Abstract
Zinc (Zn) deficiency hinders growth and development in tomato. This study unveils the responses of how Zn starvation affects physiological and molecular processes in tomato. Zn deficiency negatively affected the biomass, cellular integrity, and chlorophyll synthesis in tomato. Also, Zn deficiency decreased the maximum yield of PSII, photosynthesis performance index and dissipation energy per active reaction center, although the antenna size, trapping energy efficiency and electron transport flux were stable in Zn-starved leaves. Further, Zn shortage caused a substantial reduction in Zn and Fe concentrations in both roots and shoots along with decreased root Fe-reductase activity accompanied by the downregulation of Fe-regulated transporter 1, Zn transporter-like (LOC100037509), and Zn transporter (LOC101255999) genes predicted to be localized in the root plasma membrane. The interactome partners of these Zn transporters are predominantly associated with root-specific metal transporter, ferric-chelate reductase, BHLH transcriptional regulator, and Zn metal ion transporters, suggesting that Zn homeostasis may be tightly linked to the Fe status along with BHLH transcription factor in Zn-deficient tomato. We also noticed elevated O2.− and H2O2 due to Zn deficiency which was consistent with the inefficient antioxidant properties. These findings will be useful in the downstream approach to improve vegetable crops sensitive to Zn-deficiency.
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Introduction
Zinc (Zn) shortage, a scarcity of plant nutrition has an inauspicious consequence on vegetables1. With high pH or alkaline stress, there are many lands in different regions across the globe are Zn-deficient2. The soil, which contains a sandy texture, as well as less phosphorus, is also deficient in Zn3. The plants which are sensitive Zn-starved conditions are often exhibiting reduced growth, dwarf stems, and light chlorosis4. The functions of Zn can be found in photosynthetic and gene expression processes besides enzymatic and catalytic activities5. Inadequacy of Zn also resulted in a deterioration in plant stomatal activity1. In response to the pathogenic invasion of plants, Zn proteins also play a significant role6. The insufficiency of Zn in plant-based foods contributes to human malnutrition throughout the world7.
While generally there is a genotypic difference in Zn-deficiency responses, a plethora of plants are susceptible to Zn deprivation4,8. As Zn efficiency mechanisms are somewhat complex in their actions, research has been found a few adaptive features confer Zn deficiency in plants4,9. Since it is tough for Zn to penetrate the root cell membranes, therefore the elevation of Zn is attained through transporters at the primary stage4,8. The IRT1, known as Fe-regulated transporter, is often incited to ameliorate translocation as well as Zn exploitation whenever the plant has possibly Zn or Fe shortcomings10,11. IRT1 is responsible for transporting several minerals such as Zn, Mn, and Fe, even though the pattern of expression varies depending on mineral stresses12. Fe and Zn are strictly regulated in plants and they are typically connected owing to the chemical resemblance and IRT1 non-specificity13. Besides, ferric chelate reductase (FCR) activity along with Strategy I responses are induced in Fe-deprived plants, which are generally dicots14,15. FCR affects the availability of Fe and could influence the absorption of different types of metals16. Hereafter, improvement is declared to conferring the involvement of ZIP (Zn transporter) in Fe and Zn uptake11,17,18, though the evidence in tomato continues to be limited. Pavithra et al.19 classified as low-affinity (SlZIP5L2, SlZIP5L1, SlZIP4, and SlZIP2) and high-affinity (SlZIPL) Zn transporters in tomato. Nevertheless, these explications were tentatively claimed in the shoot instead of root tissue, in which the absorption of metal occurs in plants.
The deficiency of Zn also causes the accumulation of excessive reactive oxygen (ROS) species, which impedes photosynthesis and protein biosynthesis4,9. Therefore, elevated detoxifying ROS is one of the pioneer strategies plants often possess in response to abiotic stress. ROS scavenging enzymes play a vital part in controlling reactive radicals to restore the cell's redox balance9. Among the enzymes, catalase (CAT) and superoxide dismutase (SOD) were mainly demonstrated to underlie ROS scavenging in Zn-deprived plants20. Besides, ascorbate peroxidase (APX) is able to transform excessive H2O2 into the water in ROS-induced plants21. However, the induction of antioxidant resistance largely depends on the plant species/genotypes.
Tomato (Solanum lycopersicum L.) is known to a high-value crop all over the world22. The insufficiency of the inevitable micronutrient Zn adversely affects tomato production worldwide. However, the outcome of low Zn availability on the physiological and molecular repercussions for which tomato plants suffer from growth retardation remains entirely unfamiliar. In this manner, this study aims at unveiling the reasons for dysfunctions found in the tomato plant under Zn deficiency supported by the different biochemical and molecular investigations.
Materials and methods
Plant cultivation technique
As obvious from a previous study23, Zn-sensitive tomato cultivar (cv. Ratan) was used for this study. The periphery of the seeds was cleaned up with 70% ethyl alcohol for 3 min, followed by doubled Milli-Q water before transferred to the germination tray at room temperature. The sprouted three-days homogeneous plantlets thereafter were transferred to the hydroponic solution at pH 6.024, as it is formerly narrated (µM): KNO3 (1600), Ca(NO3)2.4H2O (600), KH2PO4 (100), MgSO4.7H2O (200), KCl (50), H3BO3 (25), Fe-EDTA (20), MnSO4.4H2O (2), Na2MoO4.2H2O (0.5) and CuSO4.5H2O (0.5). Furthermore, ZnSO4 has been used as follows8: 2.0 µM (Zn-sufficient or control) and 0.01 µM (Zn-deficient). The pH of the solution was adjusted to 6.0 using KOH/HCl and the solution was changed in every 4 days interval. The seedlings were grown in an aerated plastic container (1.2 L) for each treatment having 9 plants/container in an indoor growth chamber. Each seedling was inserted through foam attached to a cut centrifuge tube, exposing the roots to the solution. The growth chamber was strictly controlled with photoperiod 14 h dark/10 h light at ± 26 °C, 65% air humidity, and 200 μmol m−2 s−1 light intensity. The plants were cultivated for 14d before harvesting for data analysis.
Characterization of morphology, photosynthesis and Fe chelate reductase activity
A digital caliper was used for measuring the length of the root and shoot. The dry weight of root and shoot was recorded after drying for 3 days at 80 °C in an electric oven. For measuring the chlorophyll score of young leaves, a SPAD meter was used (Minolta, Japan). Furthermore, photosynthesis biophysics through chlorophyll fluorescence kinetic (OJIP), such as maximum quantum yield of PSII (FvFm), photosynthesis performance index (Pi_ABS), dissipation energy per active reaction center (DIo/RC), absorption flux/effective antenna size of an active reaction center (ABS/RC), electron transporter flux further than QA DIo/RC (ET2o/RC) and trapped energy flux leading to a reduction of QA (TRo/RC) was recorded on young leaves kept for 1 h at dark using FluorPen FP 100 (Photon Systems Instruments, Czech Republic).
Fe chelate reductase activity (FCR) activity in roots was determined in roots by ferrozine assay15. Briefly, the 0.2 mM CaSO4 followed by Milli-Q water washed off the root surface. The roots samples were then homogenized with 1 ml of assay mixture (100 mM Fe(III) EDTA, 0.10 mmol MES-NaOH (pH 5.5), 300 mM ferrozine). The samples and blank tubes (without assay mixture) were incubated in the dark for 20 min at 25 °C. Finally, aliquots were read at 562 nm. The FCR activity was determined by using the molar extinction coefficient (27.9 mM−1 cm−1) of ferrozine.
Elemental analysis in root and shoot
Tomato roots were rinsed out once with 0.1 mM CaSO4 and several instances with Milli-Q water to eliminate external components. The roots and shoots were then desiccated individually at 70 °C in an oven for 3d. The samples were then digested with HNO3/HClO4, 3:1 v/v. Afterward, the Zn and Fe were tested by means of atomic absorption spectroscopy (AA-6800, SHIMADZU).
Determination of soluble protein
In brief, fresh root and shoot were ground with tris–HCl buffer (50 mM, pH 7.5), 2 mM EDTA and 0.04% (v/v) β-mercaptoethanol. In order to separate the transparent fluid portion, the crude specimens were centrifuged for 10 min at 12,000 rpm (MicroCL 21, Thermo Scientific, United States). Subsequently, 1 ml of Coomassie Brilliant Blue (CBB) was diluted to 100 µl protein extract before measuring the absorbance at 595 nm. A bovine serum albumin (BSA) curve was modeled for estimating the total soluble protein as described by Bradford assay25.
Assessment of electrolyte leakage and cell death
The ultimate consequence of damage of the cell membrane integrity in both root and shoot were measured by using a conductivity meter with some modifications26. Root and shoot surface components were washed with deionized water. Thereafter, the freshly harvested samples were transferred into a beaker filled with 20 ml deionized water and kept at 25 °C for 2 h. Later, the solution’s electrical conductivity (EC1) was calculated. Afterward, the samples were heated in a water bath for 20 min at 95 °C then soothed at 25 °C before recording the final electrical conductivity (EC2). The electrolyte leakage was then determined as follows: = (EC1/EC2) × 100 (%).
Evans blue was determined to account for the cell death rate with some modifications27. The entire fresh root and shoot were placed into 2 mL of Evan's blue mixture for 15 min. The samples were then mixed with 80% ethyl alcohol and placed at room temperature for 10 min. Afterward, the solutions were incubated in a water bath for 15 min at 50 °C and were further centrifuged for 10 min at 12,000 rpm (MicroCL 21, Thermo Scientific, United States). The supernatant was then transferred into a new centrifuge tube before measuring the absorbance at 600 nm. Eventually, the % of cell death was calculated.
Analysis of O2 −, H2O2 and lipid peroxidase
The superoxide (O2−) was tested as mentioned previously28. Briefly, the fresh samples (0.1 g) were washed with water, homogenized with chilled K-phosphate buffer (10 mM), and centrifuged at 12,000 rpm at 4 °C for 10 min. The clear supernatant (100 µl) was mixed up with 1 ml of assay solution with 0.5 mM XTT sodium salt and 50 mM Tris–HCl (pH 7.5). Finally, the solution's absorbance was read at 580 nm and the superoxide (O2−) was calculated by the coefficient of extinction 2.16 × 104 M−1 cm−1). The H2O2 accumulation was measured, utilizing 0.1% C2HCl3O229. The fresh samples (0.1 g) were ground with a mortar and pestle in 0.1% C2HCl3O2 and further centrifuged at 10,000 rpm for 15 min. The top aqueous segment (100 µl) was appended with assay solution supplemented with 10 mM K-phosphate (pH 7.0), 1 M KI. The samples were then adapted to dark for 1 h before recording the absorbance at 390 nm (Shimadzu UV-1650PC). For the determination of lipid peroxidase activity, the fresh samples (0.1 g) were homogenized in 20% C2HCl3O2 incorporated with 0.5% C4H4N2O2S mixture. The clear supernatant (1 ml) was warmed at 95 °C for 30 min in a water bath and subsequently chilled on ice. The samples were then centrifuged at 12,000 rpm for 10 min. The optical density of the samples was monitored 532 and 600 nm (60S UV–Visible Spectrophotometer, Thermo Scientific, United States). The lipid peroxidase activity was determined according to the coefficient of molar extinction 155 mmol L−1 cm30.
Gene expression and bioinformatics analysis
The total RNA was isolated from the fresh roots as described by SV total RNA isolation system (Promega, USA). The quantified RNA was then converted to cDNA using the cDNA synthesis kit (Promega, USA) before performing real-time PCR analysis in an ECO real-time PCR system (Illumina, USA) using gene-specific primers for iron-regulated transporter 1, zinc transporter-like (LOC100037509) and zinc transporter (LOC101255999) (Table S1). The PCR reactions were set as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 57 °C for 30 s. The relative expression of candidate genes was calculated by the dd − ∆Ct method considered Actin as an internal control. Further, the sub-cellular localization of these genes was predicted by CELLO v.2.5. The interactome networks of iron-regulated transporter 1, zinc transporter-like (LOC100037509) and zinc transporter (LOC101255999) were generated based on the physical interactions among molecules (protein–protein interactions) using the STRING server (http://string-db.org) visualized in Cytoscape31.
Activities of antioxidant enzymes
In brief, the root and shoot samples (0.1 g) were independently homogenized with 100 mM phosphate (pH 7.0) in a mortar and pestle. The homogenate was centrifuged at 8000 rpm for 10 min (MicroCL 21, Thermo Scientific, United States)., and the transparent portion was collected for analysis. We first analyzed the SOD activity by mixing the plant extract (100 µl) with 0.1 mM of EDTA, 50 mM of NaHCO3 (pH 9.8), and 0.6 mM of epinephrine32. After 4 min, the adrenochrome confirmation was read at 475 in a spectrophotometer. Secondly, the plant extract (100 µl) was supplemented with 1 ml of assay solution with 0.1 mM EDTA, 0.1 mM H2O2, 0.5 mM ascorbic acid, and 50 mM phosphate33. The absorbance of the mixture was then observed at 290 nm and thereafter calculated for APX activity by extinction coefficient (2.8 mM−1 cm−1). Besides, 100 µl extract was mixed up with 100 mM phosphate buffer and 6% hydrogen peroxide solution. The absorbance was read twice in 30 s interval and thereafter calculated for the activity of CAT using a coefficient of 0.036 mM−1 cm−1). Lastly, 100 mM phosphate, 1 mM EDTA, 20 mM GSSG and 0.2 mM NADPH added individually to 100 μL of plant extract. The reaction was initiated by GSSG, and absorption was reduced by NADPH-oxidation at 340 nm. The calculation of GR activity was then performed by the extinction coefficient (6.12 mM−1 cm−1)34.
Statistical analysis
We opted for a completely randomized block design (CRBD) in plant cultivation. Three independent biological replications were considered for data analysis. Each experiment was performed at least three times to check whether the results were reproducible. The statistical significance of mean was evaluated by Microsoft Excel 2010 at p ≤ 0.05 by t-test. Figures were plotted by using GraphPad Prism (V. 6.0) software.
Results
Changes in morphology, photosynthetic parameters and FCR activity
Zn deficiency showed a significant reduction in the root (root length, dry root weight) and in the shoot characteristics (shoot height, dry shoot weight) in Ratan than untreated controls (Fig. 1a–e). In addition, Zn-deprivation led to a significant reduction in the SPAD score as opposed to control plants (Fig. 3a). Besides, the leaf chlorophyll score and root FCR activity significantly decreased owing to Zn-deprivation relative to controls (Fig. 1f,g). The Fv/Fm and Pi_ABS values showed a large decrease in young leaves as a result of Zn-shortage as opposed to controls (Fig. 2a,b). However, the DIo/RC value considerably increased in Zn-starved leaves in contrast to controls (Fig. 2c). OJIP analysis showed no changes in ABS/RC, ET2o/RC, and TRo/RC values between Zn + and Zn− conditions (Fig. 2d–f).
Changes in stress indicators
In Ratan, the total soluble protein in the root and shoot remained unchanged due to Zn-deprivation in contrast to plants grown with optimal Zn (Fig. 3a). Furthermore, cell death (%) and electrolyte leakage in either root or shoot increased significantly following Zn shortage relative to controls in Ratan (Fig. 3b,c). Although the O2− showed no changes in the root, the accumulation of this ROS significantly increased following Zn starvation relative to Zn-sufficient plants (Fig. 3d). The H2O2 in root and shoot of tomato significantly increased owing to Zn-deficiency relative to controls (Fig. 3e). However, the lipid peroxidase activity remained unchanged between Zn-sufficient and Zn-deficiency conditions (Fig. 3f).
Metal analysis, gene expression pattern and in silico validation
The concentrations of Fe and Zn showed a significant decrease in root and shoot because of Zn insufficiency relative to Zn-sufficient plants (Table 1). To validate the metal analysis, we analyzed the expression pattern of key Zn transporters in tomato roots. In real-time PCR analysis, Fe-regulated transporter 1 showed a substantial downregulation in roots subjected to Zn-shortage as opposed to Zn-sufficient plants (Fig. 4a). Additionally, the expressions of Zn transporter-like (LOC100037509) and Zn transporter (LOC101255999) genes were significantly downregulated due to Zn shortage in roots compared with Zn sufficient controls (Fig. 4a).
CELLO predictor showed that these genes are localized in the plasma membrane of the root resulting highest reliability score (Fig. 4b). STRING showed five putative interaction partners of iron-regulated transporter 1, which include NRAMP1 (root-specific metal transporter), NRAMP3 (metal transporter), FRO1 (ferric-chelate reductase), fer (BHLH transcriptional regulator) and CHLN (nicotianamine synthase) genes (Fig. 5). In addition, zinc transporter-like (LOC100037509) are closely linked in a partnership with Solyc05g008340.2.1 (ZIP metal ion transporter family), 101,253,965 (ZIP metal ion transporter family), 101,253,075 (ATPase EI), 101,255,936 (WCRKC thioredoxin) and 101,262,011 (Zn-binding dehydrogenase family protein). Interactome analysis also showed close interactions of Zn transporter (LOC101255999) with Solyc05g008340.2.1 (ZIP metal ion transporter family), 101,253,965 (ZIP metal ion transporter family), Solyc01g087530.2.1 (ZIP metal ion transporter family), fer (BHLH transcriptional regulator) and 101,245,241 (major facilitator superfamily protein) genes (Fig. 5).
Changes in antioxidant enzymes
We have analyzed the different antioxidant enzymes, whether this tomato cultivar does have antioxidant properties to withstand oxidative damage. Results showed that CAT, SOD, APX, and GR activities remained unchanged in roots, but these enzymes significantly reduced due to Zn-starvation in comparison with Zn-sufficient plants (Fig. 6a–d).
Discussion
Understanding the mechanistic consequence of mineral deficiency responses is of great importance to initiate crop improvement steps through breeding or genome editing approaches. Long ago35, gave a tentative sign that tomato Zn uptake is restricted to the root system rather than the aerial part. Since then, extensive studies related to Zn-starved tomato have not been elucidated. This research offers some new insight into improvements in photosynthesis, Zn transporters, and the antioxidant ability for which tomato plants cannot overcome Zn deficiency.
Morphological and physiological responses
Zn is essential for metabolism and plant growth. Nevertheless, in Zn inefficient Ratan, the absence of adaptive systems to fight Zn deficiency decreased the root and shoot morphological characteristics. This result is consistent with earlier accounts of Zn deficiency in crop plants8. In the research of ours, reduced chlorophyll, PSII capability, and photosynthesis performance index were correlated with the morphological features in Ratan. The chlorophyll value decreases along with Fv/Fm and Pi ABS, indicating that Ratan plants were unable to regulate PSII mechanisms under Zn deficiency. Furthermore, the increase of DIo/RC suggests that Zn-deficiency results in ineffective energy management, which is correlated with the reduced photosynthetic efficiency in leaves of Zn-deprived tomato. Zn functions during photosynthesis when CO2 is fixed, although chloroplast and photosynthesis can be impaired under deficiency36,37. Several cellular dysfunctions and damages related to plant abiotic stress. In this particular research, while low Zn did not affect overall soluble protein-rich, Ratan displayed a tremendous rise in cell death as well as membrane damage and accumulation of H2O2 in reaction to Zn deficiency. Membrane leakage and cell death are typical effects of abiotic stress8,38. Therefore, our results suggest that an inadequate supply of Zn is one of the features for which Ratan cannot maintain the cellular integrity, morphological, and photosynthesis efficiency subjected to Zn deficiency.
Changes of Zn and Fe status
In this study, the concentration of Zn and Fe were simultaneously reduced in response to Zn deficiency in tomato, indicating a complicated Zn and Fe homeostasis response. The rapid decrease of root and shot Zn and Fe concentration in Ratan shows that Zn homeostasis cannot be held under Zn hunger by this genotype. Differential tissue Zn levels have been displayed in a few species of plants that are different in response to Zn scarcity39. In the cytosol, IRT and ZIP proteins are mostly associated with Zn delivery40,41. Previous studies demonstrate that Zn deficiency also caused Fe deficiency as plants prevent the transfer of Fe from root to shoot42,43. Pavithra et al.19 emphasized the involvement of shoot showing differential expression of Zn transporters. However, it is slightly confusing as most of the Zn-acquisition genes are localized in the plasma membrane of roots as predicted by the localization predictor. We have therefore carried out a focused analysis on the pattern of expression of IRT and ZIP in tomato roots. Fe-regulated carrier, Fe-regulated transporter 1, has shown major downregulation in the roots of Ratan, inducing its susceptibility to Zn deficiency. Originally, IRT1 was identified as a Fe transporter, but complementation and uptake studies revealed its involvement in Mn and Zn in addition to Fe10,44. In this study, both Zn transporter-like (LOC100037509) and Zn transporter (LOC101255999) genes showed a substantial decrease in Ratan root expression patterns that could help to render the Zn root system inefficient for Zn regulation. On the contrary, the induction of ZIP transporter occurs in root or leaf, depending on the plant species/sensitivity45. These genes can be good candidates for further functional characterization of Zn-transporter in tomato through genome editing and yeast complementation assay.
The localization of Zn transporters was further validated by the computational prediction. The CELLO analysis predicts that all of these tomato Zn transporters are localized in the plasma membrane which is in agreement with wet-lab experiments reported previously in various plants46,47. Ion and solute fluxes underpin inorganic mineral nutrient uptake at the plasma membrane, thereby triggering changes in second messengers such as cytosolic-free Ca2+ concentrations in cell48. The interactome map further suggests that the Zn transporters in tomato are associated with some genes related to metal ion transporter and transcription factor. The Fe-regulated transporter 1 in tomato is in close partnership with metal transporter (NRAMP1, NRAMP2), Fe reductase gene (FRO1), fer transcription factor, and nicotianamine synthase (CHLN) genes. Previous studies also demonstrated that the BHLH068 transcription factor via interaction with FER along with IRT1, NRAMP1 and FRO1 is involved in iron homeostasis in tomato49. Further, CHLN encodes nicotianamine forming a complex with Fe2+ for the distribution of Fe in plants50. The defect in FER mutant caused dramatic downregulation of Fe-uptake genes (IRT1, FRO1) in tomato; however, the molecular mechanism remains unclear46. However, FER-like transcription factor FIT, expressed in roots was associated with FRO2 and IRT1 genes in Arabidopsis51. In order to bind to the promoters of IRT1 and FRO2, this FIT is not able to act alone but must form a heterodimer with other bHLH proteins, as shown in yeast52,53. It is, therefore, possible that Zn-deficiency is associated with the coordination of Fe homeostasis in tomato. Consistently, this study also suggests the involvement of ZIP metal ion transporter and BHLH transcription factor with Zn transporters in tomato. Overall, this interactome analysis might provide essential background for functional genomics studies in characterizing transporters and transcription factors underlying Zn-deficiency responses in tomatoes.
Oxidative damage and antioxidant response
In this study, the O2.− was higher in the roots than in the shoot but significantly decreased in the shoot due to Zn-deficiency, which may be linked to elevated SOD activity. Further, H2O2 is a key ROS indication substantially induced in tomato following Zn starvation. The increases in ion flux and gene expression are part of H2O2 in plants43,54. The degradation of elevated H2O2 into less reactive molecules is frequently correlated with CAT55. Hacisalihoglu et al.56 reported that Zn-efficiency is allied with the Zn-SOD enzyme in wheat. In this study, Ratan showed a consistent decline in antioxidant enzymes that are known to inhibit H2O2 in the shoot in response to Zn deficiency. Further, APX and GR also showed no induction in tomato subjected to Zn-starvation, suggesting that inefficiency to ROS mitigation is possibly related to the damage of chloroplasts and cellular proteins. Cells are also prone to ROS pools due to abiotic stresses, not just chloroplasts involved in energy production57. It implies that ROS scavenging is not present actively in this Zn-deficiency sensitive tomato, especially in the shoot, to combat Zn deficiency. Thus, it pinpoints that the increase of ROS and the decrease in chlorophyll synthesis in Ratan is correlated with the photosynthesis inefficiency in leaves. However, the ROS regulation in plant cells may vary on the species and cultivar of species under Zn deficiency.
Conclusion
Zn deficiency showed a substantial reduction in plant biomass, photosynthetic efficiency, and cellular damage in Zn-deficiency sensitive cultivar (Ratan). The expression of iron-regulated transporter 1, zinc transporter-like (LOC100037509) and zinc transporter (LOC101255999) significantly decreased following Zn-deprivation resulted in a substantial decrease in Zn and Fe status in either root or shoot of tomato. Although the metal uptake in plants depends on many genes and the activity of the encoded protein, these two Zn-transporters may encourage further research regulating Zn-efficiency in tomato. Furthermore, the morpho-physiological retardation was consistent with the inefficiency of antioxidant defense to cope with the elevated oxidative injuries. This outcome will help to clarify our understanding of Zn deficiency more effectively to accelerate breeding or genome-editing program to improve Zn-deficiency tolerance in tomato for human nutrition.
Data availability
All data of the manuscript are available.
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We would like to show our gratitude to Invent Technologies, Bangladesh.
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This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic grant number (S grant of MSMT CR).
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Conceptualization, A.H.K. and M.S.A.; methodology, A.H.K.; software, U.D. and M.B.; validation, M.S.A., G.S. and M.S.; formal analysis, A.H.K.; investigation, A.H.K., M.S.A. and U.D.; resources, A.H.K.; data curation, A.H.K., M.S. and G.G.; writing—original draft preparation, A.H.K.; writing—review and editing, A.H.K., M.S., G.G., M.M.H.; visualization, A.H.K. and G.G.; supervision, A.H.K. and M.M.H.; project administration, A.H.K. and M.S., funding acquisition, A.H.K., M.S. and M.B. All of the authors contributed to the writing of the final version and approved the manuscript.
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Kabir, A.H., Akther, M.S., Skalicky, M. et al. Downregulation of Zn-transporters along with Fe and redox imbalance causes growth and photosynthetic disturbance in Zn-deficient tomato. Sci Rep 11, 6040 (2021). https://doi.org/10.1038/s41598-021-85649-w
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DOI: https://doi.org/10.1038/s41598-021-85649-w
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