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Systematic mining and characterization of metal transporter families regulating zinc homeostasis provide insights into metal homeostasis in Camellia sinensis.

BACKGROUND AND AIMS: Zinc is essential for tea plant growth and quality formation, yet its homeostatic mechanisms remain poorly understood. This study identified metal transporter families regulating zinc homeostasis, analyzed their evolution, structure, and expression, and clarified zinc uptake, transport, detoxification networks, and their links to metabolism. METHODS: This study identified zinc homeostasis-related metal transporter families in the tea plant genome, characterized their structural features and expression profiles across tissues and developmental stages through integrative bioinformatics and transcriptomic analyses, and delineated the molecular mechanisms underlying zinc uptake, translocation, and detoxification by systematically integrating published evidence. RESULTS: This study identified 74 metal transporter genes from six families: 13 CsZIPs, 12 CsNRAMPs, 10 CsHMAs, 10 CsYSLs, 14 CsMTPs, and 15 CsCAXs in the 'Shuchazao2' genome, revealing closer affinity to woody species than to Arabidopsis. These proteins exhibit conserved domains, diverse subcellular localizations (cell membrane, vacuole, chloroplast, and Golgi apparatus), and tissue-specific expression with abundant stress/hormone-responsive cis-elements. At the plant-soil interface, tea plants mobilize rhizospheric zinc via proton and organic acid secretion; CsYSLs, CsNRAMPs, and CsZIPs mediate zinc uptake, aided by arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) that expand root absorption zones. Xylem CsHMAs and phloem CsYSLs coordinate root-to-shoot zinc translocation, and vacuolar transporters (CsMTPs, CsCAXs), cell wall immobilization, and antioxidant systems alleviate high-zinc stress injury. CONCLUSIONS: These findings collectively delineate an integrated zinc "acquisition-distribution-buffering" network in tea plants, offering a repertoire of candidate genes with potential utility in zinc biofortification breeding and improving acid soil adaptation. Further experimental validation, including tea transgenesis, zinc-stress qRT-PCR, and heterologous functional complementation, is essential to substantiate their biological roles.

Camellia sinensis

Zinc absorption in the rat determined by radioisotope dilution.

Zinc absorption in rats was examined by use of an isotope dilution technique. Young rats and mature rats fed varying levels of dietary zinc were injected intramuscularly with 65Zn. Nine days later, a zinc balance study was begun and continued for 5 days. The rats were then decapitated and the intestines and kidneys were removed and analyzed for zinc and 65Zn content. The feces were analyzed for zinc and 65Zn content and the specific activities of the intestines and kidneys were used to compute the contribution of endogenous zinc to total daily zinc excretion and thus calculate the true daily absorption of zinc. The zinc concentration of the intestines and kidneys did not differ significantly among groups but the specific radioactivity in these organs decreased as dietary zinc intake increased. Endogenous zinc excretion was greatest in mature rats and young rats fed the highest level of dietary zinc. Mature rats actually absorbed as much or more zinc than young rats fed the same dietary level of zinc. The results demonstrate that zinc homeostasis in rats is maintained by zinc secretion from the intestine rather than by regulation of zinc absorption.

Aging

Involvement of hepatic metallothioneins in hypozincemia associated with bacterial infection.

Hypozincemia was induced in rats by Salmonella typhimurium and live vaccine strain Francisella tularensis (LVS) infections. Hepatic synthesis of zinc-binding proteins (ZBP) was studied in order to elucidate the mechanisms involved in the redistribution of zinc from plasma to liver occurring during infectious illness. ZBP, labeled in vivo with 65Zn, were isolated and identified as metallothioneins based, in part, on their heat stability, dimorphism, and amino acid composition. Cysteine was the major amino acid found in both forms of metallothionein and constituted 28-31% of total residues. The apparent half-life of these proteins as measured by disappearance of 65Zn was determined to be 19 h in a relatively mild infection (LVS) and 38 h in a more severe S. typhimurium infection. Results provide evidence that metallothioneins not only have the previously postulated regulatory role in normal zinc homeostasis but are intimately involved in the zinc redistribution occurring during the acute stage of infectious illness.

Amino Acids

Accumulation and depletion of zinc in chick tissue metallothioneins.

Two experiments were conducted with Hubbard broiler chicks on the metabolism of high levels of dietary zinc. In the first experiment, chicks were fed a basal diet or the basal diet plus 500, 1,000, 2,000 or 4,000 ppm zinc, and in the second experiment chicks were fed the basal diet or the basal diet plus 1,000, 2,000, 4,000, 8,000 or 16,000 ppm zinc, using zinc acetate. Zinc was found to be associated with 4 peaks when the cytosols from tissues (liver, kidney, pancreas and intestinal mucosa) were eluted through columns of G-75 Sephadex. Although the zinc content of all four peaks increased with increased zinc content of the diet, the greatest elevation occurred in metallothionein (MT). This protein was purified from all four tissues (liver, kidney, pancreas and intestinal mucosa) and the amino acid analysis revealed the presence of about 30% cysteine. Zinc accumulated to the greatest extent (microgram in MT/g tissue) in MT of the pancreas. The zinc in MT disappeared very rapidly when chicks which had been fed a diet containing high levels of zinc were fed a low zinc diet, indicating the extreme lability of this metal in this protein. The results suggest the involvement of MT in zinc homeostasis.

Amino Acids

DNA Methylation and Proteomic Profiling of Postmortem Brain Tissue Reveals Epigenetic Dysregulation and Neuroinflammatory in Fragile X-associated Tremor/Ataxia Syndrome (FXTAS).

BACKGROUND: Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by FMR1 premutation CGG repeat expansions (55-200 repeats). The epigenetic landscape of the FXTAS brain remains uncharacterized. We performed genome-wide DNA methylation profiling of postmortem prefrontal cortex tissue to identify differentially methylated positions (DMPs) and candidate genes, and sought protein-level support for a neuroinflammatory signal. METHODS: DNA methylation was profiled in postmortem prefrontal cortex (Brodmann area 9) from 27 male FXTAS cases and 29 male controls using the Illumina MethylationEPIC array (EPICv1 and EPICv2 platforms), merging 721,802 common probes. Surrogate variable analysis (SVA) controlled for confounders. DMPs were defined by |&#x394;&#x3b2;| > 0.10 and FDR < 0.05; exploratory Reactome 2024 pathway analysis was performed on the DMP-associated gene list. Targeted proteomic profiling was performed in the same brain region using the Olink (proximity extension assay) Inflammation panel in 9 FXTAS cases and 12 controls, with SVA-adjusted differential abundance analysis, and concordance assessment against a prior mass spectrometry dataset. RESULTS: We identified 108 significant cg-type DMPs mapping to 80 genes (50 hypermethylated, 58 hypomethylated in FXTAS). The strongest signal was CYP2E1 (7 concordant hypomethylated DMPs, mean &#x394;&#x3b2; = -0.143), an oxidative stress gene also implicated in Parkinson's disease. FTCD, a one-carbon cycle enzyme, carried 5 hypermethylated DMPs (mean &#x394;&#x3b2; = +0.210). A cluster of DMP-associated genes with established roles in innate immune and NF-&#x3ba;B signaling, TRAF3 (the single most significant DMP among the inflammation genes, hypermethylated), BATF, RCOR1, and MSI2; they pointed toward neuroinflammatory dysregulation. Additional genes included LINGO1 (myelination inhibitor), SYT3 (synaptic vesicle), and SLC39A4 (zinc transporter). Exploratory Reactome enrichment using the DMP-associated gene set nominated themes including neuroinflammation resolution, axonal growth inhibition, zinc homeostasis, and CYP2E1 metabolism at nominal significance (p<0.05); however, the gene-to-pathway mapping rate was low and no pathway survived correction for multiple testing. Olink proteomic analysis independently identified 60 significantly altered inflammation proteins (59 downregulated), including CXCL8, CXCL10, IL6, IL15, IL18, TLR3, IRAK1/4, and complement C1QA, which were directionally concordant with prior mass spectrometry data. CONCLUSIONS: This integrated study reveals a genome-wide epigenetic signature in the FXTAS prefrontal cortex implicating oxidative stress, myelination failure, zinc dysregulation, one-carbon cycle disruption, and most notably a coordinated set of epigenetically altered genes governing innate immune and NF-&#x3ba;B signaling. Convergence of TRAF3 hypermethylation with independent downregulation of TLR3 and NF-&#x3ba;B-pathway proteins at the protein level supports a coherent, cross-platform model of dysregulated neuroinflammatory signaling in FXTAS, identified here through individual gene- and protein-level convergence rather than formal pathway enrichment. FTCD hypermethylation proposes a self-reinforcing epigenetic loop via SAM depletion. These multi-omic findings establish FXTAS as a disorder of pervasive epigenetic reprogramming and nominate candidate genes for future mechanistic and therapeutic investigation.

CYP2E1

EWAS in a polyphenol dense, DNA methylation-targeted, controlled diet and lifestyle study.

BACKGROUND: Dietary and lifestyle factors can influence DNA methylation patterns. We previously reported epigenetic age attenuation following a controlled study using an 8-week polyphenol-dense, DNA methylation-targeted diet and lifestyle intervention in healthy males (Methylation Diet and Lifestyle Study), with phytonutrient/polyphenol-rich foods (green tea, oolong tea, curcumin, garlic, and berries) being most predictive of this effect. METHODS: Here we conducted an epigenome-wide association study (EWAS) in 38 participants from the Methylation Diet and Lifestyle Study. The intervention included a dietary pattern intentionally rich in substrate and cofactor nutrients for methylation pathways, and components known to alter DNA-methyltransferase (DNMT) enzyme activity. In line with prior EWAS studies with small sample sizes where FDR-significant findings are unlikely, we used pre-specified nominal P-value thresholds (0.001, 0.0001) for the exploratory analyses. RESULTS: At P < 0.001 (unadjusted), 676 differentially methylated loci (DML) were identified in the intervention group versus 286 in controls. At P < 0.0001 (unadjusted), 50 DML were identified in the intervention group compared to 13 in controls. Fifteen DML were in transcription start site-proximal regions of genes including those involved in zinc homeostasis and nutrient sensing, development and pluripotency, proteostasis and genome stability, tumor suppression, and synaptic function. A group-by-time interaction analysis identified 70 intervention-specific DML at P < 0.0001, with nominal enrichment including autophagy, mTOR signaling, and chromatin remodeling pathways. A regional DMR analysis identified 128 within-group and 129 interaction-specific DMRs. DMR functional enrichment analyses revealed convergent nominal associations with lipid metabolism (alpha-linolenic acid, lipoic acid, biosynthesis of unsaturated fatty acids, PPAR signaling, cholesterol homeostasis), central energy metabolism (TCA cycle, glycolysis/gluconeogenesis, pentose phosphate, pyruvate), and nutrient sensing (PI3K-Akt, mTOR, AMPK, autophagy as well as other pathways). As expected for the limited cohort size and short intervention duration, none of the single CpG findings or enrichment analyses survived multiple test correction and are therefore considered exploratory and hypothesis-generating only. CONCLUSION: This EWAS identified a larger number of nominally changing CpGs in the intervention group compared to controls as well as biologically coherent methylation changes. These findings provide mechanistic hypotheses for previously observed epigenetic age attenuation. Replication in larger cohorts, longer intervention durations, and functional validation remain essential.

DNA methylation

The transcription factor AtANAC070 enhances zinc tolerance by promoting AtMTP1 expression in Arabidopsis thaliana.

The NAC transcription factor AtANAC070 functions in zinc tolerance by directly activating AtMTP1 transcription to promote vacuolar zinc sequestration and homeostasis in A. thaliana. Zinc (Zn) is an essential micronutrient for plant growth, but it becomes toxic when present in excess. An initial screen of Arabidopsis thaliana T-DNA insertion mutants suggested a positive role of AtANAC070 in tolerance to excess Zn. AtANAC070 expression was induced under excess Zn, and loss of function of AtANAC070 led to increased Zn sensitivity and higher Zn accumulation. Conversely, AtANAC070 overexpression enhanced Zn tolerance and reduced Zn accumulation. Yeast one-hybrid assays identified Metal Tolerance Protein 1 (AtMTP1), which encodes a key transporter mediating vacuolar sequestration of excess Zn, as a downstream target of AtANAC070. Dual-luciferase reporter and real-time quantitative PCR (RT-qPCR) assays confirmed that AtANAC070 directly binds to the AtMTP1 promoter to activate its expression. The atmtp1 mutant accumulated more Zn than the WT and was more sensitive to excess Zn, whereas AtMTP1-overexpressing lines showed the opposite phenotype. The atanac070 atmtp1 double mutant displayed Zn sensitivity comparable to that of atmtp1 mutant, while AtMTP1 overexpression in the atanac070 background reduced Zn accumulation and restored Zn tolerance. These results indicate that AtANAC070 contributes to Zn homeostasis under excess Zn by promoting AtMTP1 expression.

Arabidopsis

Metallothionein: an exceptional metal thiolate protein.

Metallothioneins are unusual, low molecular weight proteins of extremely high sulphur and metabl content. They occur in substantial quantity and in multiple variant forms in parenchymatous tissues (liver, kidney, intestines) of vertebrates and certain microorganisms (Neurospora crassa, yeast). They are though to play a central role in the cellular metabolism of metals such as zinc, copper and cadmium. All mammalian forms studied are single chains with 20 cysteinyl residues among a total of 61 amino acid residues and highly characteristic amino acid sequences. Their most conspicuous common features are seven -Cys-X-Cys- sequences where X stands for an alphatic residue other than Cys. Together with additional cysteinyl residues located elsewhere in the chain and brought into juxtaposition by appropriate chain folding, these dithiol sequences are believed to form the basis of the trithiolate chelating structures typical of most of the six or seven metal-binding sites of the mammalian cadium- and/or zinc-containing metallothioneins. The positions of the cysteinyl residues are preserved in evolution: the copper-containing metallothionein from Neurospora crassa, containing only 25 amino acid residues, has a distribution of metal-binding cysteinyl residues identical to that of the N-terminal portion of the mammalian chains. The detailed physiological role of metallothionein remains to be clarified but its biosynthesis is known to be modulated by nutritional and endocrine factors. Recent evidence suggests that metallothionein is a critical determinant in the homeostasis of zinc.

Amino Acid Sequence

Nutritional agents which affect metabolic zinc status.

As the number of factors which affect zinc absorption and homeostasis increase, it becomes more apparent that the relative zinc status of the population cannot be estimated from the analyzed zinc content of foods. The environment of the small intestine at the time of absorption can only be estimated from the diet fed. Even then since both endogenous and exogenous factors determine the zinc absorbed, the precision of these estimations should allow for large individual variation.

6-Phytase

A novel DNA-protective function of Escherichia coli thioredoxin 2 mediated by its N-terminal zinc-binding domain.

Thioredoxins are ubiquitous thiol-disulfide oxidoreductases that maintain intracellular redox homeostasis. In addition to its conserved catalytic domain, Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain whose physiological function remains largely unknown. Here, we identify a previously unrecognized DNA-binding activity of EcTrx2 and demonstrate its role in protecting DNA during oxidative stress. Electrophoretic mobility shift assays showed that EcTrx2 bound plasmid DNA in a concentration-dependent and GST-tag-independent manner, whereas EcTrx1 exhibited no detectable DNA-binding activity. DNA binding was abolished by deletion of the N-terminal zinc-binding domain and was blocked by zinc occupancy, indicating that this unique domain is essential for DNA interaction. Consistent with these findings, EcTrx2 significantly protected plasmid DNA from DNase I digestion and hydroxyl radical-mediated oxidative damage in vitro. Furthermore, EcTrx2 enhanced bacterial tolerance to the DNA-damaging agents zeocin and diamide, supporting the physiological relevance of its DNA-binding activity. Our results reveal a DNA-binding role for EcTrx2 and identify its N-terminal zinc-binding domain as a key determinant of DNA binding and protection against oxidative DNA damage.

DNA binding

Rice LSD1-like Genes: Genome-Wide Characterization and Evidence Linking OsLSD3 to Plant Height.

LSD1-like zinc-finger proteins participate in programmed cell death, redox homeostasis, and stress responses in plants, but their functional diversification and contributions to agronomic variation in rice remain poorly defined. This study aimed to characterize the rice LSD1-like gene family and evaluate the potential agronomic roles of selected members, with particular emphasis on OsLSD3. Genome-wide analyses were integrated with OsLSD3 natural variation and haplotype analyses in 4666 rice accessions, CRISPR/Cas9 mutant phenotyping in the ZH11 background, and subcellular localization assays. Seven LSD1-like genes were identified and showed substantial divergence in protein architecture, gene organization, promoter cis-element profiles, and tissue- and stress-responsive expression. OsLSD3 formed six population-structured haplotypes, and two common Japonica haplotypes differed significantly in plant height. Consistently, two independent oslsd3 mutant lines were taller than the wild type, whereas additional changes in grain-related traits were line-specific. OsLSD2 and OsLSD3 localized mainly to the nucleus, while OsLSD4 was predominantly nuclear with weak cytoplasmic localization. These results identify OsLSD3 as the strongest candidate among the examined members for further investigation of plant height- and grain-related traits, while OsLSD2 and OsLSD4 represent additional candidates for grain-trait regulation. Further validation using additional alleles and environments is required.

LSD1-like

Homeostatic adjustments in zinc digestion to widely varying dietary zinc intake.

A 15-day Zn balance study was conducted with 36 growing rats maintained on a casein diet with 18.2 microgram Zn/g from day 0 to 3. Thereafter, six rats each were given the diet with the following variates of Zn content (microgram Zn/g): 5.6, 10.6, 18.2, 39, 70, 141. Zn digestion adjusted to the change in dietary Zn intake within the first 6 days via control of the extent of intestinal Zn absorption and fecal excretion of endogenous Zn. From day 9 to 15, mean apparent Zn digestion ranged from 92 to 18% and Zn absorption from 100 to 34% in an inverse relation to Zn intake. At the supply of 39 and 70 microgram Zn/g of diet, fecal excretion of endogenous Zn amounted to nearly one fourth of the dietary Zn intake. Reduction of intestinal absorption became the major factor to confine Zn accretion by the body when Zn intake was in great excess above requirement. Endogenous fecal Zn excretion exhibited its greatest homeostatic response in the range of optimum Zn intake. Zn excretion via the kidneys did not partake in Zn homeostasis, except that it was a major drain of body Zn compared to the intestinal route at low Zn intakes.

Animals

The transcription factor NO TRANSMITTING TRACT/WIP2 modulates cytokinin homeostasis in Arabidopsis.

The transcription factor WIP2/NO TRANSMITTING TRACT (WIP2/NTT) belongs to the WIP zinc finger family. Loss of WIP/NTT function in Arabidopsis thaliana causes alterations in specific tissues in the gynoecium. It also impairs root development, but only when combined with the loss of WIP4 and WIP5 function, due to redundancy. Certain mutant loss-of-function phenotypes can be recovered by cytokinin application, NTT interacts with cytokinin signaling components, and the phenotypes displayed by plants with increased WIP2/NTT expression also suggest a possible interaction with this pathway. Therefore, the objective of this study was to investigate the relationship between WIP2/NTT and the cytokinin pathway. To overcome the issue of genetic redundancy, we used a commonly used inducible system. We found that WIP2/NTT induction alters cytokinin levels and signaling in a tissue-specific manner, as shown by cytokinin content measurements and TCSn::GFP reporter analysis. Transcriptome analyses revealed candidate target genes related to the cytokinin pathway. Yeast one-hybrid and transactivation assays demonstrated direct NTT binding to regulatory regions of the cytokinin genes ISOPENTENYL TRANSFERASE 5 (IPT5), ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), and CYTOKININ OXIDASE/DEHYDROGENASE 7 (CKX7) involved in cytokinin biosynthesis, signaling, and degradation, respectively. Moreover, immunolocalization assays revealed that cytokinin distribution was altered in loss of function mutants and after NTT induction. The results of this work indicate that WIP2/NTT modulates cytokinin homeostasis.

Cytokinins

Effect of acute disease and ACTH on serum zinc proteins.

The effect of acute disease and ACTH infusion on serum zinc proteins was studied in serums from 156 healthy and diseased subjects. The mean (+/-2 S.D.) zinc content of 20 normal serums was 96 +/- 20 microng per 100 ml. In 87 serums from acutely ill patients the zinc ranged from 92 to 40 microng per 100 ml. The mean values for nearly all categories of disease studied were lower than normal (P is less than 0.001). Chromatography of normal serum on Sephadex G-100 separates two protein fractions, I and II, containing 37.8+/-8.8 and 76+/-10 microng of zinc per 100 ml, respectively. In serum from diseased patients the zinc in fraction I is unaltered whereas that in fraction II decreases to 29.8+/-7.5 microng per 100 ml (P is less than 0.001). ACTH administration reduces secrum zinc from 10 to 60 microng per 100 ml, the decrements being due to changes in the zinc content of fraction II. Thus, ACTH may have an important role in the reduction of zinc content associated with pathologic states.

Acute Disease

Genome-wide interactions with cadmium exposure in dysglycemia: Populational effects and molecular insights.

Dysglycemia is a complex metabolic disorder governed by the interplay between environmental exposures and genetic factors, yet the precise molecular mechanisms driving these gene-environment (G&#xd7;E) interactions remains poorly understood. Here, we characterized the population-level landscape and molecular causality underlying the interactions between cadmium (Cd), a widespread environmental toxicant, and genetic susceptibility loci in dysglycemia. By conducting a Genome-wide Environmental Interaction (GWEI) study within a sub-cohort of the China National Human Biomonitoring (CNHBM) cohort (N&#x202f;=&#x202f;1298), we identified 29 genetic risk loci that significantly interact with Cd burden to exacerbate elevated fasting plasma glucose levels. Functional enrichment integrated with metabolomic profiling unmasked a profound multi-omics convergence, positioning epigenetic modifications (e.g. H3K27me3) and zinc-finger transcription factors (e.g. OVOL2, KLFs) as central regulatory hubs that disrupt metabolic homeostasis. To establish causality, we demonstrated that the rs11743277 A>T variant at the lead G&#xd7;E locus functions as a Cd-responsive enhancer element, facilitating recruitment of TEAD3 and upregulating TICAM2 expression in CRISPR/Cas9-edited HepG2 cells, especially upon Cd exposure. This initiates a TICAM2-mediated inflammatory response, with elevated pro-inflammatory cytokines (IFN-&#x3b2;, TNF-&#x3b1;, IL-6) impairing downstream insulin signaling and glucose utilization. Collectively, these findings establish a robust paradigm for G&#xd7;E interactions in complex metabolic disorders, revealing how environmental stressors reprogram genetic susceptibilities through molecular checkpoints and paving the way for tailored, precision-prevention strategies in environmental health.

CRISPR/Cas9 editing

Strigolactones constrain rice drought acclimation by suppressing ROS scavenging through the D53-OsWRKY31-ZFP36 module.

Strigolactones (SLs) are a class of plant hormones essential for tiller development and yield under diverse environmental conditions. Drought is a major limiting factor for rice yields. Although SLs contribute to drought resistance, mechanisms and practical applications of SL pathway in drought acclimation of rice remain poorly understood. Our study shows that short-term dehydration represses SL biosynthesis in rice roots. Genetic assays indicate that disruption of SL biosynthesis or signaling elevates rice drought resistance, whereas SL signaling activation or supplementation with the SL analog GR244DO impairs drought resistance. SLs negatively regulate drought acclimation by promoting degradation of the repressor protein DWARF53 (D53). D53 interacts with the transcription factor OsWRKY31 via its N-terminal domain and suppresses the protein level of OsWRKY31, which binds to and represses transcription of the ZFP36 promoter. ZFP36 encodes a zinc-finger transcription factor that promotes H2O2 scavenging to sustain reactive oxygen species (ROS) homeostasis during drought stress. Notably, the drought-resistant upland rice variety IRAT109 exhibits lower SL levels in root exudates than the lowland rice variety Nipponbare (NP). Genome editing of key components in SL pathway enhances drought resistance in NP, Huazhan (HZ), and IRAT109. The agronomic potential of tuning SL biosynthesis is further supported by the elite D17/HTD1 allele, which weakens SL biosynthesis and improves drought resistance and grain yield in Nekken 2 (NK2) under field conditions. These findings uncover a key mechanism underlying SL-repressed drought acclimation in rice and provide an effective strategy to improve drought resistance in diverse rice varieties amid ongoing climate change.

D53