truncatula) were introduced in the NCBI (National Center for Biotechnology Information) Blast (Basic Local Alignment Search Tool) algorithm in order to find similar sequences in tomato (S. lycopersicum L.). Retrieved sequences were compared against the MiBASE Micro-Tom Database Genome (http://www.kazusa.or.jp/jsol/microtom/) using the blast tool to obtain specific sequences of ZIP at the cultivar Micro-Tom. Corresponding amino acid sequences were deduced and analyzed against the NCBI database of conserved domains to confirm
62 the presence of the putative domain Zip superfamily (cl00437) present in zinc transporter members of the ZIP family. The E-value cut-off was set to 0.1.
Phylogenetic analysis of SlZIP proteins. For the determination of the evolutionary relationship between the ZIP proteins, a phylogenetic tree was constructed using MEGA 7.0 with the Neighbor Joining algorithm and a bootstrap test with 1000 replications. The resulting tree was edited with the software FigTree 1.4.3.
Analysis of conserved domains and sequence alignment in SlZIP proteins. The signal peptides of the proteins coded by the genes evaluated were identified using targetP 1.1 to determine their localization: chloroplastic, mitochondrial or secretory pathway (Emanuelsson et al., 2007). The transmembrane domains were predicted with HMMTOP (Tusnady and Simon, 2001), the isoelectric point and molecular weight were estimated with Compute pI/Mw (Bjellqvist et al.
1993) and the conserved domains with Pfam 28.0 (El-Gebali et al., 2018). Multiple amino acid sequence alignment was performed using the software Clustal X (2.0).
Molecular modeling de ZIP4, ZIP5 y ZIP8. The amino acid sequences of ZIPs 4, 5 y 8 were used to obtain their tridimensional theoretical model with the Phyre2 server (Kelley et al., 2015). The model used was the crystal structure of the Bordetella bronchiseptica with bound Zn2+ Zrt-/lrt- like protein (PDB 5TSA) and the figure was built using PyMol (Schrodinger, 2019).
Analysis of cis-elements within the promoter region of the ZIP genes. Genomic sequences (1500 bp) upstream of the initiation codon were obtained from NCBI database. To identify the cis regulatory elements the MatInspector program was used with the general core promoter elements and plant matrix groups. Moreover, the analysis of overrepresented transcription factors binding sites was done utilizing genomic and promoter background of A. thaliana, TAIR 10 (Cartharius et al., 2005).
63 Plant material. Tomato seeds (Solanum lycopersicum cv. Micro-Tom) were surface sterilized with detergent, followed by alcohol 30% (v/v), and sodium hypochlorite 30% (w/v). Once washed, the seeds were transferred to 13 cm plates containing solid MS (Murashige and Skoog medium) supplemented with 3% sucrose. After three weeks, plants were moved to a hydroponic system under controlled environment: photoperiod of 18 h light/ 6 h dark, temperature (27 °C) and relative humidity (70 %), under the same conditions of nutrition (1.13 mM Ca(NO3)2•4H2O; 0.75 mM KNO3; 0.38 mM K2SO4; 0.50 mM MgSO4•7H2O; 0.25 mM KH2PO4; 17 µM H3BO3; 4.74 µM Cl2Mn•4H2O; 0.38 µM Cl2Cu; 0.14 µM MoO3; 0.57 µM ZnSO4•7H2O and 20 µM Fe-EDTA) for three weeks. After this adaptation period, the plants were divided into two batches, one was treated with 0 µM (-Zn) and the other with 0.57 µM (Zn), which was used as control. Three replicates of two plants each were used. The leaves from both Zn deficient and control plants were collected after 0, 6 and 48 h, frozen in liquid nitrogen and stored at -80°C until use for gene expression analysis. Plants of both treatments were grown up to 60 days on separate trays to obtain visual verification of symptoms of Zn deficiency and the following parameters were measured: number of leaves, number of fruits, fruit diameter, shoot length, root length, number of internodes, and internode distance (n=3).
RNA extraction. Total RNA was prepared from leaves of tomato plants using Tri Reagent (Sigma), integrity was analyzed by gel electrophoresis under denaturing conditions and concentration was assayed with a Nanodrop 1000 (Thermo Scientific). Genomic DNA was removed with DNase I (Roche); then, cDNAs were synthesized from 5 µg of RNA with the SuperScript III First-Strand Synthesis System (Invitrogen). For qPCR analysis, PCR primers specific for each of the ZIP genes were designed (Table 1) and tested using the online tool Primer-Blast and in test reactions (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The reaction mix (20 µL) contained 20 ng of cDNA, 10 µL SYBR Green 2X (BIO-RAD) and 250 nM of the primers (Table 1). The
64 amplification was performed using a real time system model StepOne (Applied Biosystems) using the following conditions, 95 °C for 10 min, followed by 40 cycles at 95 °C (15 s) and 60 °C (1 min) with a melt curve from 60°C to 96°C with a temperature increment of 0.3°C. The EF1α gene was used as control and the expression values of the ZIP genes were calculated relative to the control plants (0 h) using the method of Pfaffl (2001). The results were analyzed by one way ANOVA and the differences between means were determined with the Tukey's test (p ≤ 0.05) with the software Minitab 17. The assays were replicated three times.
RESULTS AND DISCUSSION
In silico analyses of amino acid sequences
The characteristics of the putative proteins from the ZIP family, such as transmembrane domains predicted, molecular weight, isoelectric point, and signal peptide are shown in Table 2. Five zinc transporters were identified with E values below 0. The length of the five proteins varied between 337 and 407 amino acids with isoelectric points between 5.8 and 8.4, which are similar to those reported for ZIP proteins previously characterized in silico (Guerinot, 2000; Pedas et al., 2009).
The conserved domain cl00437 is present in zinc transporters, like the ZIP superfamily of proteins (Marchler-Bauer et al., 2017). All the tomato ZIPs analyzed in the present study contained the cl00437 domain, predicting them as putative Zn transporters. On the other hand, the main consensus about ZIP proteins indicates that they contain eight transmembrane domains, although some of them have between 6 and 9 (Li et al., 2013). In our study, all the putative ZIP proteins had eight transmembrane domains, except for SlZIP2 that had nine. Moreover, all of them contain a signal peptide, which is the main target of the ZIP proteins evaluated in other species (Moreau et al., 2002; Lee et al., 2010; Li et al., 2013).
65 The multiple alignment revealed several conserved regions (Figure 2) between the putative ZIP proteins from tomato. It was possible to find the conserved domains, those that function as transmembrane domains and the histidine-rich region characteristic of ZIP proteins. The similarities and differences specifically between ZIP4, ZIP5 and ZIP8 were also observed in their theoretical tridimensional model (Figure 3). Overall the structure is consistent to the sequence identity to known zinc transporters containing eight transmembrane domains. However the differences on the extracellular loops may lead to changes in zinc affinity or overall channel regulation. In general these proteins are structurally conserved.
Phylogenetic analysis of ZIP proteins from tomato
Phylogenetic analysis showed that SlZIP2 groups more closely with Medicago truncatula MtZIP2 and more distantly within the same clade with Arabidopsis thaliana AtZIP2, Oriza sativa OsZIP2 and Zea mays ZmZIP2. SlZIP4 formed a clade with AtZIP4 and several other ZIP proteins from species of monocots and dicots. SlZIP3, SlZIP5 and SlZIP8 also associated with monocots and dicots (Figure 4). The grouping of the proteins in clades of both monocots and dicots could be an indication of their conserved functions (Tiong et al., 2015). However, it is worth mentioning that the temporal and tissue specific expression of the genes encoding these proteins is associated with regulatory mechanisms that involve the non-coding sequences.
The phylogenetic analyses of the ZIP candidate proteins from tomato showed the presence of clades with amino acid sequences from another species based on sequence homology; for example, SlZIP2 was grouped with MtZIP2 while SlZIP3 was in a clade with MtZIP1 and AtZIP1. SlZIP4 was predicted to be a chloroplastic protein and did not form a defined clade; however, it shares characteristics with other members of the ZIP family of proteins.
66 Morphological traits
The number of leaves and fruits, fruit diameter, shoot length, root length, number of internodes, and internode distances were measured in 60 days-old plants to identify symptoms of Zn deficiency (Figure 1). The most important differences were observed for internodes; Zn deficient plants had more internodes (7.6) when compared with the control plants (6.6), which corresponded with the differences observed in the distance between internodes with 1.8 cm vs 2.3 cm, respectively. The shorter internodes observed in the Zn deficient plants might be the result of reduced synthesis of gibberellins and indol-acetic acid, caused for impaired translation or transcription of 3β-hidroxilase, this two hormones necessary for stem elongation (Sekimoto et al., 1997).
Expression of ZIP genes under Zn deficiency
Three weeks old tomato plants (Solanum lycopersicum L. cv. Micro-Tom) grown under hydroponic system with a photoperiod of 18 h light/ 6 h dark, 27°C and 70% relative humidity were put under Zn nutrition of 0 µM (Zn-) for three additional weeks, and the expression of the genes ZIP4, ZIP5, and ZIP8 was evaluated in sampled leaves after 0, 6 and 48 h (Figure 5). These genes were selected based on the phylogenetic analysis where ZIP5 and ZIP8 share a clade while ZIP4 is in a distant clade.
Some members of the ZIP family of proteins are overexpressed under Zn deficiency as a response to adverse conditions (Grotz et al., 1998; Jain et al., 2013). In this research, the relative expression of SlZIP4 in leaves from tomato plants grown under Zn deficiency was constant after 6 and 48 h (Figure 6a). In Arabidopsis thaliana, ZIP4 was detected in shoots (~8-fold) and roots (~6-fold) 7 days after Zn deficiency whereas in Zea mays the induction in the expression of ZmZIP4 in shoots
67 was found until 96 h, although no changes in the expression of this gene were observed in roots (Li et al., 2013). These results suggest that ZIP4 is constantly expressed in tomato leaves independently of the Zn condition, as previously observed for ZmZIP1 and ZmZIP2 in Zea mays, as well as for MtZIP6 and MtZIP7 in Medicago truncatula. A larger list of ZIP members and their expression patterns under Zn deficiency can be found in Lira-Morales et al. (2019).
SlZIP5 was up-regulated after 6 and 48 h (60-fold and 30-fold, respectively) of Zn-deficiency in the leaves of tomato (Figure 6b). Zn is necessary in the leaves for the photosynthetic process in order to maintain the optimal nutrimental status in plants. In Arabidopsis, a ZIP5 homolog showed overexpression (~3x) after 7 days of Zn deficiency in both shoots and roots (Jain et al., 2013), whereas in Hordeum vulgare there was evidence of constant induction of ZIP5 in the same tissues even 28 days post-treatment (Tiong, et al., 2015); similarly, in Zea mays ZIP5 was induced in shoots 7 days after the induction of Zn deficiency (Li et al., 2013). These results can be related to the participation of ZIP5 in the early stages of stress resistance helping plants to maintain cellular homeostasis.
SlZIP8 changed its expression until 48 h after the beginning of treatment (Figure 6c). Similar results were observed in Arabidopsis thaliana where longer times were related with the overexpression of this gene in shoot and root (Jain et al., 2013). In Zea mays, ZIP8 showed two expression bursts in the shoots, one after 6h and the other after 96 h (Tiong et al., 2015). This gene can be related to the early response to a mineral deficiency.
The results showed different expression patterns in each of the ZIP genes evaluated. These patterns seem to be dependent of the time elapsed from the beginning of the stress, but they can also be
68 related to the mineral nutrition level, meaning they could be activated by a threshold, all as part of the plant´s strategy to maintain homeostasis.
The three Zinc transporters, ZIP4, ZIP5 and ZIP8, shared the cis-regulatory element identified as bZIP element; aditionally, ZIP 4 and 5 contain a phosphate starvation response (PHR1-like 2) element. ZIP8 contains a MYB and ZIP5 a GBOX element. These elements are common regulators of zinc homeostasis (Briat et al., 2015) and these results suggest that ZIP proteins might be regulated by transcription factors that bind to the predicted cis elements. Further studies should be done to prove these asseverations.
CONCLUSIONS
Several studies have shown an increase in the transcript levels of genes encoding mineral transporters under mineral deficiency, including some members of the ZIP proteins in several species. In this work, two members of the ZIP family (ZIP5 and ZIP8) were overexpressed under Zn deficiency (0 µM) in leaves of tomato (Solanum lycopersicum cv. Micro-Tom), and the fold- change was related to the time the plants were exposed to the deficiency. Such information besides the roles of individual proteins, uptake kinetics, and specific localization on tissues could be useful for crop improvement. The full understanding of the behavior of ZIP proteins in different tissues, cultivars and even species is still unclear; however, the design of more robust experiments with controlled conditions for many species at the same time and vegetative state to widen the current knowledge about ZIP proteins.