Zinc Translocation from Zn-Sufficient to Zn-Deficient Roots as an Adaptation to Heterogeneous Zn Availability

Zinc Translocation from Zn-Sufficient to Zn-Deficient Roots as an Adaptation to Heterogeneous Zn Availability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zinc Translocation from Zn-Sufficient to Zn-Deficient Roots as an Adaptation to Heterogeneous Zn Availability Magdalena Pypka, Diana Davydenko, Katarzyna Sowa, Julia Maksymiuk, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6802346/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Oct, 2025 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Zinc is essential for plant development and human health. While the effects of soil nutrient heterogeneity on plant growth were studied for macronutrients, adaptive mechanisms for micronutrients like Zn remain largely unexplored. We investigate Zn homeostasis in plants grown in a transparent soil medium mimicking natural soil conditions with spatially heterogeneous Zn availability. Our findings suggests that Zn is translocated between lateral roots, moving from Zn-sufficient to Zn-deficient ones, mitigating Zn deficiency responses and reducing Zn uptake. Under heterogeneous Zn conditions, the expression of key Zn homeostasis-related genes (Zn importer - NtZIP4B, Zn exporter - NtHMA4a/b and Zn chelator – NtNAS) was significantly altered. NtHMA4a/b expression was influenced by the vertical positioning of the Zn-sufficient medium, while NtZIP4B and NtNAS showed suppressed expression in roots under heterogeneous conditions compared to homogeneous Zn-sufficient conditions. This suggests a systemic regulatory mechanism coordinating Zn allocation depending on whole root system Zn access. Elemental analysis revealed reduced overall Zn concentrations in plants grown in heterogeneous Zn media, with elevated Zn levels in leaf veins. This study uncovers a novel mechanism of Zn translocation within the root system in response to heterogeneous Zn supply, highlighting the complexity of micronutrient homeostasis and its adaptive regulation. zinc transport soil heterogeneity microelements xylem phloem micro-XRF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction It is projected that almost 582 million people will be chronically undernourished by 2030. Although the number is declining, insufficient nutrient intake/absorption still affects 6.8 percent (45.0 million) of children under 5 years old( 1 , 2 ). Zinc deficiency in soils is a significant issue, potentially affecting as much as 50% of the world’s agricultural lands( 3 ). Zinc is a divalent transition metal and an essential micronutrient for plants, playing critical roles in numerous biological processes. Approximately 9% of the eukaryotic proteome utilizes zinc( 4 ). Over 300 enzymes, spanning all six major enzyme classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases), require zinc as a cofactor. Furthermore, zinc serves as a crucial structural component in proteins, including alcohol dehydrogenases, protein kinases, and transcription factors (TFs)( 5 ). Symptoms of zinc deficiency in plants typically manifest as stunted growth and chlorosis, leading to substantial reductions in crop yields. As plants are the primary source of zinc in human diets, reduced zinc content in crops can result in zinc malnutrition, a condition that currently affects an estimated 17.3% of the global population( 3 , 6 ). In light of the growing global population, there is an urgent need to increase agricultural productivity by 70% over the next four decades( 7 ). Achieving this while maintaining or improving the nutritional quality of crops presents a dual challenge, as efforts to enhance yield (primarily carbohydrate content) often compromise the accumulation of essential micronutrients, including zinc. Plants, as sessile organisms, rely entirely on the nutrients available in their surrounding environment. However, under natural conditions, root systems frequently encounter nutrient heterogeneity in soil( 8 ). For instance, some roots may experience Zn deficiency, while others access Zn-sufficient zones. Plants have evolved intricate mechanisms to tightly regulate their responses to fluctuating Zn availability( 9 , 10 ). Central to this regulation is the control of Zn transport within and between cells, tissues, and organs. This is achieved primarily by modulating the expression of Zn transporters, their localization to the plasma membrane, and their subsequent removal or recycling( 10 , 11 ). Major families of Zn transporters include ZIPs (ZRT/IRT-like proteins), NRAMP (Natural Resistance-Associated Macrophage Protein), and YSL (Yellow Stripe-Like, Zn-ligand transport), which facilitate Zn influx into the cytoplasm. Conversely, Zn efflux is mediated by CDF/MTP (Cation Diffusion Facilitator/Metal Tolerance Protein), HMA (Heavy Metal ATPase), CAX (Cation/proton eXchanger), ZIF (Zinc-Induced Facilitator), and VIT (Vacuolar Iron Transporters)( 12 , 13 ). Zn homeostasis is further supported by Zn chelators such as malate, citrate, histidine, and nicotianamine (NA), which prevent undesirable Zn binding and facilitate efficient transport( 11 ). Zn uptake occurs predominantly in its ionic form (Zn²⁺) from the soil solution via roots( 14 ). Once absorbed, Zn is distributed through both symplastic and apoplastic pathways( 15 ). In the root’s central cylinder, transporters such as NtHMA4a/b in tobacco mediate Zn efflux into the xylem, enabling root-to-shoot translocation( 16 , 17 ). In leaves, Zn is retrieved from the xylem (apoplast) by ZIPs and NRAMPs, such as NtZIP1-like, NtZIP4, NtZIP5, NtZIP11, and NtNRAMP3 in tobacco( 18 ). Studies on Zn transporters have mainly been conducted under homogeneous growth conditions, where nutrients are evenly distributed in the medium (e.g., hydroponics). Under these conditions, it was shown that Zn transporters NtZIP4 and NtZIP5 are expressed mostly in the central cylinder in the middle part of the roots when Zn is sufficient and in the epidermis/cortex of most roots, including the root apical part, under Zn deficiency( 9 , 19 ). Research on nutrient heterogeneity has largely focused on root architecture responses; for instance, Giehl (2012) demonstrated that heterogeneous Fe distribution promoted lateral root growth in Fe-rich zones to sustain shoot Fe content( 20 ). However, investigations into the dynamics of micronutrient distribution, particularly under heterogeneous Zn conditions, remain limited. In this study, we grew plants in a transparent soil medium designed to mimic soil conditions( 21 ). This three-dimensional setup allowed the application of alternating nutrient concentrations to different regions. Compared to the split-root system, our approach allows for the creation of both vertical and horizontal Zn heterogeneity within a single root system. In contrast, the split-root system enables only vertical heterogeneity where the main roots are often cut to create two evenly sized root systems (from lateral roots) that grow in separate containers. The connection between roots is than provided through the hypocotyl only and root to root transport would be limited. Using transparent soil system, we demonstrated that Zn can be translocated between lateral roots, from Zn-sufficient to Zn-deficient regions. This surprising finding suggests the existence of previously unknown Zn homeostasis mechanisms in roots, likely involving an active and complex process mediated by Zn transporters. These mechanisms would necessitate the unloading of Zn from the xylem and its subsequent loading into the phloem, a process supported by Zn status sensing and signaling to ensure precise execution within specific root or shoot regions, independent of Zn concentration in the surrounding medium. Furthermore, we analyzed how different Zn distribution scenarios affected Zn content and distribution in shoots. This work provides new insights into plant Zn homeostasis, particularly regarding Zn translocation between tissues and organs, and may inform future agricultural practices to address Zn deficiency. Results Plants adjust Zn homeostasis when grown in Zn heterogeneous conditions. We were interested in identifying the Zn-related control of NtZIP4B expression (Zn importer involved in Zn uptake to cytoplasm( 9 , 18 )) in plants grown in a medium that mimics soil conditions, including its heterogeneous Zn distribution. For this, we tested plants with GUS expression under the NtZIP4B promoter ( promNtZIP4B::GUS ( 9 )) grown in transparent soil( 21 ), where the upper or lower half of the medium had either Zn-sufficient or Zn-deficient conditions ( -Zn /+1µMZn and + 1µMZn/ -Zn ; Fig. 1 ). As a control, we used plants grown in homogeneous Zn distribution both halves prepared with the same Zn concentration in the medium ( -Zn / -Zn , + 1µMZn/+1µMZn). As expected, in the homogeneous Zn-deficient medium, NtZIP4B promoter activity in roots increased (Fig. 2 a) compared to the roots grown in homogeneous Zn-sufficient medium (Fig. 2 b). This confirms that plants grown in the transparent soil medium have a similar response to Zn deficiency as those grown in hydroponic culture. However, we noticed that when plants were grown in a heterogeneous setup of the transparent soil medium (Zn-deficient and Zn-sufficient layers in one pot), lateral roots that grew in the Zn-deficient section of the medium exhibited lower NtZIP4B promoter activity, characteristic of Zn sufficiency (Fig. 2 c, d). To confirm this observation, we quantified the characteristic Zn-deficient tissue-specific profile of the NtZIP4B promoter in the epidermis and cortex (profile alpha, Fig. 2 e) compared to its limited expression localization only in the central cylinder under Zn-sufficient conditions (profile beta, Fig. 2 f). Profile alpha dominated in roots grown in homogeneous Zn-deficient conditions (~ 70%), while profile beta dominated in homogeneous Zn-sufficient conditions (~ 90%). Nevertheless, profile beta dominated in roots that grew in the Zn-deficient sections of the heterogeneous Zn distribution setup (both: -Zn /+1µMZn and + 1µMZn/ -Zn ). It appears that roots growing in the Zn-deficient part of the medium do not present a Zn deficiency response like plants grown in a homogeneously Zn-deficient medium. It is worth noting that, in this experiment, we ensured that the Zn concentration in the Zn-sufficient half of the heterogeneous treatment matched the concentration in the homogeneous Zn-sufficient medium (1 µM ZnSO₄). This means the total Zn amount (within whole pot) in the heterogeneous treatment was half that of the homogeneous Zn-sufficient treatment. Despite this, roots in the Zn-deficient portion of the heterogeneous setup exhibited Zn sufficiency-like behavior. In conclusion, these unexpected results suggest that the lack of a Zn-deficiency response in the NtZIP4B promoter in roots growing in the Zn-deficient region of a heterogeneously distributed Zn medium may be due to a relatively increased Zn level in those roots. This could be explained by Zn translocation from roots in Zn-sufficient areas to those in Zn-deficient regions. This finding could provide new insights into Zn homeostasis and highlights potential mechanisms of inter-root communication and nutrient redistribution that could play an important role in plant adaptation to heterogeneous environments. Plants appear to relocate Zn from Zn-sufficient to Zn-deficient regions. To further investigate whether Zn levels increase in roots growing in the Zn-deficient region of a heterogeneously distributed Zn medium, we analyzed differences in Zn concentration between roots grown under homogeneous Zn conditions (-Zn/-Zn, + 1µMZn/+1µMZn) and heterogeneous Zn conditions (-Zn/+1µMZn, + 1µMZn/-Zn), dividing the root system into halves for each treatment (Fig. 3 ). As expected, in homogeneous Zn conditions, Zn concentration was significantly higher in the Zn-sufficient treatment compared to the Zn-deficient treatment, regardless of whether the upper or lower part of the root system was analyzed (Fig. 3 a, homogeneous Zn distribution). However, in heterogeneous Zn conditions, no significant difference in Zn concentration was observed between any of the treatments (-Zn/+1µMZn, + 1µMZn/-Zn) or between the roots growing in Zn-sufficient and Zn-deficient sections of the medium (Fig. 3 a, heterogeneous Zn distribution). Interestingly, all of the samples from the heterogeneous treatments exhibited higher Zn concentrations compared to samples from homogeneous Zn-deficient treatments. There was no difference in root biomass between those plants (SI Fig. 1 ). These results indicate that plants growing in media with heterogeneous Zn distribution have increased Zn level in roots that grow in Zn-deficient medium compare to plants that grow in homogenous only Zn deficient medium. We propose that this is a result of translocation of Zn within root system from roots growing in Zn sufficient to roots growing in Zn deficient medium. We also suggest that this may be due to the transport between roots rather than redistribution from stored Zn in older roots or shoots( 22 ), as plants were grown under given conditions with only primary roots, a few short lateral roots and fraction of the shoot at the start of the experiment (SI Fig. 2 ). Therefore, Zn stored in the seedling is diluted during 14 days of biomass and organ growth happening directly under treatments. The Zn level in organs of plants growing in Zn deficient homogenous medium would be a representative of the maximal potential if any Zn would be redistributed from storage gained in pre-treatment growth (or seeds). As a control, we analyzed Zn concentrations in transparent soil before and after 14 days of homogeneous Zn distribution treatment, as well as after 14 days of heterogeneous Zn treatment. As expected, Zn concentrations in the beads remained similar at the beginning and end of the experiment with homogeneous Zn distribution. This suggests that Zn-sufficient conditions remained stable for 14 days and that there was no external Zn input in Zn-deficient conditions. Interestingly, after heterogeneous treatment, we observed a small but statistically significant decrease in Zn content in the Zn-sufficient region compared to the initial Zn-sufficient medium. It is important to remember that there is only half of Zn amount in medium with heterogenous Zn distribution compared to Zn-sufficient homogenous conditions. Therefore, this result could indicate increased Zn depletion due to increased Zn uptake by roots growing in the Zn-sufficient half, potentially to support Zn-deficient part. Similarly, we observed a small but statistically significant increase in Zn content in Zn-deficient regions compared to the initial Zn-deficient medium (Fig. 3 b). This increase might be due to Zn leakage from the Zn-sufficient part. However, we also considered other explanations, as similar results were observed for Zn-deficient beads in the upper half of the pot. Solute movement against gravity is limited to capillary spaces, which are absent in this system, and the relatively long distance over a short time scale would constrain diffusion effects( 23 , 24 ). Instead, the increase in Zn content in beads without added Zn may indirectly support our hypothesis that Zn is translocated between lateral roots, moving from those in the Zn-sufficient region to those in the Zn-deficient region. This process could contribute to additional Zn in the local environment, possibly through exudates. Further testing is needed to confirm these observations. Zn distribution differs in roots of plants grown in Zn heterogeneous medium If Zn is translocated from roots with access to a Zn-sufficient medium to roots growing in Zn-deficient conditions, it is important to determine its spatial localization to identify Zn sinks in root system. However, most analytical methods used to study elemental distribution in plant samples face limitations, including low sensitivity, sample handling challenges, and pretreatment requirements that can alter metal distribution and speciation ( 25 , 26 ). Synchrotron X-ray-based techniques can overcome these limitations by providing direct, highly sensitive, spatially resolved information on the distribution of the metal and its speciation within plants, while also requiring only a limited level of sample manipulation ( 7 , 8 )( 27 , 28 ). To show the Zn distribution patterns within root systems of plants grown in a medium with heterogeneous Zn distribution, we performed µXRF (micro-X-ray Fluorescence) analysis using synchrotron radiation (SOLARIS, POLYX beamline). In the following experiments we doubled Zn concentration in one section of the container in heterogenous treatment to maintain a consistent total Zn concentration throughout the treatments. For example, by having 2µM ZnSO 4 in only half of the container, the average concentration across the entire volume became 1µM ZnSO 4 , matching the control condition (1µMZn). We used that strategy across treatments ranging from 1 to 5 µM Zn. This ensures equivalence in Zn exposure across treatments and allows us to compare potential Zn localization. Interestingly, across all Zn treatments (ranging from 1 to 5 µM Zn), we observed a consistent pattern of Zn distribution within the root system (Fig. 4 ). In plants grown in homogeneous Zn-sufficient conditions, Zn was evenly distributed between the upper and lower halves of the root system (Fig. 4 a–c). As expected, in plants grown under heterogeneous Zn conditions, Zn was distributed relatively evenly (Zn0/Zn5 µM Zn and Zn0/Zn10 µM Zn have slightly increased signal in lower parts), including in roots located in the Zn-deficient part of the medium, regardless of whether the Zn-sufficient part was in the upper or lower section of the pot (Fig. 4 a–c). However, an intriguing observation emerged: when Zn was localized in the upper part of the medium, its concentration in the roots appeared relatively higher compared to plants where the Zn-sufficient part was in the lower section of the pot (Fig. 4 a–c). Given that the total Zn amount in these treatments was identical, this suggests that upper and lower roots may differ in their capacity for Zn uptake. In summary, µXRF analysis corroborates that Zn could be relocated to roots growing in Zn-deficient regions. Moreover, the data indicates that Zn uptake is facilitated when increased amounts of Zn is localized in the upper part of the medium under heterogeneous Zn conditions. Zn localization in the medium (upper/lower) impacts Zn levels in leaves Given the observed differences in Zn levels in roots grown under heterogeneous Zn localization, we mapped Zn distribution and analyzed Zn levels in leaves (excluding the hypocotyl) using µXRF. From the µXRF analysis, we found that the highest relative Zn levels were present in plants grown in homogeneous Zn-sufficient medium, while the lowest levels were observed in plants grown in homogeneous Zn-deficient medium (Fig. 5 a, b). The semi-quantitative comparison of µXRF fluorescence intensities between samples (Fig. 5 b) was feasible because we used leaves with specific characteristics (2 and 3 leaves) that have similar thickness, density and were analyzed in the same µXRF run. These findings were further corroborated by Zn concentration analysis in whole shots (Fig. 6 a). In leaves from plants grown in homogeneous Zn-sufficient medium, Zn was distributed throughout the entire leaf blade (Fig. 5 a). In contrast, in plants grown in medium with heterogeneous Zn distribution, Zn localization was primarily restricted to the leaf veins, accompanied by significantly lower Zn levels compared to plants grown in homogeneous Zn-sufficient medium, despite a similar total Zn content in the medium for both treatments. This indicates that heterogeneous Zn localization in the soil affects Zn distribution within the leaves. Zn concentration analysis also revealed that shoots from plants grown in medium with heterogeneous Zn distribution exhibited lower Zn concentrations compared to those grown in the same total Zn amount under homogeneous conditions. Notably, Zn levels were higher in shoots when the Zn-sufficient medium was placed in the upper part of the pot (Fig. 6 a). However, this difference in Zn distribution and concentration was not associated with significant differences in biomass, suggesting an overall lower Zn content in plants grown in medium with heterogeneously distributed Zn (Fig. 6 b). Control over expression of Zn homeostasis related genes may play a role in Zn distribution changes in plants growing under heterogeneous Zn conditions Finally, we analyzed the expression of key genes involved in Zn homeostasis: NtHMA4a/b , encoding a major Zn exporter responsible for Zn loading into the xylem( 17 , 29 , 30 ); NtNAS , encoding tobacco nicotianamine (NA) synthase ( 31 – 33 ), which produces the Zn chelator NA involved in Zn transport in the phloem( 31 ) and vacuolar storage( 34 ); and NtZIP4B , encoding a critical Zn transporter responsible for Zn uptake under Zn-deficient conditions( 9 , 33 , 35 ). Interestingly, NtHMA4a/b expression was significantly reduced in roots grown in homogeneous Zn-deficient medium compared to those grown in Zn-sufficient medium. In plants grown in media with heterogeneous Zn distribution, NtHMA4a/b expression was similar to that observed under homogeneous Zn-deficient conditions (Fig. 7 a). However, the expression of NtHMA4a/b varied depending on whether Zn was localized in the upper (increased expression) or lower (decreased expression) part of the medium. This pattern correlated with the Zn concentration in shoots of plants grown in heterogeneous media, suggesting a relationship between NtHMA4a/b activity, Zn content in shoot and potentially Zn distribution in the medium. The expression of NtNAS was similar between roots grown in homogeneous Zn-sufficient and Zn-deficient media, with a tendency for increased expression under Zn deficiency. However, in roots of plants grown in heterogeneous Zn conditions, NtNAS expression was significantly lower than in plants grown in Zn-sufficient homogenous medium. This reduced expression may indicate a decreased requirement for Zn chelation in plants grown in media with heterogeneous Zn distribution. For NtZIP4B , the expression pattern was consistent with the promoter-GUS studies (Fig. 2 ), showing increased expression under Zn deficiency. However, in plants grown under heterogeneous Zn conditions, NtZIP4B expression was significantly lower than in plants grown in homogeneous Zn-deficient media. Similarly, to NtNAS , the reduced expression of NtZIP4B in heterogeneous Zn conditions may reflect a mechanism by which plants coordinate Zn distribution to lateral roots. Earlier research from our group showed that NtZIP4B and NtNAS expression depends on Zn level in medium and increase under Zn deficiency( 9 , 33 ). However, both NtZIP4B and NtNAS expression is suppressed in plants grown under heterogeneous Zn conditions compare to homogenous Zn-sufficient media (Fig. 7 ) regardless of similar Zn presence and distribution within roots (Fig. 4 a). Such a result may suggest alternative regulatory mechanism for those genes that could be independent from local Zn cellular status ( 36 ) and that these mechanisms could be related to observed Zn distribution changes in plants grown in Zn heterogenous medium. In summary, the expression of Zn homeostasis-related genes suggests that the observed Zn distribution phenotype within the root system of plants grown in a heterogeneous Zn medium may involve coordinated regulation of Zn translocation (HMA4), uptake (ZIP4B), and storage or phloem transport (NAS). Further studies are needed to reveal if those mechanisms are involved in the movement of Zn from Zn-sufficient roots to Zn-deficient roots. Discussion Nutrient medium heterogeneity is known to influence plant growth and nutrient allocation, with effects varying based on plant species and root system architecture, as well as patch characteristics such as size and contrast in nutrient concentration( 37 ). Plants typically optimize nutrient acquisition from heterogeneous patches by altering root growth, particularly through increased lateral root elongation in nutrient-rich zones( 20 , 37 , 38 ). However, studies addressing impact of micronutrient heterogeneity in medium and how it impacts plant homeostasis, particularly Zn, are limited. In this study, we demonstrated that a medium with heterogeneous Zn distribution, comprising alternating Zn-sufficient and Zn-deficient layers, significantly affects Zn homeostasis in plants, altering Zn distribution patterns within roots and leaves. We observed that Zn from the Zn-sufficient medium is distributed to roots growing in Zn-deficient zones. This distribution mitigates Zn deficiency responses (e.g., expression of Zn-responsive promoters) in Zn-deficient roots and limits transport of Zn to the shoot, findings not previously reported. Our results suggest that Zn is transported between roots in heterogeneous Zn media. In homogeneous conditions, Zn uptake occurs across the entire root system (Fig. 2 a, b and schematic representation in Fig. 8 a, b). In heterogeneous conditions, Zn-sufficient roots act as "Zn source roots," while Zn-deficient roots function as "Zn sink roots," receiving Zn from Zn source roots. This redistribution does not appear to inhibit root growth, unlike deficiencies of macronutrients such as nitrogen, which are known to restrict higher-order root growth( 39 ). Zn translocation between roots likely involves both apoplastic (water stream) and symplastic pathways, overcoming apoplastic barriers to load Zn into the xylem, which then transports it to the shoot( 40 ). Given the unidirectional nature of xylem flow, this implies that nutrients may be relocated between xylem and phloem at lateral root junctions (Fig. 8 c, d). We hypothesize that Zn redistribution may also occurs within shoot, e.g. leaf veins, as our µXRF results show higher Zn concentrations in veins compared to the rest of the leaf blade in plants grown in heterogeneous Zn conditions (Fig. 5 ). Experiments with foliar application of Zn radioisotopes (Zn 65 ) in wheat and Sedum alfredii showed Zn translocation to leaves above and below the treated leaf, as well as to the root tips via phloem( 41 , 42 ). The potential regulation of Zn transfer between roots could be related to a systemic signal derived from either Zn-deficient roots or shoot, similarly to proposed control over AtMTP2 and AtHMA2 expression in A. thaliana ( 43 ). Key Zn-transporters appear to play distinct roles in mediating Zn distribution in heterogeneous conditions. For instance, in tobacco, there are two variants of the HMA4 gene, NtHMA4a and NtHMA4b, which may facilitate Zn loading into the xylem. Their expression has been shown to be triggered by Zn toxicity but not Zn deficiency( 17 ). Interestingly, foliar Zn application reduced AtHMA2 (also loads Zn to xylem) but not AtHMA4 expression in the roots of wild-type plants grown in a Zn-deficient hydroponic solution, suggesting systemic, shoot-derived regulation of AtHMA2 expression, but not AtHMA4( 43 ). This shows that control over Zn root-to-shoot transport is complex. In this context, it is important to notice that in our study, we used primers that detect the expression of both NtHMA4a and NtHMA4b, referred to as NtHMA4a/b. We observed that the expression of tobacco HMA4a/b was reduced in both heterogeneous Zn treatments and the homogeneous Zn-deficient treatment (Fig. 7 a). Interestingly, previous studies from our group using homogeneous hydroponic conditions showed that Zn deficiency led to reduced NtHMA4a/b expression across the apical, middle, and basal root regions( 44 ). This result was accompanied by a significant (5-fold) reduction in Zn levels in roots and shoots( 44 ). However, in this study, Zn levels in roots did not show a dramatic difference (Fig. 4 a), and slightly (less than 1-fold) lower Zn levels in shoot of plants growing in the heterogeneous Zn treatment compared to the homogeneous Zn-sufficient condition (Fig. 5 a, 6 a). Nevertheless, in shoots, we observed a statistically significant reduction in Zn concentration under both heterogeneous Zn conditions compared to homogeneous Zn sufficiency (Fig. 6 a). Interestingly, under Zn heterogeneous conditions in medium, NtHMA4a/b expression was influenced by the placement of the Zn-sufficient medium part. NtHMA4a/b expression was higher when Zn was localized in the upper layer and lower when Zn was present in the lower layer (Fig. 7 a). This could suggest that upper roots, often associated with nutrient acquisition( 45 ), may have a greater capacity for Zn uptake and translocation to the shoot, eg. even just by a fact that they are closer to hypocotyl. Based on these findings, we hypothesize that tobacco HMA4a/b control of expression may rely on systemic shoot control or its regulation is unrelated to Zn concentration. Further investigation whether the regulation of HMA4a/b expression plays a specific role in trafficking Zn to lateral roots is needed. The expression of NtNAS, which encodes nicotianamine synthase involved in Zn binding (storage in vacuole)( 34 ) and Zn phloem transport( 46 ), was significantly reduced in roots grown in heterogeneous Zn media. This reduction may be need for limiting Zn storage or phloem transport in plants that relocate Zn between their roots. We also observed a reduced Zn level in the shoots of plants grown under Zn heterogeneous conditions suggesting limited Zn translocation, which would be reasonable effect of downregulation of NtNAS. This may highlight the role of control over NAS expression as regulator of how much Zn may be available for Zn distribution within root system. Finally, NtZIP4B, a Zn uptake transporter that was activated under Zn-deficient conditions was suppressed in heterogeneous Zn media, despite the presence of Zn-deficiency. In our study, we examined the expression pattern of pNtZIP4B:GUS under both Zn heterogeneous and Zn-sufficient homogeneous conditions. We observed that its expression was limited to the cortex and central cylinder (Fig. 2 ). It is possible that reduced Zn transport to cell in central cylinder could impact Zn apolastic/symplastic balance which itself is known to regulate Zn homeostasis ( 47 , 48 ). This would also facilitate apoplastic transport and potentially xylem loading reducing Zn retention in Zn-sufficient roots. This however do not explain how Zn would be redirected to Zn-deficient lateral roots as the same mechanisms would reduce potential for phloem loading in part of the root where Zn have to be transferred from xylem to phloem (Fig. 8 d). This might suggest that Zn relocation to Zn-deficient parts that should involve Zn phloem transport would involve xylem-to-phloem reloading in shoot. We plant to investigate that in our future studies. Previous studies on Arabidopsis and tobacco, show that Zn transport and homeostasis are tightly regulated by collective functioning of different Zn transporters/chelators like HMA, NAS and ZIP family transporters( 9 , 18 , 29 , 30 , 33 , 35 , 47 , 49 – 51 ). The process by which Zn-sufficient roots in heterogeneous media provide Zn to Zn-deficient roots likely involves a complex Zn transport mechanism that requires coordinated regulation. This process to function efficiently is probably limited to specific part of root or shoot where xylem-phloem Zn relocation occurs (Fig. 8 d). Interestingly, both NtZIP4B and NtNAS promoters have Zinc Deficiency Response Element (ZDRE) motif ( 9 ), regulated by bZIP19 and bZIP23 transcription factors which induce expression of these genes under Zn cellular deficiency. This mechanism would allow to alter expression of genes between Zn-sufficient and Zn-deficient parts of the root system ( 9 , 52 ). We also anticipate that some kind of signaling between different root levels, or shoot-to-root, may be involved in Zn partitioning within the root system (Fig. 8 d). However, further studies on signaling, systemic responses and cellular mechanisms that facilitate Zn-dependent control over Zn supply are needed. Our study demonstrates that plants grown in media with heterogeneous Zn distribution exhibit a unique mechanism for Zn allocation, providing the first evidence that Zn may be delivered from Zn-sufficient roots to Zn-deficient roots within the same root system. This finding highlights the potential for studying previously unknown Zn homeostasis mechanisms in roots, driven by dynamic and intricate processes likely involving Zn transporters. Additionally, our results indicate that Zn distribution within the root system limits Zn translocation to the shoot. We propose that this distribution involves the downregulation of Zn-deficiency-related genes and the movement of Zn to Zn-deficient regions, potentially mediated by xylem-phloem interactions and nicotianamine transport. Uncovering these mechanisms, which appear to operate independently of external Zn concentrations, will offer new opportunities to manipulate Zn distribution within plants to enhance nutrient use efficiency and adaptability to suboptimal growth conditions. Our findings provide valuable insights into the complex regulation of Zn homeostasis and open avenues for further exploration. Future studies should focus on tissue-specific expression analysis, the role of phloem transporters, and the molecular mechanisms underlying sensing, signaling and Zn allocation in heterogeneous environments. Material and Methods Plant Material, Growth Conditions, and Treatments The experiments were conducted using tobacco ( Nicotiana tabacum var. Xanthi) plants. Seeds were obtained from the stock of the Institute of Biochemistry and Biophysics PAS, Warsaw, Poland, in 2002 and have since been propagated in the greenhouse of the University of Warsaw for experimental purposes. Plants were grown in a controlled environment chamber under the following conditions: 23/16°C day/night temperature, 40–50% relative humidity, 16 h photoperiod, and photosynthetically active radiation (PAR) of 250 µmol m⁻² s⁻¹ provided by fluorescent Flora tubes. Seeds were surface sterilized using 8% (w/v) sodium hypochlorite for 2 minutes, rinsed thoroughly, and germinated on vertically positioned Petri dishes containing quarter-strength Knop’s medium supplemented with 2% (w/v) sucrose and 1% (w/v) agar. After three weeks of growth (SI Fig. 2 ), seedlings were transferred to transparent soil medium in 7 × 10 cm Magenta® boxes and grown for an additional two weeks. Preparation of Transparent Soil Transparent soil was prepared by creating gel beads through the dropwise addition of a sodium alginate:Phytogel® (Sigma) mixture (1:4) into a 10 mM MgCl₂ solution, which immediately polymerized the outer layer to form spherical beads, as described previously( 21 ). A sterile, custom-made system was used to facilitate the production of large quantities of transparent soil (Fig. 1 , SI Fig. 3 ). The bead diameter was controlled by the size of the tip opening; beads approximately 0.5 cm in diameter were used in this study. Beads were crosslinked for 4 hours, rinsed with deionized sterile water, and then transferred to an equal volume of liquid half-strength Knop’s medium adjusted to the desired Zn concentration (1, 2, 5, or 10 µM ZnSO₄ or without added Zn). Beads were left in the medium overnight to allow nutrient diffusion. For experimental setups, two layers of beads (~ 100 g each) were placed in Magenta® boxes, with layer boundaries marked on the box side to identify root sections exposed to each treatment. Residual fluid accumulating at the bottom was removed with a sterile pipette. Seedlings were positioned in holes made with a pipette tip to ensure root growth through both medium layers. GUS Assay Plants expressing promNtZIP4B::GUS and wild-type (WT) plants (as staining controls) ( 9 )were fixed in 90% ice-cold acetone for 25 minutes with gentle rotation. Samples were washed four times in reaction buffer (50 mM Na₂HPO₄, pH 7.0, and 0.2% Triton X-100), with the third wash performed under mild vacuum (0.04 bar). Samples were then transferred to reaction buffer containing 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl β-d-glucuronic acid), infiltrated under vacuum for 15 minutes, and incubated at 37°C in the dark for 2.5 hours with gentle shaking. Samples were cleared in ethanol solutions of increasing concentration (50%, then 75%) before scanning the entire root system (EPSON V850 Pro). To visualize NtZIP4B expression at the tissue/cellular level, stained root fragments were embedded in 3% agarose and sectioned at 50–150 µm thickness using a vibratome (Leica VT1000S). Sections were analyzed microscopically (OPTA-TECH microscope). Differences in staining intensity were quantified using ImageJ. Based on the observations, Zn-deficient (alpha) and Zn-sufficient (beta) NtZIP4B expression patterns were identified (Fig. 2 e, f). The frequency of each expression pattern was determined by analyzing scans of entire root systems. Data came from 3 independent experiments (biological) with at least 14 plants for each heterogenous treatment (excluding plants that did not grew in both layers) and at least 9 plants for each homogenous setup. Determination of Metal Concentrations At the end of the transparent soil experiment, upper and lower root sections were separated. Shoots were rinsed briefly with deionized water. Roots were washed sequentially with Milli-Q water, 5 mM CaCl₂ (4°C, 15 minutes under agitation) to remove unbound and weakly bound metals from the apoplast, and finally with water. Samples were dried at 55°C, and dry biomass was measured. Digestion was performed using 65% HNO₃ and 39% H₂O₂ (9:1, v:v) in a closed microwave mineralizer (Milestone Ethos 900, Milestone, Bergamo, Italy)( 30 ). Zn concentrations were measured using flame atomic absorption spectrophotometry (AAS; TJA Solution Solar M, Thermo Electron Manufacturer Ltd, Cambridge, UK). Certified reference material (Virginia tobacco leaves, CTA-VTL-2) was included in each analysis. At least five biological replicates were analyzed per treatment. Data came from at least 7 plants from each treatment (that roots penetrated both layers) grown in two independent experiments. µXRF Analysis Micro-XRF 2D mapping experiments were carried out at the PolyX beamline( 53 ) of the SOLARIS National Synchrotron Radiation Centre( 54 ). Monochromatic beam from SOLARIS bending magnet (1.3T) was generated with a Mo/B 4 C multilayer monochromator with 1.3% bandwidth. The beam was focused with an ellipsoidal monocapillary optics (Sigray, 20 nm thick Pt inner coating, 20 mm working distance) to focal spot of 5µm. Samples were placed on a system of translation stages to perform 2D scans. Spectra were acquired with two Hitachi Vortex EM360 silicon drift detectors (ML3.3 extreme and 25 µm thick Be windows, 0.5 mm thick Si sensor, active area 100mm 2 ) coupled to XGlab Dante digital pulse processor. Detectors were placed in backscattering geometry (45 degree from the sample surface) and the incident X-ray beam was normal to the sample surface. 2D maps were acquired using mapping mode of the Dante DPP with a fast continuous horizontal and vertical point-by-point motions. In 2D maps the pixel size was 100 µm (one instance of 50 µm for Zn5/Zn5 image) with dwell time 12.5 ms per single spectrum. To create the 2D maps, count-rates in energetic region-of-interests were normalized by detector’ live time and incident beam intensity measured with ionization chamber. Data from two detectors were summed up taking into account the ratio between their response to the Zn signal. Samples of root systems and leaves (2nd and 3rd ) were exercised, rinsed with deionized water, and dried using paper towels. Samples were mounted in a 3D-printed polymer holder with a 5 × 5 cm mounting area. Roots were sandwiched between 3.6 µm foil (Spectro-Film™, DuPont) and gel was added on sides to prevent desiccation during analysis. At least three biological replicates were analyzed per treatment, with each replicate including three root systems per beamtime. Leaves (3 replicates) were analyzed during single beamtime. RNA Extraction Total RNA was extracted from samples stored at -80°C using the Universal RNA Purification Kit (EURx, #E3598) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA), with 260/280 ratios ranging between 1.8 and 2.0. RNA integrity was verified by agarose gel electrophoresis. RNA was isolated from at least 9 plants (that roots penetrated both layers) grown in two independent experiments, roots were divided into two technical samples. Quantitative Real-Time PCR cDNA was synthesized from total RNA using the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. RT-qPCR was conducted using the LightCycler® 480 System (Roche) and SYBR Green Master Mix (Roche, #0488735001). Primers (Supplementary Table 1) were designed using IDT OligoAnalyzer and OligoCalc tools. The reference gene NtPP2A (protein phosphatase 2A; AJ007496) was co-amplified with the target genes to normalize expression levels. Reactions were performed in triplicate for each independent biological replicate. Relative transcript levels were calculated using the ΔCt method. The quality of qPCR results was assessed using amplification and melting curves, with non-template controls included in each assay. Primers are listed in SI. Declarations Acknowledgments The authors thank Anna Barabasz, Nina Adamek-Siwirykow, Martyna Siwik, Małgorzata Palusińska for help especially during μXRF analysis. The authors also thank Danta Marii Antosiewicz for seeds of plant with promNtZIP4B::GUS expression and helpful discussions. Funding This research was supported by the National Science Centre, Poland, under project no. 2023/51/B/NZ9/02518 This publication was partially developed under the provision of the Polish Ministry and Higher Education project "Support for research and development with the use of research infra-structure of the National Synchrotron Radiation Centre SOLARIS” under contract no 1/SOL/2021/2. Data sharing Data are available from the corresponding author upon reasonable request. Conflict of interest The author declares no competing financial interests or personal relationships that could have influenced the work reported in this study . Author contributions OS conceived the idea and OS, MP amd DD designed the experiments. MP, DD, OS, performed and analyze data for GUS, qPCR, metal concentration analysis, MP, DD, JM, OS, KS, PW, PK, TK performed and analyze data for μXRF analysis, OS wrote the paper with contributions from all authors. All authors reviewed it and accepted. Corresponding author Correspondence to Oskar Siemianowski References WHO. Food Security and Nutrition in the World. 2024. WHO. The State of Food Security and Nutrition in the World 2023: Urbanization, agrifood systems transformation and healthy diets across the rural–urban continuum. Food & Agriculture Org; 2023. White PJ, Broadley MR. Physiological limits to zinc biofortification of edible crops. Front Plant Sci. 2011;2:80. Andreini C, Bertini I, Rosato A. Metalloproteomes: a bioinformatic approach. Acc Chem Res. 2009;42(10):1471–9. Broadley MR, White PJ, Hammond JP, Zelko I, Lux A. Zinc in plants. New Phytol. 2007;173(4):677–702. Alloway BJ. Zinc in soils and crop nutrition. 2008. Alexandratos N, Bruinsma J. World agriculture towards 2030/2050: the 2012 revision. 2012. Lima JE, Kojima S, Takahashi H, von Wirén N. Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1; 3-dependent manner. Plant Cell. 2010;22(11):3621–33. Barabasz A, Palusińska M, Papierniak A, Kendziorek M, Kozak K, Williams LE, et al. Functional analysis of NtZIP4B and Zn status-dependent expression pattern of tobacco ZIP genes. Front Plant Sci. 2019;9:1984. Sinclair SA, Krämer U. The zinc homeostasis network of land plants. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2012;1823(9):1553–67. Stanton C, Sanders D, Krämer U, Podar D. Zinc in plants: Integrating homeostasis and biofortification. Mol Plant. 2022;15(1):65–85. Palmgren MG, Clemens S, Williams LE, Krämer U, Borg S, Schjørring JK, et al. Zinc biofortification of cereals: problems and solutions. Trends Plant Sci. 2008;13(9):464–73. Ricachenevsky FK, Menguer PK, Sperotto RA, Fett JP. Got to hide your Zn away: molecular control of Zn accumulation and biotechnological applications. Plant Sci. 2015;236:1–17. Kisko M, Bouain N, Rouached A, Choudhary SP, Rouached H. Molecular mechanisms of phosphate and zinc signalling crosstalk in plants: Phosphate and zinc loading into root xylem in Arabidopsis. Environ Exp Bot. 2015;114:57–64. Caldelas C, Weiss DJ. Zinc homeostasis and isotopic fractionation in plants: a review. Plant Soil. 2017;411:17–46. Liedschulte V, Laparra H, Battey JND, Schwaar JD, Broye H, Mark R, et al. Impairing both HMA4 homeologs is required for cadmium reduction in tobacco. Plant Cell Environ. 2017;40(3):364–77. Hermand V, Julio E, Dorlhac de Borne F, Punshon T, Ricachenevsky FK, Bellec A, et al. Inactivation of two newly identified tobacco heavy metal ATPases leads to reduced Zn and Cd accumulation in shoots and reduced pollen germination. Metallomics. 2014;6(8):1427–40. Kozak K, Antosiewicz DM. Tobacco as an efficient metal accumulator. Biometals. 2023;36(2):351–70. Palusińska M, Barabasz A, Kozak K, Papierniak A, Maślińska K, Antosiewicz DM. Zn/Cd status-dependent accumulation of Zn and Cd in root parts in tobacco is accompanied by specific expression of ZIP genes. BMC Plant Biol. 2020;20:1–19. Giehl RF, Lima JE, von Wirén N. Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution. Plant Cell. 2012;24(1):33–49. Ma L, Shi Y, Siemianowski O, Yuan B, Egner TK, Mirnezami SV et al. Hydrogel-based transparent soils for root phenotyping in vivo. Proceedings of the National Academy of Sciences. 2019;116(22):11063-8. Marschner H. Marschner's mineral nutrition of higher plants. Academic; 2011. Lind KR, Siemianowski O, Yuan B, Sizmur T, VanEvery H, Banerjee S et al. Evidence for root adaptation to a spatially discontinuous water availability in the absence of external water potential gradients. Proceedings of the National Academy of Sciences. 2021;118(1):e2012892118. Siemianowski O, Lind KR, Tian X, Cain M, Xu S, Ganapathysubramanian B, et al. HOMEs for plants and microbes–A phenotyping approach with quantitative control of signaling between organisms and their individual environments. Lab Chip. 2018;18(4):620–6. Siemianowski O, Rongpipi S, Del Mundo JT, Freychet G, Zhernenkov M, Gomez ED, et al. Flexible Pectin Nanopatterning Drives Cell Wall Organization in Plants. JACS Au. 2024;4(1):177–88. Rongpipi S, Barnes WJ, Siemianowski O, Del Mundo JT, Wang C, Freychet G et al. Measuring calcium content in plants using NEXAFS spectroscopy. Front Plant Sci. 2023;14. Terzano R, Al Chami Z, Vekemans B, Janssens K, Miano T, Ruggiero P. Zinc distribution and speciation within rocket plants (Eruca vesicaria L. Cavalieri) grown on a polluted soil amended with compost as determined by XRF microtomography and micro-XANES. J Agric Food Chem. 2008;56(9):3222–31. van Der Ent A, Przybyłowicz WJ, de Jonge MD, Harris HH, Ryan CG, Tylko G, et al. X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants. New Phytol. 2018;218(2):432–52. Siemianowski O, Barabasz A, Kendziorek M, Ruszczyńska A, Bulska E, Williams LE, et al. HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier. J Exp Bot. 2014;65(4):1125–39. Siemianowski O, Mills RF, Williams LE, Antosiewicz DM. Expression of the P1B-type ATPase AtHMA4 in tobacco modifies Zn and Cd root to shoot partitioning and metal tolerance a. Plant Biotechnol J. 2011;9(1):64–74. Clemens S, Deinlein U, Ahmadi H, Höreth S, Uraguchi S. Nicotianamine is a major player in plant Zn homeostasis. Biometals. 2013;26:623–32. Lu J, Ye R, Qu M, Wang Y, Liang T, Lin J, et al. Combined transcriptome and proteome analysis revealed the molecular regulation mechanisms of zinc homeostasis and antioxidant machinery in tobacco in response to different zinc supplies. Plant Physiol Biochem. 2023;202:107919. Barabasz A, Klimecka M, Kendziorek M, Weremczuk A, Ruszczyńska A, Bulska E et al. The ratio of Zn to Cd supply as a determinant of metal-homeostasis gene expression in tobacco and its modulation by overexpressing the metal exporter AtHMA4. J Exp Bot. 2016:erw389. Haydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, Krämer U. Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis. Plant Cell. 2012;24(2):724–37. Palusińska M, Barabasz A, Kozak K, Papierniak A, Maślińska K, Antosiewicz DM. Zn/Cd status-dependent accumulation of Zn and Cd in root parts in tobacco is accompanied by specific expression of ZIP genes. BMC Plant Biol. 2020;20(1):37. Krämer U. Metal Homeostasis in Land Plants: A Perpetual Balancing Act Beyond the Fulfilment of Metalloproteome Cofactor Demands. Annu Rev Plant Biol. 2024;75. Wang P, Mou P, Hu L, Hu S. Effects of nutrient heterogeneity on root foraging and plant growth at the individual and community level. J Exp Bot. 2022;73(22):7503–15. Giehl RF, Lima JE, von Wirén N. Regulatory components involved in altering lateral root development in response to localized iron: evidence for natural genetic variation. Plant Signal Behav. 2012;7(7):711–3. Wang G, Xue S, Liu F, Liu G. Nitrogen addition increases the production and turnover of the lower-order roots but not of the higher-order roots of Bothriochloa ischaemum. Plant Soil. 2017;415:423–34. Scharwies JD, Dinneny JR. Water transport, perception, and response in plants. J Plant Res. 2019;132(3):311–24. Lu L, Tian S, Zhang J, Yang X, Labavitch JM, Webb SM, et al. Efficient xylem transport and phloem remobilization of Z n in the hyperaccumulator plant species S edum alfredii. New Phytol. 2013;198(3):721–31. Haslett BS, Reid RJ, Rengel Z. Zinc mobility in wheat: uptake and distribution of zinc applied to leaves or roots. Ann Botany. 2001;87(3):379–86. Sinclair SA, Senger T, Talke IN, Cobbett CS, Haydon MJ, Krämer U. Systemic upregulation of MTP2-and HMA2-mediated Zn partitioning to the shoot supplements local Zn deficiency responses. Plant Cell. 2018;30(10):2463–79. Maślińska-Gromadka K, Barabasz A, Palusińska M, Kozak K, Antosiewicz DM. Suppression of NtZIP4A/B changes Zn and Cd root-to-shoot translocation in a Zn/Cd status-dependent manner. Int J Mol Sci. 2021;22(10):5355. Giehl RF, von Wirén N. Root nutrient foraging. Plant Physiol. 2014;166(2):509–17. Seregin IV, Kozhevnikova AD. Nicotianamine: a key player in metal homeostasis and hyperaccumulation in plants. Int J Mol Sci. 2023;24(13):10822. Siemianowski O, Barabasz A, Weremczuk A, RUSZCZYŃSKA A, Bulska E, Williams LE, et al. Development of Z n-related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Z n levels. Plant Cell Environ. 2013;36(6):1093–104. Weremczuk A, Papierniak A, Kozak K, Willats WG, Antosiewicz DM. Contribution of NtZIP1-like, NtZIP11 and a WAK-pectin based mechanism to the formation of Zn-related lesions in tobacco leaves. Environ Exp Bot. 2020:104074. Nouet C, Charlier J-B, Carnol M, Bosman B, Farnir F, Motte P, et al. Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri. J Exp Bot. 2015;66(19):5783–95. Kozak K, Papierniak A, Barabasz A, Kendziorek M, Palusińska M, Williams LE, et al. NtZIP11, a new Zn transporter specifically upregulated in tobacco leaves by toxic Zn level. Environ Exp Bot. 2019;157:69–78. Antosiewicz DM, Barabasz A, Siemianowski O. Phenotypic and molecular consequences of overexpression of metal-homeostasis genes. Front Plant Sci. 2014;5:80. Assunção AG, Herrero E, Lin Y-F, Huettel B, Talukdar S, Smaczniak C et al. Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency. Proceedings of the National Academy of Sciences. 2010;107(22):10296-301. Szlachetko J, Szade J, Beyer E, Błachucki W, Ciochoń P, Dumas P, et al. SOLARIS national synchrotron radiation centre in Krakow, Poland. Eur Phys J Plus. 2023;138(1):1–10. Sowa K, Wróbel P, Kołodziej T, Błachucki W, Kosiorowski F, Zając M, et al. PolyX beamline at SOLARIS—Concept and first white beam commissioning results. Nucl Instrum Methods Phys Res Sect B. 2023;538:131–7. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.pdf Cite Share Download PDF Status: Published Journal Publication published 08 Oct, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 16 Jul, 2025 Reviews received at journal 15 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers agreed at journal 25 Jun, 2025 Reviewers agreed at journal 12 Jun, 2025 Reviewers invited by journal 11 Jun, 2025 Editor invited by journal 10 Jun, 2025 Editor assigned by journal 09 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 First submitted to journal 02 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6802346","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470438897,"identity":"bee625c3-2d84-4b99-8d37-549110c22e5f","order_by":0,"name":"Magdalena Pypka","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Pypka","suffix":""},{"id":470438900,"identity":"3d935e73-3dde-484f-9592-b0af19d0614e","order_by":1,"name":"Diana Davydenko","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Diana","middleName":"","lastName":"Davydenko","suffix":""},{"id":470438903,"identity":"38eadd36-f06a-4083-a784-af436018f92d","order_by":2,"name":"Katarzyna Sowa","email":"","orcid":"","institution":"National Synchrotron Radiation Centre SOLARIS, Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Sowa","suffix":""},{"id":470438905,"identity":"940579af-8237-4b85-8567-4287ed2e081e","order_by":3,"name":"Julia Maksymiuk","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Maksymiuk","suffix":""},{"id":470438908,"identity":"301c5a09-7a3a-4e3a-8960-1d119b7f56a9","order_by":4,"name":"Paweł Wróbel","email":"","orcid":"","institution":"AGH University of Krakow","correspondingAuthor":false,"prefix":"","firstName":"Paweł","middleName":"","lastName":"Wróbel","suffix":""},{"id":470438911,"identity":"792e4f7c-bee5-4165-ab2f-7a2476fae136","order_by":5,"name":"Tomasz Kołodziej","email":"","orcid":"","institution":"National Synchrotron Radiation Centre SOLARIS, Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Kołodziej","suffix":""},{"id":470438912,"identity":"8de4b451-36d6-401f-81e0-f0494d79273f","order_by":6,"name":"Pawel Korecki","email":"","orcid":"","institution":"Institute of Physics, Jagiellonian University in Krakow","correspondingAuthor":false,"prefix":"","firstName":"Pawel","middleName":"","lastName":"Korecki","suffix":""},{"id":470438914,"identity":"16756ce9-fbcb-47e5-bc9c-29376f524e5c","order_by":7,"name":"Oskar Siemianowski","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYJCCAwwMEmAGY4OBDZgB4soQqyUNroWHKOsYGxgOE9bCz8D+8MDHHAt5c/Ye448zCs4nbjjAfPA2D8MdnFokG3gMDs7cJmG4s+eMmeQGg9tALWzJ1jwMz3BqMTjAw3CYd5sE44YbudsYHwC1bDvAYyYNFMSjhf3B4b/bJOyBWjZ/fGBwDqiF/xsBLQwGhxm3SSQCtWwAOuwAyBY2vFokm4F+6d0mkbzhzPlvkjMMko33H2YztpxjgNsv/Oztjz/83FZnu+F4W/LHnj92sjPbmx/eeFNxRw6XFgZm7CJAB5MMyNAyCkbBKBgFwxUAAAnTW4LT/IZJAAAAAElFTkSuQmCC","orcid":"","institution":"University of Warsaw","correspondingAuthor":true,"prefix":"","firstName":"Oskar","middleName":"","lastName":"Siemianowski","suffix":""}],"badges":[],"createdAt":"2025-06-02 12:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6802346/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6802346/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07391-z","type":"published","date":"2025-10-08T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84527248,"identity":"3111ee6e-4365-430f-b417-ac312346ad44","added_by":"auto","created_at":"2025-06-13 05:30:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":507387,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental workflow illustrating plant growth and treatment setup. Plants were pre-grown for 21 days on transparent soil (top left) with quarter-strength Knop’s medium supplemented with 2% (w/v) sucrose and 1% (w/v) agar, followed by 14 days of treatment in customized growth chambers with defined conditions (top right). Transparent soil preparation and applied treatments are detailed with schematics (bottom left). Representative images at the end of the experiment (bottom right).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/97d79eb1ba37724923def716.png"},{"id":84528811,"identity":"100ea1c9-9eff-4105-9c9c-ef17863e6814","added_by":"auto","created_at":"2025-06-13 05:46:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1581165,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of homogeneous and heterogeneous Zn distribution on root development and pNtZIP4B::GUS expression profiles in growth medium. \u003cstrong\u003e(a–d)\u003c/strong\u003e Homogeneous Zn conditions (-Zn, +Zn) and heterogeneous Zn conditions (-Zn/+Zn, +Zn/-Zn) are depicted alongside representative root images and GUS expression in roots. Insets show detailed views of root zones with varying GUS activity. \u003cstrong\u003e(e, f)\u003c/strong\u003eQuantitative analysis of pNtZIP4B::GUS expression profiles (alpha and beta) in Zn-deficient and Zn-sufficient conditions. Box plot showing data distribution with whiskers extending to the outermost points within the upper and lower inner fences (1.5 × IQR) depict the percentage of roots exhibiting specific expression profiles under different treatments. Statistical differences (pairwise t-test) between treatments are indicated (letters denote significance, p \u0026lt; 0.05). Black rhomboid shows data point. Pictograms above the graph indicate the region of the medium where samples were taken (red rectangles) and the corresponding treatment applied. Dark blue represents Zn-sufficient zones, light blue indicates Zn-deficient zones, and half/half represents heterogeneous treatments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/2a2ec5933363fd1efb985862.png"},{"id":84527240,"identity":"26c7f1b3-cbed-47a7-a8a9-bc05b41cd3f8","added_by":"auto","created_at":"2025-06-13 05:30:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":276770,"visible":true,"origin":"","legend":"\u003cp\u003eZn concentration in plants grown in medium with heterogeneous (0/1 µM Zn; 1/0 µM Zn) Zn distribution was different from plants grown in homogeneous Zn medium (1/1 µM Zn; 0/0 µM Zn). \u003cstrong\u003ea)\u003c/strong\u003e Zn accumulation in upper and lower parts of root system (µg/g DW), \u003cstrong\u003eb)\u003c/strong\u003e Zn concentration (µM) in transparent soil before and after (14d) homogenous Zn distribution treatment and after (14d) heterogenous Zn treatment. Box plot showing data distribution with whiskers extending to the outermost points within the upper and lower inner fences (1.5 × IQR). Significant differences between treatments are indicated by pairwise t-tests (p \u0026lt; 0.05). Root system dry weight did not differ significantly. Black rhomboid shows data point. Pictograms above the graphs indicate the region of the medium where samples were taken (red rectangles) and the corresponding treatment applied. Dark blue represents Zn-sufficient zones, light blue indicates Zn-deficient zones, and half/half represents heterogeneous treatments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/237c112573dfd56be519ee13.png"},{"id":84528080,"identity":"10ad44a6-202c-4cba-95ca-39762d74911e","added_by":"auto","created_at":"2025-06-13 05:38:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1135429,"visible":true,"origin":"","legend":"\u003cp\u003eZn is distributed from Zn-sufficient to Zn-deficient grown roots, demonstrating adaptive Zn allocation in heterogeneous Zn conditions. Visualization using Zn Kα fluorescence (8.637 keV) with a synchrotron incident beam energy of 12.5 keV at the SOLARIS POLYX beamline. (a) Representative roots (from at least 3 biological replicates) grown under heterogeneous (a1) 0/2 µM Zn and (a3) 2/0 µM Zn, and homogeneous (a2) 1/1 µM Zn and (a4) 0/0 µM Zn conditions. (b, c) Set of roots grown in homogenous and heterogeneous media with 2.5× and 5× higher Zn concentrations. The color bars represent normalized count rates and the lower limits of color bars were set to the background level. 3 out of 4 root systems in a) and c) were analyzed simultaneously, and other (Zn0/Zn2 and Zn5/Zn5) in consecutive run (same settings) and images were merged. Set of roots in (b) was analyzed at the same time The color bar represents normalized count rates. Pictograms below the images are corresponding to the treatment applied. Dark blue represents Zn-sufficient zones, light blue indicates Zn-deficient zones, and half/half represents heterogeneous treatments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/df6b12be777bf015f6777da9.png"},{"id":84527249,"identity":"1f4c9c36-af45-4c6f-ad45-de6f0e34dbe8","added_by":"auto","created_at":"2025-06-13 05:30:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":857383,"visible":true,"origin":"","legend":"\u003cp\u003eZn translocation to leaves depends on homogenous or heterogenous Zn distribution. Visualization with Zn Kα fluorescence (8.637 keV). \u003cstrong\u003e(a)\u003c/strong\u003e Representative leaves (from at least 3 biological replicates) grown under heterogeneous (0/2 µM Zn; 2/0 µM Zn), and homogeneous (1/1 µM Zn; 0/0 µM Zn) conditions. Leaves were analyzed simultaneously and the colorbar represents normalized count rates and the lower limits of color bars were set to the background level. \u003cstrong\u003e(b)\u003c/strong\u003e Comparison of relative Zn level by analysis of average pixel intensity within the leaf blade (3 replicates). Box plot showing data distribution with whiskers extending to the outermost points within the upper and lower inner fences (1.5 × IQR). Significant differences between treatments are indicated by pairwise t-tests (p \u0026lt; 0.05). Black rhomboid shows data point. Pictograms above the graph indicate the corresponding treatment applied. Dark blue represents Zn-sufficient zones, light blue indicates Zn-deficient zones, and half/half represents heterogeneous treatments.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/96385fafbf4c4fe82d5e9b92.png"},{"id":84527232,"identity":"fac0c861-8704-427f-bc36-3dfab204dca6","added_by":"auto","created_at":"2025-06-13 05:30:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":172681,"visible":true,"origin":"","legend":"\u003cp\u003eZn concentration in shoots varies depending on whether the upper or lower root system has access to Zn. (a) Zn concentration and (b) dry weight of plant shoot grown in medium with homogeneous (1/1 µM Zn; 0/0 µM Zn) or heterogeneous (0/2 µM Zn; 2/0 µM Zn) Zn distribution. Box plot showing data distribution with whiskers extending to the outermost points within the upper and lower inner fences (1.5 × IQR). Significant differences between treatments are indicated by pairwise t-tests (p \u0026lt; 0.05). Black rhomboid shows data point. Pictograms above the graph indicate the corresponding treatment applied. Dark blue represents Zn-sufficient zones, light blue indicates Zn-deficient zones, and half/half represents heterogeneous treatments.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/790f5a85d4ba800ea5995399.png"},{"id":84527231,"identity":"219f7740-652a-463a-8764-e4e999be20d0","added_by":"auto","created_at":"2025-06-13 05:30:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":126880,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of chosen Zn transporters is altered in plants grown in medium with heterogenous Zn distribution. Expression of (a) \u003cem\u003eNtHMA4\u003c/em\u003e; (b) \u003cem\u003eNtNAS\u003c/em\u003e; c) \u003cem\u003eNtZIP4B\u003c/em\u003e in roots of plants grown in medium with homogeneous (1/1 µM Zn; 0/0 µM Zn) or heterogeneous (0/2 µM Zn; 2/0 µM Zn) Zn distribution. Each root system yields 2 samples (upper and lower roots), no significant difference between those parts were detected. Box plot showing data distribution with whiskers extending to the outermost points within the upper and lower inner fences (1.5 × IQR). Significant differences between treatments are indicated by pairwise t-tests (p \u0026lt; 0.05). Black rhomboid shows data point. Pictograms above the graph indicate the corresponding treatment applied. Dark blue represents Zn-sufficient zones, light blue indicates Zn-deficient zones, and half/half represents heterogeneous treatments.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/e63bd47cc9350b5770ff3605.png"},{"id":84528813,"identity":"d67c9e14-09e8-4e69-8a2f-af9da6a498ad","added_by":"auto","created_at":"2025-06-13 05:46:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":654911,"visible":true,"origin":"","legend":"\u003cp\u003eHypothesis on how Zn\u003csup\u003e+\u003c/sup\u003e is distributed between lateral roots. a) and b) plants grown in medium with homogeneous Zn distribution, c) and d) plants grown in medium with heterogenous Zn distribution in upper or lower part.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/4d69b343e6f0532706747c18.png"},{"id":93419724,"identity":"178203e6-fda9-4f10-834f-b709f8f77174","added_by":"auto","created_at":"2025-10-13 16:06:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5556147,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/b2ea998f-18a1-4ed8-a954-00dc6afadbc2.pdf"},{"id":84528086,"identity":"d5e54b6c-e0d8-4080-b139-68ee26aa391e","added_by":"auto","created_at":"2025-06-13 05:38:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":324783,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6802346/v1/50b0995971d67f6850ddc3e9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zinc Translocation from Zn-Sufficient to Zn-Deficient Roots as an Adaptation to Heterogeneous Zn Availability","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIt is projected that almost 582\u0026nbsp;million people will be chronically undernourished by 2030. Although the number is declining, insufficient nutrient intake/absorption still affects 6.8 percent (45.0\u0026nbsp;million) of children under 5 years old(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Zinc deficiency in soils is a significant issue, potentially affecting as much as 50% of the world\u0026rsquo;s agricultural lands(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Zinc is a divalent transition metal and an essential micronutrient for plants, playing critical roles in numerous biological processes. Approximately 9% of the eukaryotic proteome utilizes zinc(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Over 300 enzymes, spanning all six major enzyme classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases), require zinc as a cofactor. Furthermore, zinc serves as a crucial structural component in proteins, including alcohol dehydrogenases, protein kinases, and transcription factors (TFs)(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Symptoms of zinc deficiency in plants typically manifest as stunted growth and chlorosis, leading to substantial reductions in crop yields. As plants are the primary source of zinc in human diets, reduced zinc content in crops can result in zinc malnutrition, a condition that currently affects an estimated 17.3% of the global population(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In light of the growing global population, there is an urgent need to increase agricultural productivity by 70% over the next four decades(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Achieving this while maintaining or improving the nutritional quality of crops presents a dual challenge, as efforts to enhance yield (primarily carbohydrate content) often compromise the accumulation of essential micronutrients, including zinc.\u003c/p\u003e \u003cp\u003ePlants, as sessile organisms, rely entirely on the nutrients available in their surrounding environment. However, under natural conditions, root systems frequently encounter nutrient heterogeneity in soil(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). For instance, some roots may experience Zn deficiency, while others access Zn-sufficient zones. Plants have evolved intricate mechanisms to tightly regulate their responses to fluctuating Zn availability(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Central to this regulation is the control of Zn transport within and between cells, tissues, and organs. This is achieved primarily by modulating the expression of Zn transporters, their localization to the plasma membrane, and their subsequent removal or recycling(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Major families of Zn transporters include ZIPs (ZRT/IRT-like proteins), NRAMP (Natural Resistance-Associated Macrophage Protein), and YSL (Yellow Stripe-Like, Zn-ligand transport), which facilitate Zn influx into the cytoplasm. Conversely, Zn efflux is mediated by CDF/MTP (Cation Diffusion Facilitator/Metal Tolerance Protein), HMA (Heavy Metal ATPase), CAX (Cation/proton eXchanger), ZIF (Zinc-Induced Facilitator), and VIT (Vacuolar Iron Transporters)(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Zn homeostasis is further supported by Zn chelators such as malate, citrate, histidine, and nicotianamine (NA), which prevent undesirable Zn binding and facilitate efficient transport(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZn uptake occurs predominantly in its ionic form (Zn\u0026sup2;⁺) from the soil solution via roots(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Once absorbed, Zn is distributed through both symplastic and apoplastic pathways(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In the root\u0026rsquo;s central cylinder, transporters such as NtHMA4a/b in tobacco mediate Zn efflux into the xylem, enabling root-to-shoot translocation(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In leaves, Zn is retrieved from the xylem (apoplast) by ZIPs and NRAMPs, such as NtZIP1-like, NtZIP4, NtZIP5, NtZIP11, and NtNRAMP3 in tobacco(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Studies on Zn transporters have mainly been conducted under homogeneous growth conditions, where nutrients are evenly distributed in the medium (e.g., hydroponics). Under these conditions, it was shown that Zn transporters NtZIP4 and NtZIP5 are expressed mostly in the central cylinder in the middle part of the roots when Zn is sufficient and in the epidermis/cortex of most roots, including the root apical part, under Zn deficiency(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch on nutrient heterogeneity has largely focused on root architecture responses; for instance, Giehl (2012) demonstrated that heterogeneous Fe distribution promoted lateral root growth in Fe-rich zones to sustain shoot Fe content(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, investigations into the dynamics of micronutrient distribution, particularly under heterogeneous Zn conditions, remain limited.\u003c/p\u003e \u003cp\u003eIn this study, we grew plants in a transparent soil medium designed to mimic soil conditions(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This three-dimensional setup allowed the application of alternating nutrient concentrations to different regions. Compared to the split-root system, our approach allows for the creation of both vertical and horizontal Zn heterogeneity within a single root system. In contrast, the split-root system enables only vertical heterogeneity where the main roots are often cut to create two evenly sized root systems (from lateral roots) that grow in separate containers. The connection between roots is than provided through the hypocotyl only and root to root transport would be limited. Using transparent soil system, we demonstrated that Zn can be translocated between lateral roots, from Zn-sufficient to Zn-deficient regions. This surprising finding suggests the existence of previously unknown Zn homeostasis mechanisms in roots, likely involving an active and complex process mediated by Zn transporters. These mechanisms would necessitate the unloading of Zn from the xylem and its subsequent loading into the phloem, a process supported by Zn status sensing and signaling to ensure precise execution within specific root or shoot regions, independent of Zn concentration in the surrounding medium. Furthermore, we analyzed how different Zn distribution scenarios affected Zn content and distribution in shoots. This work provides new insights into plant Zn homeostasis, particularly regarding Zn translocation between tissues and organs, and may inform future agricultural practices to address Zn deficiency.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePlants adjust Zn homeostasis when grown in Zn heterogeneous conditions.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe were interested in identifying the Zn-related control of NtZIP4B expression (Zn importer involved in Zn uptake to cytoplasm(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)) in plants grown in a medium that mimics soil conditions, including its heterogeneous Zn distribution. For this, we tested plants with GUS expression under the \u003cem\u003eNtZIP4B\u003c/em\u003e promoter (\u003cem\u003epromNtZIP4B::GUS\u003c/em\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)) grown in transparent soil(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), where the upper or lower half of the medium had either Zn-sufficient or Zn-deficient conditions (\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e-Zn\u003c/span\u003e/+1\u0026micro;MZn and +\u0026thinsp;1\u0026micro;MZn/\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e-Zn\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As a control, we used plants grown in homogeneous Zn distribution both halves prepared with the same Zn concentration in the medium (\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e-Zn\u003c/span\u003e/\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e-Zn\u003c/span\u003e, +\u0026thinsp;1\u0026micro;MZn/+1\u0026micro;MZn).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, in the homogeneous Zn-deficient medium, \u003cem\u003eNtZIP4B\u003c/em\u003e promoter activity in roots increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) compared to the roots grown in homogeneous Zn-sufficient medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This confirms that plants grown in the transparent soil medium have a similar response to Zn deficiency as those grown in hydroponic culture. However, we noticed that when plants were grown in a heterogeneous setup of the transparent soil medium (Zn-deficient and Zn-sufficient layers in one pot), lateral roots that grew in the Zn-deficient section of the medium exhibited lower \u003cem\u003eNtZIP4B\u003c/em\u003e promoter activity, characteristic of Zn sufficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm this observation, we quantified the characteristic Zn-deficient tissue-specific profile of the \u003cem\u003eNtZIP4B\u003c/em\u003e promoter in the epidermis and cortex (profile alpha, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) compared to its limited expression localization only in the central cylinder under Zn-sufficient conditions (profile beta, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Profile alpha dominated in roots grown in homogeneous Zn-deficient conditions (~\u0026thinsp;70%), while profile beta dominated in homogeneous Zn-sufficient conditions (~\u0026thinsp;90%). Nevertheless, profile beta dominated in roots that grew in the Zn-deficient sections of the heterogeneous Zn distribution setup (both: \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e-Zn\u003c/span\u003e/+1\u0026micro;MZn and +\u0026thinsp;1\u0026micro;MZn/\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e-Zn\u003c/span\u003e). It appears that roots growing in the Zn-deficient part of the medium do not present a Zn deficiency response like plants grown in a homogeneously Zn-deficient medium. It is worth noting that, in this experiment, we ensured that the Zn concentration in the Zn-sufficient half of the heterogeneous treatment matched the concentration in the homogeneous Zn-sufficient medium (1 \u0026micro;M ZnSO₄). This means the total Zn amount (within whole pot) in the heterogeneous treatment was half that of the homogeneous Zn-sufficient treatment. Despite this, roots in the Zn-deficient portion of the heterogeneous setup exhibited Zn sufficiency-like behavior.\u003c/p\u003e \u003cp\u003eIn conclusion, these unexpected results suggest that the lack of a Zn-deficiency response in the \u003cem\u003eNtZIP4B\u003c/em\u003e promoter in roots growing in the Zn-deficient region of a heterogeneously distributed Zn medium may be due to a relatively increased Zn level in those roots. This could be explained by Zn translocation from roots in Zn-sufficient areas to those in Zn-deficient regions. This finding could provide new insights into Zn homeostasis and highlights potential mechanisms of inter-root communication and nutrient redistribution that could play an important role in plant adaptation to heterogeneous environments.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlants appear to relocate Zn from Zn-sufficient to Zn-deficient regions.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate whether Zn levels increase in roots growing in the Zn-deficient region of a heterogeneously distributed Zn medium, we analyzed differences in Zn concentration between roots grown under homogeneous Zn conditions (-Zn/-Zn, +\u0026thinsp;1\u0026micro;MZn/+1\u0026micro;MZn) and heterogeneous Zn conditions (-Zn/+1\u0026micro;MZn, +\u0026thinsp;1\u0026micro;MZn/-Zn), dividing the root system into halves for each treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As expected, in homogeneous Zn conditions, Zn concentration was significantly higher in the Zn-sufficient treatment compared to the Zn-deficient treatment, regardless of whether the upper or lower part of the root system was analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, homogeneous Zn distribution). However, in heterogeneous Zn conditions, no significant difference in Zn concentration was observed between any of the treatments (-Zn/+1\u0026micro;MZn, +\u0026thinsp;1\u0026micro;MZn/-Zn) or between the roots growing in Zn-sufficient and Zn-deficient sections of the medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, heterogeneous Zn distribution). Interestingly, all of the samples from the heterogeneous treatments exhibited higher Zn concentrations compared to samples from homogeneous Zn-deficient treatments. There was no difference in root biomass between those plants (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results indicate that plants growing in media with heterogeneous Zn distribution have increased Zn level in roots that grow in Zn-deficient medium compare to plants that grow in homogenous only Zn deficient medium. We propose that this is a result of translocation of Zn within root system from roots growing in Zn sufficient to roots growing in Zn deficient medium. We also suggest that this may be due to the transport between roots rather than redistribution from stored Zn in older roots or shoots(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), as plants were grown under given conditions with only primary roots, a few short lateral roots and fraction of the shoot at the start of the experiment (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, Zn stored in the seedling is diluted during 14 days of biomass and organ growth happening directly under treatments. The Zn level in organs of plants growing in Zn deficient homogenous medium would be a representative of the maximal potential if any Zn would be redistributed from storage gained in pre-treatment growth (or seeds).\u003c/p\u003e \u003cp\u003eAs a control, we analyzed Zn concentrations in transparent soil before and after 14 days of homogeneous Zn distribution treatment, as well as after 14 days of heterogeneous Zn treatment. As expected, Zn concentrations in the beads remained similar at the beginning and end of the experiment with homogeneous Zn distribution. This suggests that Zn-sufficient conditions remained stable for 14 days and that there was no external Zn input in Zn-deficient conditions.\u003c/p\u003e \u003cp\u003eInterestingly, after heterogeneous treatment, we observed a small but statistically significant decrease in Zn content in the Zn-sufficient region compared to the initial Zn-sufficient medium. It is important to remember that there is only half of Zn amount in medium with heterogenous Zn distribution compared to Zn-sufficient homogenous conditions. Therefore, this result could indicate increased Zn depletion due to increased Zn uptake by roots growing in the Zn-sufficient half, potentially to support Zn-deficient part. Similarly, we observed a small but statistically significant increase in Zn content in Zn-deficient regions compared to the initial Zn-deficient medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This increase might be due to Zn leakage from the Zn-sufficient part. However, we also considered other explanations, as similar results were observed for Zn-deficient beads in the upper half of the pot. Solute movement against gravity is limited to capillary spaces, which are absent in this system, and the relatively long distance over a short time scale would constrain diffusion effects(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Instead, the increase in Zn content in beads without added Zn may indirectly support our hypothesis that Zn is translocated between lateral roots, moving from those in the Zn-sufficient region to those in the Zn-deficient region. This process could contribute to additional Zn in the local environment, possibly through exudates. Further testing is needed to confirm these observations.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eZn distribution differs in roots of plants grown in Zn heterogeneous medium\u003c/h2\u003e \u003cp\u003eIf Zn is translocated from roots with access to a Zn-sufficient medium to roots growing in\u003c/p\u003e \u003cp\u003eZn-deficient conditions, it is important to determine its spatial localization to identify Zn sinks in root system. However, most analytical methods used to study elemental distribution in plant samples face limitations, including low sensitivity, sample handling challenges, and pretreatment requirements that can alter metal distribution and speciation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Synchrotron X-ray-based techniques can overcome these limitations by providing direct, highly sensitive, spatially resolved information on the distribution of the metal and its speciation within plants, while also requiring only a limited level of sample manipulation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). To show the Zn distribution patterns within root systems of plants grown in a medium with heterogeneous Zn distribution, we performed \u0026micro;XRF (micro-X-ray Fluorescence) analysis using synchrotron radiation (SOLARIS, POLYX beamline).\u003c/p\u003e \u003cp\u003eIn the following experiments we doubled Zn concentration in one section of the container in heterogenous treatment to maintain a consistent total Zn concentration throughout the treatments. For example, by having 2\u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e in only half of the container, the average concentration across the entire volume became 1\u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e, matching the control condition (1\u0026micro;MZn). We used that strategy across treatments ranging from 1 to 5 \u0026micro;M Zn. This ensures equivalence in Zn exposure across treatments and allows us to compare potential Zn localization.\u003c/p\u003e \u003cp\u003eInterestingly, across all Zn treatments (ranging from 1 to 5 \u0026micro;M Zn), we observed a consistent pattern of Zn distribution within the root system (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In plants grown in homogeneous Zn-sufficient conditions, Zn was evenly distributed between the upper and lower halves of the root system (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, in plants grown under heterogeneous Zn conditions, Zn was distributed relatively evenly (Zn0/Zn5 \u0026micro;M Zn and Zn0/Zn10 \u0026micro;M Zn have slightly increased signal in lower parts), including in roots located in the Zn-deficient part of the medium, regardless of whether the Zn-sufficient part was in the upper or lower section of the pot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c). However, an intriguing observation emerged: when Zn was localized in the upper part of the medium, its concentration in the roots appeared relatively higher compared to plants where the Zn-sufficient part was in the lower section of the pot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c). Given that the total Zn amount in these treatments was identical, this suggests that upper and lower roots may differ in their capacity for Zn uptake.\u003c/p\u003e \u003cp\u003eIn summary, \u0026micro;XRF analysis corroborates that Zn could be relocated to roots growing in Zn-deficient regions. Moreover, the data indicates that Zn uptake is facilitated when increased amounts of Zn is localized in the upper part of the medium under heterogeneous Zn conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eZn localization in the medium (upper/lower) impacts Zn levels in leaves\u003c/h3\u003e\n\u003cp\u003eGiven the observed differences in Zn levels in roots grown under heterogeneous Zn localization, we mapped Zn distribution and analyzed Zn levels in leaves (excluding the hypocotyl) using \u0026micro;XRF. From the \u0026micro;XRF analysis, we found that the highest relative Zn levels were present in plants grown in homogeneous Zn-sufficient medium, while the lowest levels were observed in plants grown in homogeneous Zn-deficient medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). The semi-quantitative comparison of \u0026micro;XRF fluorescence intensities between samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) was feasible because we used leaves with specific characteristics (2 and 3 leaves) that have similar thickness, density and were analyzed in the same \u0026micro;XRF run. These findings were further corroborated by Zn concentration analysis in whole shots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn leaves from plants grown in homogeneous Zn-sufficient medium, Zn was distributed throughout the entire leaf blade (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In contrast, in plants grown in medium with heterogeneous Zn distribution, Zn localization was primarily restricted to the leaf veins, accompanied by significantly lower Zn levels compared to plants grown in homogeneous Zn-sufficient medium, despite a similar total Zn content in the medium for both treatments. This indicates that heterogeneous Zn localization in the soil affects Zn distribution within the leaves.\u003c/p\u003e \u003cp\u003eZn concentration analysis also revealed that shoots from plants grown in medium with heterogeneous Zn distribution exhibited lower Zn concentrations compared to those grown in the same total Zn amount under homogeneous conditions. Notably, Zn levels were higher in shoots when the Zn-sufficient medium was placed in the upper part of the pot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). However, this difference in Zn distribution and concentration was not associated with significant differences in biomass, suggesting an overall lower Zn content in plants grown in medium with heterogeneously distributed Zn (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003cb\u003eControl over expression of Zn homeostasis related genes may play a role in Zn distribution changes in plants growing under heterogeneous Zn conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, we analyzed the expression of key genes involved in Zn homeostasis: \u003cem\u003eNtHMA4a/b\u003c/em\u003e, encoding a major Zn exporter responsible for Zn loading into the xylem(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e); \u003cem\u003eNtNAS\u003c/em\u003e, encoding tobacco nicotianamine (NA) synthase (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), which produces the Zn chelator NA involved in Zn transport in the phloem(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and vacuolar storage(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e); and \u003cem\u003eNtZIP4B\u003c/em\u003e, encoding a critical Zn transporter responsible for Zn uptake under Zn-deficient conditions(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, \u003cem\u003eNtHMA4a/b\u003c/em\u003e expression was significantly reduced in roots grown in homogeneous Zn-deficient medium compared to those grown in Zn-sufficient medium. In plants grown in media with heterogeneous Zn distribution, \u003cem\u003eNtHMA4a/b\u003c/em\u003e expression was similar to that observed under homogeneous Zn-deficient conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). However, the expression of \u003cem\u003eNtHMA4a/b\u003c/em\u003e varied depending on whether Zn was localized in the upper (increased expression) or lower (decreased expression) part of the medium. This pattern correlated with the Zn concentration in shoots of plants grown in heterogeneous media, suggesting a relationship between NtHMA4a/b activity, Zn content in shoot and potentially Zn distribution in the medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eNtNAS\u003c/em\u003e was similar between roots grown in homogeneous Zn-sufficient and Zn-deficient media, with a tendency for increased expression under Zn deficiency. However, in roots of plants grown in heterogeneous Zn conditions, \u003cem\u003eNtNAS\u003c/em\u003e expression was significantly lower than in plants grown in Zn-sufficient homogenous medium. This reduced expression may indicate a decreased requirement for Zn chelation in plants grown in media with heterogeneous Zn distribution.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eNtZIP4B\u003c/em\u003e, the expression pattern was consistent with the promoter-GUS studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), showing increased expression under Zn deficiency. However, in plants grown under heterogeneous Zn conditions, \u003cem\u003eNtZIP4B\u003c/em\u003e expression was significantly lower than in plants grown in homogeneous Zn-deficient media. Similarly, to \u003cem\u003eNtNAS\u003c/em\u003e, the reduced expression of \u003cem\u003eNtZIP4B\u003c/em\u003e in heterogeneous Zn conditions may reflect a mechanism by which plants coordinate Zn distribution to lateral roots. Earlier research from our group showed that \u003cem\u003eNtZIP4B\u003c/em\u003e and \u003cem\u003eNtNAS\u003c/em\u003e expression depends on Zn level in medium and increase under Zn deficiency(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). However, both \u003cem\u003eNtZIP4B\u003c/em\u003e and \u003cem\u003eNtNAS\u003c/em\u003e expression is suppressed in plants grown under heterogeneous Zn conditions compare to homogenous Zn-sufficient media (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) regardless of similar Zn presence and distribution within roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Such a result may suggest alternative regulatory mechanism for those genes that could be independent from local Zn cellular status (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and that these mechanisms could be related to observed Zn distribution changes in plants grown in Zn heterogenous medium.\u003c/p\u003e \u003cp\u003eIn summary, the expression of Zn homeostasis-related genes suggests that the observed Zn distribution phenotype within the root system of plants grown in a heterogeneous Zn medium may involve coordinated regulation of Zn translocation (HMA4), uptake (ZIP4B), and storage or phloem transport (NAS). Further studies are needed to reveal if those mechanisms are involved in the movement of Zn from Zn-sufficient roots to Zn-deficient roots.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNutrient medium heterogeneity is known to influence plant growth and nutrient allocation, with effects varying based on plant species and root system architecture, as well as patch characteristics such as size and contrast in nutrient concentration(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Plants typically optimize nutrient acquisition from heterogeneous patches by altering root growth, particularly through increased lateral root elongation in nutrient-rich zones(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). However, studies addressing impact of micronutrient heterogeneity in medium and how it impacts plant homeostasis, particularly Zn, are limited.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that a medium with heterogeneous Zn distribution, comprising alternating Zn-sufficient and Zn-deficient layers, significantly affects Zn homeostasis in plants, altering Zn distribution patterns within roots and leaves. We observed that Zn from the Zn-sufficient medium is distributed to roots growing in Zn-deficient zones. This distribution mitigates Zn deficiency responses (e.g., expression of Zn-responsive promoters) in Zn-deficient roots and limits transport of Zn to the shoot, findings not previously reported.\u003c/p\u003e \u003cp\u003eOur results suggest that Zn is transported between roots in heterogeneous Zn media. In homogeneous conditions, Zn uptake occurs across the entire root system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b and schematic representation in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b). In heterogeneous conditions, Zn-sufficient roots act as \"Zn source roots,\" while Zn-deficient roots function as \"Zn sink roots,\" receiving Zn from Zn source roots. This redistribution does not appear to inhibit root growth, unlike deficiencies of macronutrients such as nitrogen, which are known to restrict higher-order root growth(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eZn translocation between roots likely involves both apoplastic (water stream) and symplastic pathways, overcoming apoplastic barriers to load Zn into the xylem, which then transports it to the shoot(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Given the unidirectional nature of xylem flow, this implies that nutrients may be relocated between xylem and phloem at lateral root junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, d). We hypothesize that Zn redistribution may also occurs within shoot, e.g. leaf veins, as our \u0026micro;XRF results show higher Zn concentrations in veins compared to the rest of the leaf blade in plants grown in heterogeneous Zn conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Experiments with foliar application of Zn radioisotopes (Zn\u003csup\u003e65\u003c/sup\u003e) in wheat and \u003cem\u003eSedum alfredii\u003c/em\u003e showed Zn translocation to leaves above and below the treated leaf, as well as to the root tips via phloem(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The potential regulation of Zn transfer between roots could be related to a systemic signal derived from either Zn-deficient roots or shoot, similarly to proposed control over AtMTP2 and AtHMA2 expression in \u003cem\u003eA. thaliana\u003c/em\u003e(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKey Zn-transporters appear to play distinct roles in mediating Zn distribution in heterogeneous conditions. For instance, in tobacco, there are two variants of the HMA4 gene, NtHMA4a and NtHMA4b, which may facilitate Zn loading into the xylem. Their expression has been shown to be triggered by Zn toxicity but not Zn deficiency(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Interestingly, foliar Zn application reduced AtHMA2 (also loads Zn to xylem) but not AtHMA4 expression in the roots of wild-type plants grown in a Zn-deficient hydroponic solution, suggesting systemic, shoot-derived regulation of AtHMA2 expression, but not AtHMA4(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). This shows that control over Zn root-to-shoot transport is complex. In this context, it is important to notice that in our study, we used primers that detect the expression of both NtHMA4a and NtHMA4b, referred to as NtHMA4a/b. We observed that the expression of tobacco HMA4a/b was reduced in both heterogeneous Zn treatments and the homogeneous Zn-deficient treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Interestingly, previous studies from our group using homogeneous hydroponic conditions showed that Zn deficiency led to reduced NtHMA4a/b expression across the apical, middle, and basal root regions(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). This result was accompanied by a significant (5-fold) reduction in Zn levels in roots and shoots(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). However, in this study, Zn levels in roots did not show a dramatic difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and slightly (less than 1-fold) lower Zn levels in shoot of plants growing in the heterogeneous Zn treatment compared to the homogeneous Zn-sufficient condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Nevertheless, in shoots, we observed a statistically significant reduction in Zn concentration under both heterogeneous Zn conditions compared to homogeneous Zn sufficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Interestingly, under Zn heterogeneous conditions in medium, NtHMA4a/b expression was influenced by the placement of the Zn-sufficient medium part. NtHMA4a/b expression was higher when Zn was localized in the upper layer and lower when Zn was present in the lower layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). This could suggest that upper roots, often associated with nutrient acquisition(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), may have a greater capacity for Zn uptake and translocation to the shoot, eg. even just by a fact that they are closer to hypocotyl.\u003c/p\u003e \u003cp\u003eBased on these findings, we hypothesize that tobacco HMA4a/b control of expression may rely on systemic shoot control or its regulation is unrelated to Zn concentration. Further investigation whether the regulation of HMA4a/b expression plays a specific role in trafficking Zn to lateral roots is needed.\u003c/p\u003e \u003cp\u003eThe expression of NtNAS, which encodes nicotianamine synthase involved in Zn binding (storage in vacuole)(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and Zn phloem transport(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), was significantly reduced in roots grown in heterogeneous Zn media. This reduction may be need for limiting Zn storage or phloem transport in plants that relocate Zn between their roots. We also observed a reduced Zn level in the shoots of plants grown under Zn heterogeneous conditions suggesting limited Zn translocation, which would be reasonable effect of downregulation of NtNAS. This may highlight the role of control over NAS expression as regulator of how much Zn may be available for Zn distribution within root system. Finally, NtZIP4B, a Zn uptake transporter that was activated under Zn-deficient conditions was suppressed in heterogeneous Zn media, despite the presence of Zn-deficiency. In our study, we examined the expression pattern of pNtZIP4B:GUS under both Zn heterogeneous and Zn-sufficient homogeneous conditions. We observed that its expression was limited to the cortex and central cylinder (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is possible that reduced Zn transport to cell in central cylinder could impact Zn apolastic/symplastic balance which itself is known to regulate Zn homeostasis (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This would also facilitate apoplastic transport and potentially xylem loading reducing Zn retention in Zn-sufficient roots. This however do not explain how Zn would be redirected to Zn-deficient lateral roots as the same mechanisms would reduce potential for phloem loading in part of the root where Zn have to be transferred from xylem to phloem (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). This might suggest that Zn relocation to Zn-deficient parts that should involve Zn phloem transport would involve xylem-to-phloem reloading in shoot. We plant to investigate that in our future studies.\u003c/p\u003e \u003cp\u003ePrevious studies on Arabidopsis and tobacco, show that Zn transport and homeostasis are tightly regulated by collective functioning of different Zn transporters/chelators like HMA, NAS and ZIP family transporters(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The process by which Zn-sufficient roots in heterogeneous media provide Zn to Zn-deficient roots likely involves a complex Zn transport mechanism that requires coordinated regulation. This process to function efficiently is probably limited to specific part of root or shoot where xylem-phloem Zn relocation occurs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Interestingly, both \u003cem\u003eNtZIP4B\u003c/em\u003e and \u003cem\u003eNtNAS\u003c/em\u003e promoters have Zinc Deficiency Response Element (ZDRE) motif (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), regulated by bZIP19 and bZIP23 transcription factors which induce expression of these genes under Zn cellular deficiency. This mechanism would allow to alter expression of genes between Zn-sufficient and Zn-deficient parts of the root system (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). We also anticipate that some kind of signaling between different root levels, or shoot-to-root, may be involved in Zn partitioning within the root system (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). However, further studies on signaling, systemic responses and cellular mechanisms that facilitate Zn-dependent control over Zn supply are needed.\u003c/p\u003e \u003cp\u003eOur study demonstrates that plants grown in media with heterogeneous Zn distribution exhibit a unique mechanism for Zn allocation, providing the first evidence that Zn may be delivered from Zn-sufficient roots to Zn-deficient roots within the same root system. This finding highlights the potential for studying previously unknown Zn homeostasis mechanisms in roots, driven by dynamic and intricate processes likely involving Zn transporters.\u003c/p\u003e \u003cp\u003eAdditionally, our results indicate that Zn distribution within the root system limits Zn translocation to the shoot. We propose that this distribution involves the downregulation of Zn-deficiency-related genes and the movement of Zn to Zn-deficient regions, potentially mediated by xylem-phloem interactions and nicotianamine transport. Uncovering these mechanisms, which appear to operate independently of external Zn concentrations, will offer new opportunities to manipulate Zn distribution within plants to enhance nutrient use efficiency and adaptability to suboptimal growth conditions.\u003c/p\u003e \u003cp\u003eOur findings provide valuable insights into the complex regulation of Zn homeostasis and open avenues for further exploration. Future studies should focus on tissue-specific expression analysis, the role of phloem transporters, and the molecular mechanisms underlying sensing, signaling and Zn allocation in heterogeneous environments.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material, Growth Conditions, and Treatments\u003c/h2\u003e \u003cp\u003eThe experiments were conducted using tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e var. Xanthi) plants. Seeds were obtained from the stock of the Institute of Biochemistry and Biophysics PAS, Warsaw, Poland, in 2002 and have since been propagated in the greenhouse of the University of Warsaw for experimental purposes.\u003c/p\u003e \u003cp\u003ePlants were grown in a controlled environment chamber under the following conditions: 23/16\u0026deg;C day/night temperature, 40\u0026ndash;50% relative humidity, 16 h photoperiod, and photosynthetically active radiation (PAR) of 250 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; provided by fluorescent Flora tubes.\u003c/p\u003e \u003cp\u003eSeeds were surface sterilized using 8% (w/v) sodium hypochlorite for 2 minutes, rinsed thoroughly, and germinated on vertically positioned Petri dishes containing quarter-strength Knop\u0026rsquo;s medium supplemented with 2% (w/v) sucrose and 1% (w/v) agar. After three weeks of growth (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), seedlings were transferred to transparent soil medium in 7 \u0026times; 10 cm Magenta\u0026reg; boxes and grown for an additional two weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Transparent Soil\u003c/h2\u003e \u003cp\u003eTransparent soil was prepared by creating gel beads through the dropwise addition of a sodium alginate:Phytogel\u0026reg; (Sigma) mixture (1:4) into a 10 mM MgCl₂ solution, which immediately polymerized the outer layer to form spherical beads, as described previously(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). A sterile, custom-made system was used to facilitate the production of large quantities of transparent soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, SI Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe bead diameter was controlled by the size of the tip opening; beads approximately 0.5 cm in diameter were used in this study. Beads were crosslinked for 4 hours, rinsed with deionized sterile water, and then transferred to an equal volume of liquid half-strength Knop\u0026rsquo;s medium adjusted to the desired Zn concentration (1, 2, 5, or 10 \u0026micro;M ZnSO₄ or without added Zn). Beads were left in the medium overnight to allow nutrient diffusion.\u003c/p\u003e \u003cp\u003eFor experimental setups, two layers of beads (~\u0026thinsp;100 g each) were placed in Magenta\u0026reg; boxes, with layer boundaries marked on the box side to identify root sections exposed to each treatment. Residual fluid accumulating at the bottom was removed with a sterile pipette. Seedlings were positioned in holes made with a pipette tip to ensure root growth through both medium layers.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGUS Assay\u003c/h3\u003e\n\u003cp\u003ePlants expressing \u003cem\u003epromNtZIP4B::GUS\u003c/em\u003e and wild-type (WT) plants (as staining controls) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)were fixed in 90% ice-cold acetone for 25 minutes with gentle rotation. Samples were washed four times in reaction buffer (50 mM Na₂HPO₄, pH 7.0, and 0.2% Triton X-100), with the third wash performed under mild vacuum (0.04 bar).\u003c/p\u003e \u003cp\u003eSamples were then transferred to reaction buffer containing 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl β-d-glucuronic acid), infiltrated under vacuum for 15 minutes, and incubated at 37\u0026deg;C in the dark for 2.5 hours with gentle shaking. Samples were cleared in ethanol solutions of increasing concentration (50%, then 75%) before scanning the entire root system (EPSON V850 Pro).\u003c/p\u003e \u003cp\u003eTo visualize \u003cem\u003eNtZIP4B\u003c/em\u003e expression at the tissue/cellular level, stained root fragments were embedded in 3% agarose and sectioned at 50\u0026ndash;150 \u0026micro;m thickness using a vibratome (Leica VT1000S). Sections were analyzed microscopically (OPTA-TECH microscope). Differences in staining intensity were quantified using ImageJ. Based on the observations, Zn-deficient (alpha) and Zn-sufficient (beta) \u003cem\u003eNtZIP4B\u003c/em\u003e expression patterns were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). The frequency of each expression pattern was determined by analyzing scans of entire root systems. Data came from 3 independent experiments (biological) with at least 14 plants for each heterogenous treatment (excluding plants that did not grew in both layers) and at least 9 plants for each homogenous setup.\u003c/p\u003e\n\u003ch3\u003eDetermination of Metal Concentrations\u003c/h3\u003e\n\u003cp\u003eAt the end of the transparent soil experiment, upper and lower root sections were separated. Shoots were rinsed briefly with deionized water. Roots were washed sequentially with Milli-Q water, 5 mM CaCl₂ (4\u0026deg;C, 15 minutes under agitation) to remove unbound and weakly bound metals from the apoplast, and finally with water.\u003c/p\u003e \u003cp\u003eSamples were dried at 55\u0026deg;C, and dry biomass was measured. Digestion was performed using 65% HNO₃ and 39% H₂O₂ (9:1, v:v) in a closed microwave mineralizer (Milestone Ethos 900, Milestone, Bergamo, Italy)(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Zn concentrations were measured using flame atomic absorption spectrophotometry (AAS; TJA Solution Solar M, Thermo Electron Manufacturer Ltd, Cambridge, UK). Certified reference material (Virginia tobacco leaves, CTA-VTL-2) was included in each analysis. At least five biological replicates were analyzed per treatment. Data came from at least 7 plants from each treatment (that roots penetrated both layers) grown in two independent experiments.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u0026micro;XRF Analysis\u003c/h2\u003e \u003cp\u003eMicro-XRF 2D mapping experiments were carried out at the PolyX beamline(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) of the SOLARIS National Synchrotron Radiation Centre(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Monochromatic beam from SOLARIS bending magnet (1.3T) was generated with a Mo/B\u003csub\u003e4\u003c/sub\u003eC multilayer monochromator with 1.3% bandwidth. The beam was focused with an ellipsoidal monocapillary optics (Sigray, 20 nm thick Pt inner coating, 20 mm working distance) to focal spot of 5\u0026micro;m. Samples were placed on a system of translation stages to perform 2D scans. Spectra were acquired with two Hitachi Vortex EM360 silicon drift detectors (ML3.3 extreme and 25 \u0026micro;m thick Be windows, 0.5 mm thick Si sensor, active area 100mm\u003csup\u003e2\u003c/sup\u003e) coupled to XGlab Dante digital pulse processor. Detectors were placed in backscattering geometry (45 degree from the sample surface) and the incident X-ray beam was normal to the sample surface. 2D maps were acquired using mapping mode of the Dante DPP with a fast continuous horizontal and vertical point-by-point motions. In 2D maps the pixel size was 100 \u0026micro;m (one instance of 50 \u0026micro;m for Zn5/Zn5 image) with dwell time 12.5 ms per single spectrum. To create the 2D maps, count-rates in energetic region-of-interests were normalized by detector\u0026rsquo; live time and incident beam intensity measured with ionization chamber. Data from two detectors were summed up taking into account the ratio between their response to the Zn signal.\u003c/p\u003e \u003cp\u003eSamples of root systems and leaves (2nd and 3rd ) were exercised, rinsed with deionized water, and dried using paper towels. Samples were mounted in a 3D-printed polymer holder with a 5 \u0026times; 5 cm mounting area. Roots were sandwiched between 3.6 \u0026micro;m foil (Spectro-Film\u0026trade;, DuPont) and gel was added on sides to prevent desiccation during analysis. At least three biological replicates were analyzed per treatment, with each replicate including three root systems per beamtime. Leaves (3 replicates) were analyzed during single beamtime.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from samples stored at -80\u0026deg;C using the Universal RNA Purification Kit (EURx, #E3598) according to the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA), with 260/280 ratios ranging between 1.8 and 2.0. RNA integrity was verified by agarose gel electrophoresis. RNA was isolated from at least 9 plants (that roots penetrated both layers) grown in two independent experiments, roots were divided into two technical samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003ecDNA was synthesized from total RNA using the RevertAid\u0026trade; First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s protocol. RT-qPCR was conducted using the LightCycler\u0026reg; 480 System (Roche) and SYBR Green Master Mix (Roche, #0488735001).\u003c/p\u003e \u003cp\u003ePrimers (Supplementary Table\u0026nbsp;1) were designed using IDT OligoAnalyzer and OligoCalc tools. The reference gene \u003cem\u003eNtPP2A\u003c/em\u003e (protein phosphatase 2A; AJ007496) was co-amplified with the target genes to normalize expression levels. Reactions were performed in triplicate for each independent biological replicate. Relative transcript levels were calculated using the ΔCt method.\u003c/p\u003e \u003cp\u003eThe quality of qPCR results was assessed using amplification and melting curves, with non-template controls included in each assay. Primers are listed in SI.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Anna Barabasz, Nina Adamek-Siwirykow, Martyna Siwik, Małgorzata Palusińska for help especially during μXRF analysis. The authors also thank Danta Marii Antosiewicz for seeds of plant with \u003cem\u003epromNtZIP4B::GUS\u003c/em\u003e expression and helpful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Science Centre, Poland, under project no. 2023/51/B/NZ9/02518\u003c/p\u003e\n\u003cp\u003eThis publication was partially developed under the provision of the Polish Ministry and Higher Education project \"Support for research and development with the use of research infra-structure of the National Synchrotron Radiation Centre SOLARIS” under contract no 1/SOL/2021/2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no competing financial interests or personal relationships that could have influenced the work reported in this study\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOS\u0026nbsp;conceived the idea and OS, MP amd DD designed the experiments.\u003c/p\u003e\n\u003cp\u003eMP, DD, OS, performed and analyze data for GUS, qPCR, metal concentration analysis,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMP, DD, JM, OS, KS, PW, PK, TK performed and analyze data for μXRF analysis,\u003c/p\u003e\n\u003cp\u003eOS\u0026nbsp;wrote the paper with contributions from all authors.\u0026nbsp;All authors reviewed it and accepted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Oskar Siemianowski\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO. Food Security and Nutrition in the World. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. The State of Food Security and Nutrition in the World 2023: Urbanization, agrifood systems transformation and healthy diets across the rural\u0026ndash;urban continuum. Food \u0026amp; Agriculture Org; 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite PJ, Broadley MR. Physiological limits to zinc biofortification of edible crops. Front Plant Sci. 2011;2:80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndreini C, Bertini I, Rosato A. Metalloproteomes: a bioinformatic approach. Acc Chem Res. 2009;42(10):1471\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroadley MR, White PJ, Hammond JP, Zelko I, Lux A. Zinc in plants. New Phytol. 2007;173(4):677\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlloway BJ. Zinc in soils and crop nutrition. 2008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexandratos N, Bruinsma J. World agriculture towards 2030/2050: the 2012 revision. 2012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLima JE, Kojima S, Takahashi H, von Wir\u0026eacute;n N. Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1; 3-dependent manner. Plant Cell. 2010;22(11):3621\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarabasz A, Palusińska M, Papierniak A, Kendziorek M, Kozak K, Williams LE, et al. Functional analysis of NtZIP4B and Zn status-dependent expression pattern of tobacco ZIP genes. Front Plant Sci. 2019;9:1984.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinclair SA, Kr\u0026auml;mer U. The zinc homeostasis network of land plants. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2012;1823(9):1553\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanton C, Sanders D, Kr\u0026auml;mer U, Podar D. Zinc in plants: Integrating homeostasis and biofortification. Mol Plant. 2022;15(1):65\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmgren MG, Clemens S, Williams LE, Kr\u0026auml;mer U, Borg S, Schj\u0026oslash;rring JK, et al. Zinc biofortification of cereals: problems and solutions. Trends Plant Sci. 2008;13(9):464\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicachenevsky FK, Menguer PK, Sperotto RA, Fett JP. Got to hide your Zn away: molecular control of Zn accumulation and biotechnological applications. Plant Sci. 2015;236:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKisko M, Bouain N, Rouached A, Choudhary SP, Rouached H. Molecular mechanisms of phosphate and zinc signalling crosstalk in plants: Phosphate and zinc loading into root xylem in Arabidopsis. Environ Exp Bot. 2015;114:57\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaldelas C, Weiss DJ. Zinc homeostasis and isotopic fractionation in plants: a review. Plant Soil. 2017;411:17\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiedschulte V, Laparra H, Battey JND, Schwaar JD, Broye H, Mark R, et al. Impairing both HMA4 homeologs is required for cadmium reduction in tobacco. Plant Cell Environ. 2017;40(3):364\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHermand V, Julio E, Dorlhac de Borne F, Punshon T, Ricachenevsky FK, Bellec A, et al. Inactivation of two newly identified tobacco heavy metal ATPases leads to reduced Zn and Cd accumulation in shoots and reduced pollen germination. Metallomics. 2014;6(8):1427\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozak K, Antosiewicz DM. Tobacco as an efficient metal accumulator. Biometals. 2023;36(2):351\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalusińska M, Barabasz A, Kozak K, Papierniak A, Maślińska K, Antosiewicz DM. Zn/Cd status-dependent accumulation of Zn and Cd in root parts in tobacco is accompanied by specific expression of ZIP genes. BMC Plant Biol. 2020;20:1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiehl RF, Lima JE, von Wir\u0026eacute;n N. Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution. Plant Cell. 2012;24(1):33\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa L, Shi Y, Siemianowski O, Yuan B, Egner TK, Mirnezami SV et al. Hydrogel-based transparent soils for root phenotyping in vivo. Proceedings of the National Academy of Sciences. 2019;116(22):11063-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarschner H. Marschner's mineral nutrition of higher plants. Academic; 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLind KR, Siemianowski O, Yuan B, Sizmur T, VanEvery H, Banerjee S et al. Evidence for root adaptation to a spatially discontinuous water availability in the absence of external water potential gradients. Proceedings of the National Academy of Sciences. 2021;118(1):e2012892118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemianowski O, Lind KR, Tian X, Cain M, Xu S, Ganapathysubramanian B, et al. HOMEs for plants and microbes\u0026ndash;A phenotyping approach with quantitative control of signaling between organisms and their individual environments. Lab Chip. 2018;18(4):620\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemianowski O, Rongpipi S, Del Mundo JT, Freychet G, Zhernenkov M, Gomez ED, et al. Flexible Pectin Nanopatterning Drives Cell Wall Organization in Plants. JACS Au. 2024;4(1):177\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRongpipi S, Barnes WJ, Siemianowski O, Del Mundo JT, Wang C, Freychet G et al. Measuring calcium content in plants using NEXAFS spectroscopy. Front Plant Sci. 2023;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerzano R, Al Chami Z, Vekemans B, Janssens K, Miano T, Ruggiero P. Zinc distribution and speciation within rocket plants (Eruca vesicaria L. Cavalieri) grown on a polluted soil amended with compost as determined by XRF microtomography and micro-XANES. J Agric Food Chem. 2008;56(9):3222\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Der Ent A, Przybyłowicz WJ, de Jonge MD, Harris HH, Ryan CG, Tylko G, et al. X-ray elemental mapping techniques for elucidating the ecophysiology of hyperaccumulator plants. New Phytol. 2018;218(2):432\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemianowski O, Barabasz A, Kendziorek M, Ruszczyńska A, Bulska E, Williams LE, et al. HMA4 expression in tobacco reduces Cd accumulation due to the induction of the apoplastic barrier. J Exp Bot. 2014;65(4):1125\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemianowski O, Mills RF, Williams LE, Antosiewicz DM. Expression of the P1B-type ATPase AtHMA4 in tobacco modifies Zn and Cd root to shoot partitioning and metal tolerance a. Plant Biotechnol J. 2011;9(1):64\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClemens S, Deinlein U, Ahmadi H, H\u0026ouml;reth S, Uraguchi S. Nicotianamine is a major player in plant Zn homeostasis. Biometals. 2013;26:623\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, Ye R, Qu M, Wang Y, Liang T, Lin J, et al. Combined transcriptome and proteome analysis revealed the molecular regulation mechanisms of zinc homeostasis and antioxidant machinery in tobacco in response to different zinc supplies. Plant Physiol Biochem. 2023;202:107919.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarabasz A, Klimecka M, Kendziorek M, Weremczuk A, Ruszczyńska A, Bulska E et al. The ratio of Zn to Cd supply as a determinant of metal-homeostasis gene expression in tobacco and its modulation by overexpressing the metal exporter AtHMA4. J Exp Bot. 2016:erw389.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, Kr\u0026auml;mer U. Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis. Plant Cell. 2012;24(2):724\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalusińska M, Barabasz A, Kozak K, Papierniak A, Maślińska K, Antosiewicz DM. Zn/Cd status-dependent accumulation of Zn and Cd in root parts in tobacco is accompanied by specific expression of ZIP genes. BMC Plant Biol. 2020;20(1):37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026auml;mer U. Metal Homeostasis in Land Plants: A Perpetual Balancing Act Beyond the Fulfilment of Metalloproteome Cofactor Demands. Annu Rev Plant Biol. 2024;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang P, Mou P, Hu L, Hu S. Effects of nutrient heterogeneity on root foraging and plant growth at the individual and community level. J Exp Bot. 2022;73(22):7503\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiehl RF, Lima JE, von Wir\u0026eacute;n N. Regulatory components involved in altering lateral root development in response to localized iron: evidence for natural genetic variation. Plant Signal Behav. 2012;7(7):711\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang G, Xue S, Liu F, Liu G. Nitrogen addition increases the production and turnover of the lower-order roots but not of the higher-order roots of Bothriochloa ischaemum. Plant Soil. 2017;415:423\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScharwies JD, Dinneny JR. Water transport, perception, and response in plants. J Plant Res. 2019;132(3):311\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu L, Tian S, Zhang J, Yang X, Labavitch JM, Webb SM, et al. Efficient xylem transport and phloem remobilization of Z n in the hyperaccumulator plant species S edum alfredii. New Phytol. 2013;198(3):721\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaslett BS, Reid RJ, Rengel Z. Zinc mobility in wheat: uptake and distribution of zinc applied to leaves or roots. Ann Botany. 2001;87(3):379\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinclair SA, Senger T, Talke IN, Cobbett CS, Haydon MJ, Kr\u0026auml;mer U. Systemic upregulation of MTP2-and HMA2-mediated Zn partitioning to the shoot supplements local Zn deficiency responses. Plant Cell. 2018;30(10):2463\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaślińska-Gromadka K, Barabasz A, Palusińska M, Kozak K, Antosiewicz DM. Suppression of NtZIP4A/B changes Zn and Cd root-to-shoot translocation in a Zn/Cd status-dependent manner. Int J Mol Sci. 2021;22(10):5355.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiehl RF, von Wir\u0026eacute;n N. Root nutrient foraging. Plant Physiol. 2014;166(2):509\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeregin IV, Kozhevnikova AD. Nicotianamine: a key player in metal homeostasis and hyperaccumulation in plants. Int J Mol Sci. 2023;24(13):10822.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiemianowski O, Barabasz A, Weremczuk A, RUSZCZYŃSKA A, Bulska E, Williams LE, et al. Development of Z n-related necrosis in tobacco is enhanced by expressing AtHMA4 and depends on the apoplastic Z n levels. Plant Cell Environ. 2013;36(6):1093\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeremczuk A, Papierniak A, Kozak K, Willats WG, Antosiewicz DM. Contribution of NtZIP1-like, NtZIP11 and a WAK-pectin based mechanism to the formation of Zn-related lesions in tobacco leaves. Environ Exp Bot. 2020:104074.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNouet C, Charlier J-B, Carnol M, Bosman B, Farnir F, Motte P, et al. Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri. J Exp Bot. 2015;66(19):5783\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozak K, Papierniak A, Barabasz A, Kendziorek M, Palusińska M, Williams LE, et al. NtZIP11, a new Zn transporter specifically upregulated in tobacco leaves by toxic Zn level. Environ Exp Bot. 2019;157:69\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntosiewicz DM, Barabasz A, Siemianowski O. Phenotypic and molecular consequences of overexpression of metal-homeostasis genes. Front Plant Sci. 2014;5:80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAssun\u0026ccedil;\u0026atilde;o AG, Herrero E, Lin Y-F, Huettel B, Talukdar S, Smaczniak C et al. Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency. Proceedings of the National Academy of Sciences. 2010;107(22):10296-301.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzlachetko J, Szade J, Beyer E, Błachucki W, Ciochoń P, Dumas P, et al. SOLARIS national synchrotron radiation centre in Krakow, Poland. Eur Phys J Plus. 2023;138(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSowa K, Wr\u0026oacute;bel P, Kołodziej T, Błachucki W, Kosiorowski F, Zając M, et al. PolyX beamline at SOLARIS\u0026mdash;Concept and first white beam commissioning results. Nucl Instrum Methods Phys Res Sect B. 2023;538:131\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"zinc transport, soil heterogeneity, microelements, xylem, phloem, micro-XRF","lastPublishedDoi":"10.21203/rs.3.rs-6802346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6802346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZinc is essential for plant development and human health. While the effects of soil nutrient heterogeneity on plant growth were studied for macronutrients, adaptive mechanisms for micronutrients like Zn remain largely unexplored.\u003c/p\u003e \u003cp\u003eWe investigate Zn homeostasis in plants grown in a transparent soil medium mimicking natural soil conditions with spatially heterogeneous Zn availability. Our findings suggests that Zn is translocated between lateral roots, moving from Zn-sufficient to Zn-deficient ones, mitigating Zn deficiency responses and reducing Zn uptake. Under heterogeneous Zn conditions, the expression of key Zn homeostasis-related genes (Zn importer - NtZIP4B, Zn exporter - NtHMA4a/b and Zn chelator \u0026ndash; NtNAS) was significantly altered. NtHMA4a/b expression was influenced by the vertical positioning of the Zn-sufficient medium, while NtZIP4B and NtNAS showed suppressed expression in roots under heterogeneous conditions compared to homogeneous Zn-sufficient conditions. This suggests a systemic regulatory mechanism coordinating Zn allocation depending on whole root system Zn access. Elemental analysis revealed reduced overall Zn concentrations in plants grown in heterogeneous Zn media, with elevated Zn levels in leaf veins.\u003c/p\u003e \u003cp\u003eThis study uncovers a novel mechanism of Zn translocation within the root system in response to heterogeneous Zn supply, highlighting the complexity of micronutrient homeostasis and its adaptive regulation.\u003c/p\u003e","manuscriptTitle":"Zinc Translocation from Zn-Sufficient to Zn-Deficient Roots as an Adaptation to Heterogeneous Zn Availability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-13 05:29:39","doi":"10.21203/rs.3.rs-6802346/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-16T13:17:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T13:20:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T07:03:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-07T14:25:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266163301998611676686007101438770303410","date":"2025-06-26T02:45:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251023983668461890974009009258046448685","date":"2025-06-25T14:04:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152162409928553763641758829073168031099","date":"2025-06-12T14:18:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-11T08:15:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-10T12:45:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-09T12:59:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T12:56:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-06-02T12:42:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90cfb677-b917-4278-a4d8-c642ba9ca6de","owner":[],"postedDate":"June 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T16:01:16+00:00","versionOfRecord":{"articleIdentity":"rs-6802346","link":"https://doi.org/10.1186/s12870-025-07391-z","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-10-08 15:57:48","publishedOnDateReadable":"October 8th, 2025"},"versionCreatedAt":"2025-06-13 05:29:39","video":"","vorDoi":"10.1186/s12870-025-07391-z","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07391-z","workflowStages":[]},"version":"v1","identity":"rs-6802346","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6802346","identity":"rs-6802346","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}