What is the representation of racial and ethnic minority populations in studies incorporating precision oncology objectives in the US?This cross-sectional analysis evaluates breast, prostate, lung, and colorectal cancer studies in the Clinicaltrials.gov registry with precision medicine objectives and reporting race and ethnicity—a total of 93 studies with 5867 total enrollees. An underrepresentation of minority racial groups and an overrepresentation of non-Hispanic White participants relative to their incidence in the US cancer population was found in precision oncology studies.These findings demonstrate an urgent need to increase enrollment of participants from diverse racial and ethnic backgrounds onto precision oncology studies, so that meaningful precision data can be collected and stratified to traditionally underrepresented participants. This cross-sectional study evaluates the representation of racial and ethnic minority groups in breast, prostate, lung, and colorectal cancer studies with a focus on precision medicine objectives. Precision oncology is revolutionizing cancer care, allowing for personalized treatments to improve outcomes. Cancer research has benefitted from well-designed studies incorporating precision medicine objectives, but it is unclear if these studies are representative of the diverse cancer population.To evaluate racial and ethnic representation in breast, prostate, lung, and colorectal cancer studies incorporating precision oncology objectives in the Clinicaltrials.gov registry and compare with the incidence of these cancer types in racial and ethnic minority groups in the US population.This cross-sectional study identified US-based breast, prostate, lung, and colorectal cancer studies incorporating precision oncology objectives for reporting of race and ethnicity. The Surveillance, Epidemiology, and End Results and US Census databases were used to determine cancer incidence by race and ethnicity, linked with cancer type and median year of enrollment for each trial. Data were collected and analyzed between December 2020 and April 2021.The expected number of participants per study by each racial and ethnic group was calculated based on the corresponding US-based proportion. Under- and overrepresentation was defined as the ratio of the actual number of enrolled cases to the expected number of cases for each trial by cancer type. Ratios above 1 indicated overrepresentation while a ratio below 1 indicated underrepresentation. Random-effects meta-analysis of representation ratios of individual trials was performed to weigh each individual study.Of 93 studies encompassing 5867 enrollees with race and ethnicity data; 4826 participants (82.3%) were non-Hispanic White, 587 (10.0%) were Black, and 238 (4.1%) were Asian. Per observed-to-expected ratios, White participants were overrepresented in all studies, with a ratio of 1.35 , as well as Asian participants, with a ratio of 1.46 , while Black participants , Hispanic participants , and American Indian and Alaskan Native participants  were underrepresented. By individual cancer site, White participants were consistently overrepresented in all studies, while Black and Hispanic participants were underrepresented.This analysis found that precision oncology studies for breast, lung, prostate, and colorectal cancers vastly underrepresent racial and ethnic minority populations relative to their cancer incidence in the US population. It is imperative to increase diversity among enrollees so that all individuals may benefit from cancer research breakthroughs and personalized treatments. Additionally, knowledge learned from studies that are designed to collect translational data can be used as part of discovery for predictive and prognostic biomarkers, which then can be applied to personalized therapeutic regimens.Precision medicine has revolutionized oncology in the past 2 decades and is expected to continue to transform cancer management. The principle of personalized cancer therapy incorporates a multi-omic  approach along with other tumor- and patient-specific factors. The identification of biomarkers facilitates stratifying patients and guiding treatment individualization, leading to improved outcomes.2 as well as Native American ancestry.3 Separately, growing evidence suggests underlying tumor genomic differences between African American vs White men with prostate cancer.5 This information can lead to additional study and discovery as well as novel treatment options for those who harbor the specific tumor alteration or biomarker. However, the use of race and ethnicity in such analyses has multiple limitations. Race and ethnicity must be understood as social constructs. We also recognize that the traditionally broad racial and ethnic categories do not capture the heterogeneity that exists within each group.6 Consequently, even the aforementioned findings should be further explained and contextualized in terms of other social determinants of health, such as health insurance status, zip code, socioeconomic status, environment, and other characteristics. The intersection of social, environmental, and genomic and/or biologic factors and the resultant influence on disease is poorly understood, with calls to action to capture these data points more robustly in all future precision medicine studies to evaluate and understand these interactions.8Several targetable biomarkers have been found to have racial and ethnic differences in incidence for different cancer types such as epidermal growth factor receptor variants in lung cancer associating with East Asian ethnicity10 However, genetic ancestry data are not yet readily available for clinical practice or for use in clinical studies, nor have that data been historically captured.11 The historical static groupings of individuals by race and ethnicity with regards to reported health data over time have led to the acceptance and use of these categories in biomedical research.7 Stratification by various racial and ethnic groupings has identified several important differences in disease risk as well as response to certain treatments.11 In addition, the historical use of race and ethnicity as currently defined to differentiate patients in the study of disease has led to the emergence and recognition of medically underserved and underrepresented minority populations.12 Thus, although imperfect, understanding disparities by racial and ethnic groupings is of value, particularly pertaining to the study of precision medicine, as it is important to understand from which populations we are collecting our biologic and biomarker data for use in the general cancer population.The use of genetic ancestry has been lauded as a tool that can better differentiate populations in the study of biology and genomics.14 enrollment of a diverse patient population into cancer clinical trials lags behind, including for racial and ethnic minority groups.17 As more clinical studies specifically incorporate precision oncology principles into their design, it is unknown whether ethnic and racial minority populations are adequately represented, an important consideration when one critical question is whether precision oncology discoveries in clinical trials are broadly applicable to the general cancer population. Is precision oncology research missing crucial findings that could be benefiting traditionally marginalized groups, thus further widening inequality and contributing to disparities in cancer care? Moreover, any identifiable differences between groups in a single or across a collection of studies must be further evaluated, particularly if personalization of a therapeutic is identified for 1 group vs another, as sample sizes would be too small to gather appropriate safety data. Both prespecified analyses in postapproval settings as well as the use of real-world study would be necessary to interrogate safety and efficacy data.18 However, the opportunity to look for differences between various racial and ethnic groupings requires a concerted effort towards recruitment of underserved populations to these studies in their initial stages. Therefore, we sought to understand how well precision oncology studies are representative of the diverse US cancer population. Focusing on the top causes of new cancer cases in the US ,19 we evaluated clinical studies with precision medicine objectives for their reporting of race and ethnicity, and asked whether these study demographics were representative of the diverse US cancer population.Despite rapid diversification of the US cancer population,immunohistochemistry, histopathologic, DNA, RNA, DNA sequencing, RNA sequencing, sequencing, proteomics, tumor mutational burden, tumor biomarkers, biomarkers, tumor analysis, mutation testing, mutational analysis, microarray, whole exome, molecular analysis, genomics, genetics, gene expression, expression, signatures, genetic testing, and prognostic testing. Studies were excluded if they included site locations outside the US, did not include diagnosis of cancer , or did not incorporate precision oncology measures. Eligible studies were evaluated for reporting of racial and ethnic demographic data. Additional data gathered included type of clinical study  and type of funding . Participant demographics were obtained from Clinicaltrials.gov if present and corroborated with primary report journal articles or abstract and/or presentation if unpublished (per availability). Racial and ethnic groupings were mutually exclusive as follows: American Indian/Alaskan Native, Asian, Black, Hispanic, and Non-Hispanic White participants. Participants with race and ethnicity not reported or unknown were removed from the analysis. Overall, these individuals made up less than 3% of the total enrollees per each cancer type . Clinicaltrials.gov was queried in December 2020 with an update of the query and analysis in April 2021.In this cross-sectional study, the Clinicaltrials.gov registry was queried for completed US-based clinical studies by cancer type  with results incorporating precision medicine objectives based on a set of precision oncology search terms: 20 Age-standardized incidence rates adjusted to the 2000 standard US population by race and ethnicity were used to calculate cancer cases as a measure of proportion of cancer burden by race and ethnicity using US Census Bureau tables.22 This study was determined to be exempt from human participant research guidelines because it was a secondary analysis of publicly available published reports and data by the Mass General Brigham institutional review board. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional studies and the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline.Cancer incidence by race and ethnicity within the US cancer population, correlated with cancer type and median year of enrollment for each study, was collected from the National Cancer Institute Surveillance, Epidemiology, and End Result (SEER) database.For each trial, the expected number of participants of each of the 5 racial and ethnic groups was calculated by multiplying the total number of participants in a trial with the US-based proportion of each group for a given cancer type and median year of enrollment. We defined underrepresentation and overrepresentation as the ratio of the actual number of enrolled cases and the expected number of cases for each trial and per cancer type, with 95% exact binomial CIs estimated. A ratio greater than 1 indicated overrepresentation, while a ratio smaller than 1 indicated underrepresentation. Since the expected number of participants for groups other than non-Hispanic White is small, especially for smaller trials, we calculated totals of expected to enrolled ratios in all precision studies and per cancer type.2 and 2-sample Wilcoxon rank-sum tests were performed, respectively; P ≤ .05 was considered significant in 2-sided tests.Separately, we also performed random-effects meta-analysis of underrepresentation and overrepresentation ratios of individual trials , with the exclusion of trials with no enrolled participants of a given racial or ethnic group. Therefore, the weighted average of underrepresentation and overrepresentation effect sizes from the meta-analysis might be biased for minority populations with small numbers of enrolled participants . Results are presented using forest plots , followed by Black participants (587 [10.0%]), Asian participants (238 [4.1%]), Hispanic participants (200 [3.4%]), with the lowest representation of American Indian/Alaskan Native participants (16 [0.3%]). By cancer type, lung cancer studies had the highest proportion of White (2054 of 2399 [85.6%]) and Asian (116 [4.8%]) participants, and the lowest proportion of Black (199 [8.3%]), Hispanic (26 [1.1%]), and American Indian/Alaskan Native (4 [0.2%]) participants. Colorectal cancer studies had the highest proportion of Hispanic participants (59 of 999 [5.9%]). Prostate cancer studies had the highest proportion of Black participants (67 of 596 [11.2%]) and American Indian/Alaskan Native participants (5 of 596 [0.8%]), and the lowest proportion of Asian participants (6 of 596 [1.0%]). When evaluating racial and ethnic distribution by funding type, Asian individuals were statistically overrepresented on studies with NIH funding.When evaluating observed-to-expected ratios by race and ethnicity in all precision oncology studies, White and Asian participants were overrepresented with a ratio of 1.35  and 1.46 , respectively . The othIn the meta-analysis, which weighs individual studies, White participants remained overrepresented overall with a ratio of 1.34  and by cancer type . Overrep15 There is an urgent need to increase equitable recruitment of diverse participants to cancer clinical trials, as well as to understand racial and ethnic differences in treatment outcomes. However, the field of oncology has been moving toward personalized treatments based on presence of tumor molecular markers as well as genetic and genomic variation. Despite this, there is limited knowledge of underlying cancer biology in racial and ethnic minority groups. Current personalized treatments that are broadly generalized to all individuals based on the presence or absence of a biomarker without fully studying the implications of such treatment in racial and ethnic minority populations may not be appropriate. Even if biological differences are discovered that are unique to certain racial or ethnic minority subgroups, these must be further validated and evaluated for safety and efficacy. Similarly, large biorepositories from which to study precision omics are disproportionately dominated by individuals of European ancestry.24 This is a well-documented issue with genomewide association studies,10 particularly for cancer research, with European-ancestry groups making up 96% of participants, while 0.11%, 0%, and 0.5% of subjects are from African, African American and Afro-Caribbean, and Hispanic and Latin American groups in studies at the initial discovery phase in 2019,25 prompting the call for more inclusive samples and inclusive research communities. Thus, it is insufficient to take our current understanding of tumor biomarkers and biology and generalize findings to the entire diverse cancer population. Although race and ethnicity have been used historically to stratify populations, to fully understand any biological findings that differ by these social constructs, it is critical that future studies prospectively capture additional data that accounts for social determinants that influence disease, as well as data incorporating the use of genetic ancestry.11The underrepresentation of racial and ethnic minority populations in clinical trials relative to their cancer burden in the US has been previously reported.29 Yet, data now demonstrate differential biology (as it relates to somatic driver mutations) among Hispanic participants with lung adenocarcinoma, underscoring the importance of their increased representation on precision oncology lung trials, as well as capturing of social determinants of disease to analyze these associations further. Separately, Hispanic individuals also had poor representation in prostate cancer studies (by 67%). Overall, our findings data highlight the need to increase enrollment of these groups into future clinical studies. All individuals deserve to benefit from cancer research breakthroughs and deep understanding of the underlying tumor biology not only in the context of race and ethnicity, but in the context of social determinants of health as well.As contemporary clinical studies specifically incorporate precision oncology principles, it is unknown whether racial and ethnic minority populations are adequately represented overall and by cancer type relative to their cancer incidence in the US cancer population. By focusing on the most common cancer types in the US , we found that most precision oncology studies do not report the race and ethnicity of participants. Of those studies with racial and ethnic reporting, we demonstrated an underrepresentation of minority racial groups and an overrepresentation of Non-Hispanic White participants in precision oncology studies. Black and Hispanic participants were the most underrepresented on lung cancer studies by 68% and 69% per meta-analysis, respectively. On the other hand, Asian individuals were overrepresented in lung cancer studies by 196%, consistent with the prominent role of targetable oncogene drivers in this population. There is likely underlying genetic susceptibility for these alterations among the Asian population in addition to the epidemiologic association with nonsmoking status.18 There are multiple factors at play, including medical mistrust.30 Prior work has found that individuals from underrepresented racial and ethnic groups’ willingness to participate in research stems from perceived trustworthiness of the researchers, the institutions conducting the research, and the information provided about the applicable research studies.31 There may be more mistrust surrounding genetic and biological studies—for example, genetic counseling participation is low among Black women for BRCA1/2 testing.32 This finding was exacerbated among women with higher rates of medical mistrust,32 fear of discrimination by insurance companies if considered high-risk,33 and fear of carrying the mutation.34 A targeted study evaluating African American and Hispanic perspectives on the prospect of precision medicine found that, although groups believed that precision medicine can improve health outcomes, both groups were concerned that current barriers to health care would prevent their communities from benefiting from precision medicine.35There is a long history surrounding underrepresentation of medically underserved participants on trials.36 Finally, precision oncology studies should increase the reporting of race and ethnicity of participants so that improvements in diversity and representation can be observed over time, and this analysis can serve as a benchmark with which to compare future progress.Multiple strategies must be implemented to increase diverse enrollment onto precision oncology trials, including patient navigation programs, patient education about multi-omic studies, patient education about genetics and genetic counseling, unique trial designs, education for all researchers or members involved to combat bias, and other educational resources to address barriers to enrollment for trial participation. More research is needed to fully understand all barriers and fears surrounding research recruitment. An increase in racial and ethnic diversity among scientists, biomedical researchers, physicians, and clinical trialists can help to increase trust among patients from minoritized groups.14 This highlights the opportunity for future studies or trials to capture these critical nuances in the future, along with demographic, social, environmental, and other determinants of health, to create fully annotated databases to enhance the study of biology and multi-omic research with a broader lens to understand the interaction of these multiple factors.Limitations to this analysis include those associated with use of Clinicaltrials.gov. We queried studies with reported results, which could miss noncompliant and incompletely accrued studies. Our search criteria could have missed slow-accruing precision oncology studies, or those that remained open after reaching their primary end point. We may be missing crucial precision oncology studies that were not identified with the search terms used, as well as studies that did not prespecify these analyses and were instead performed post hoc. Second, no information about race and ethnicity assessment was provided—it is important to emphasize that the racial and ethnic categories as reported do not capture the cultural heterogeneity within these groups. Third, there is no information on multiracial or multiethnic patients, which fails to capture patients who might identify as more than 1 racial or ethnic group. We recognize that this is imperfect, particularly given that in the latest 2020 US Census, over 33.8 million Americans identified themselves as being multiracial, resulting in an increase of nearly 276% since the 2010 census.Increased emphasis on equitable recruitment and enrollment for precision oncology studies is essential, as resulting discoveries are used to personalize treatments, and it is unclear whether current precision medicine breakthroughs can be broadly applicable to, or safe for, our diverse cancer population. We demonstrate that precision oncology studies for breast, lung, prostate, and colorectal cancers underrepresent racial and ethnic minority populations relative to their cancer incidence in the US population. All relevant stakeholders should implement strategies to increase diverse clinical study enrollment to address this disparity. A continued lack of diversity among trial enrollees may further leave behind undeserved minority populations in the era of precision oncology.


Micronutrient malnutrition is a global health issue and needs immediate attention. Over two billion people across the globe suffer from micronutrient malnutrition. The widespread zinc (Zn) deficiency in soils, poor zinc intake by humans in their diet, low bioavailability, and health consequences has led the research community to think of an economic as well as sustainable strategy for the alleviation of zinc deficiency. Strategies like fortification and diet supplements, though effective, are not economical and most people in low-income countries cannot afford them, and they are the most vulnerable to Zn deficiency. In this regard, the biofortification of staple food crops with Zn has been considered a useful strategy. An agronomic biofortification approach that uses crop fertilization with Zn-based fertilizers at the appropriate time to ensure grain Zn enrichment has been found to be cost-effective, easy to practice, and efficient. Genetic biofortification, though time-consuming, is also highly effective. Moreover, a Zn-rich genotype once developed can also be used for many years without any recurring cost. Hence, both agronomic and genetic biofortification can be a very useful tool in alleviating Zn deficiency. Food and nutritional security are key to human health. Food insecurity, imbalanced diet, consumption of food grains with poor nutritional quality, lack of dietary diversity, etc. negatively affect human health ,2. In faLow-income countries largely trust staple foods while relying less on fruits, vegetables, and foods from animal sources compared to high-income countries. Micronutrient malnutrition, due to a lack of sufficient micronutrients in the diet results in serious but often invisible health consequences, referred to as hidden hunger. In the world, over 2 billion people are affected by micronutrient deficiency ,7. This Amongst different micronutrients, Zn inadequacy is common in both plants and animals ,16. Zn dFrom the discussion above, it is very clear that a healthy diet can not only improve human health and maximize human resource potential. It can also reduce the costs related to health. We need suitable intervention like biofortification to make nutritionally superior food available to every person in the world at an affordable cost. In this direction, biofortification of staple food grains like rice, wheat, and maize with zinc and their inclusion in the human diet can help in alleviating zinc malnutrition.Zn is a very crucial nutrient for maintaining optimal human health. The essentiality of Zn for human was established in the year 1961 . Around Though the mechanism of Zn deficiency-induced impairment of growth and development is not clearly understood, it is one of the most studied effects of Zn deficiency. The effect of Zn deficiency is more significant especially in the time of rapid growth like infancy, puberty, and pregnancy . Intake Considering the importance of Zn in human health , there iZn is an essential plant nutrient and known as a micronutrient because of its low requirement. Zn remains in plants in either free ionic form or as a complex with many low molecular weight compounds . Though Zn is a constituent of many important enzymes of great significance such as carbonic anhydrase, alcohol dehydrogenase, and superoxide dismutase ,49. CarbZn is also an integral part of the Zn finger family of transcription factors that controls cell proliferation and differentiation . Zn playThe possible role of Zn in water uptake and transport in plants and a short-term tolerance to heat and stress tolerance has been reported ,57,58,59Zn deficiency in crops limits crop yield and grain nutritional quality ,65. CereThe biofortification approach aims at enriching the grains with minerals like iron, Zn, selenium, iodine, etc., so that, their intake can be improved among people consuming those grains ,9,14. AsUnderstanding the physiological basis of micronutrient accumulation in the grains is of utmost importance for successful biofortification. Micronutrient concentration in the edible portion of the crop is decided by nutrient availability to plants and the process of absorption, translocation, and redistribution of micronutrients in the plant, which are being regulated by homeostatic mechanisms that allow accumulation of micronutrients in an adequate amount, yet at a non-toxic level ,16. The + from root cells and the release of metal complexing compounds and reductants can also be very useful root characteristics for increasing Zn uptake [The crop also plays a very important role in the absorption of micronutrients. For enhancing Zn uptake, the micronutrient level in the root–soil interface should be increased. Changing root morphology for increasing the absorptive surface area can enhance Zn uptake. Efflux of Hn uptake . Understn uptake . The micn uptake ,11. The When the soil is inherently deficient in Zn content and/or the solubility of Zn in soils is low, then the plant cannot absorb sufficient Zn to meet its physiological or metabolic need. In fact, nearly 50% of cereal growing areas in the world have been found deficient in Zn . This leAgronomic biofortification is cheap and provides the dual advantage of yield enhancement and improvement of grain Zn concentration. Though genetic biofortification also shows a lot of promise, developing a variety takes a lot of time and effort . MoreoveZn is provided to the crops by soil application, foliar application, seed application (priming), or by a combination of these methods ,70,71,723 induced rise in pH, direct sorption of Zn to the precipitated CaCO3, and formation of insoluble calcium Znate [The efficiency of soil-applied Zn fertilizer depends on soil pH. The availability of Zn is relatively higher at acidic soil pH. The solubility of soil decreases hundred-fold with each unit increase in pH . Liming um Znate . Alkalinum Znate ,74. Otheum Znate ,75,76.Arbuscular mycorrhiza in soil also plays an important role in mobilizing Zn to plants [As Zn reaches the plant root predominantly through diffusion , therefoo plants . The effo plants ,18. The The efficiency of foliar Zn application greatly depends on the type of fertilizer, crop characteristics, especially, leaf characteristics and genetic potential of the crop . The folA poor correlation between DTPA-Zn concentration of soil and grain Zn concentration has been reported . This law/v) ZnSO4. 7H2O at the heading and milk stage of wheat resulted in a significant increase in grain Zn concentration across locations in seven different countries over 2 years of an experiment. The result suggested an average increase in Zn concentration by 83.5%, while soil Zn application showed an average increase of 12.3% over no Zn application [The time of foliar application is very critical for the effectiveness of foliar-applied Zn ,92. Highlication . Foliar lication  and suchlication ,95.High seed Zn content has been found to improve seedling vigour and crop stand in the field . When plSeed priming of wheat with Zn significantly increased grain Zn concentration by 12%, while the concentration of chickpea and maize improved by 29% and 19%, respectively . Seed prDifferent combinations of application methods (soil + foliar and seed + foliar) have also been studied to show their effectiveness in improving grain zinc concentration ,70,98. CNutrient uptake from different application methods may vary due to environmental (soil and atmospheric) and crop characteristics . Agronomic management plays an important role in altering the crop environment, thus agronomic management practices other than Zn fertilization are also expected to affect Zn uptake.3− ion resulting in more release of OH- from rice roots, which ultimately precipitates Zn thus reducing its’ level [The moisture status of soil may vary depending on the irrigation practices followed. Moisture deficiency in crop field reduces the diffusion of Zn, limiting their absorption by the plant. Zn deficiency has been observed in rice under contrasting environments, i.e., flooded conditions as well as aerobic conditions . In aeros’ level .In addition to Zn fertilization, the status of other nutrients in the soil and their application also affects Zn uptake. Nutrient interaction may alter the availability of nutrients either positively or negatively. A sufficient level of nitrogen supply is very critical for increasing the grain Zn concentration of wheat. Nitrogen application improves uptake as well as remobilization of Zn in wheat . The rolIn addition to enhancing grain Zn concentration and sometimes yield, agronomic biofortification gives some additional benefits. The soil application of Zn under a Zn deficient condition has been found to reduce the uptake and accumulation of phosphorus. This Zn–phosphorus antagonism is one of the most discussed nutrient antagonisms. The reduced phosphorus uptake and accumulation may reduce the phytate content in grains . Under ZIn addition to human health benefits, grains enriched with Zn can give additional agronomic benefits. Seeds with low Zn concentration show poor tolerance to environmental stresses . When seThe second approach for enhancing grain Zn concentration in crops is genetic biofortification. Genetic biofortification follows the breeding approach to increase the concentration and bioavailability of grain Zn. Genetic biofortification can serve as a cost-effective strategy to alleviate Zn deficiency. A superior genotype, once developed, can be used for many years without any additional recurring cost.Plant breeding and/or transgenic approaches provide a hopeful and long-term strategy to overcome micronutrient malnutrition by developing genotypes with a high level of Zn in the edible plant parts ,34,67. TThe overall steps involved in breeding include the following minimum steps: finding suitable genetic variation and selection of parents, long term crossing and backcrossing, stabilization of target traits across multiple environments/climatic conditions, and adaptation of the biofortified genotypes to the regional agronomic management practices . The breA superior genotype for Zn biofortification needs to have the following characteristics: high Zn acquisition efficiency, readily translocate Zn to grain/edible part of plant, efficient remobilization of Zn from vegetative tissues to grain or edible part of the plant, and availability of Zn in the plant in a bioavailable form that can be utilized by the person consuming it ,118. SevExisting genetic variability, trait heritability, gene action, the association among traits, available screening techniques, and diagnostic tools are commonly used criteria to estimate the potential genetic gains . A largeIn addition to grain Zn concentration, the bioavailability of the Zn should be given importance in the breeding programs. Only 25% of the Zn in the staple food grains are thought to be bioavailable . The bioArabidopsis thaliana in barley roots increased the Zn concentration in grain [A transgenic approach can be followed to develop crop varieties with a high Zn content. Evidence of ZIP family iron and Zn transporter proteins in improving grain micronutrient concentration is available ,125,126.in grain .In the agronomic biofortification section, we have briefly discussed how various soil characteristics affect a plant’s availability of Zn. Under such a condition, the varieties developed to accumulate more Zn in the edible parts may not be able to show its full potential. To achieve a grain Zn concentration in the edible portions of the plant that can bring a measurable biological impact, the plant must be grown in a soil environment with sufficient plant-available Zn . As the Moreover, crop improvement to develop Zn rich variety is a fairly lengthy process and requires a lot of effort in germplasm selection/screening, crossing, and performance assessment in the multilocation trial. However, as Zn concentration is not subjected to genetic erosion, little maintenance breeding is needed after the incorporation of the desired gene into the gene pool .The success of genetic biofortification may be jeopardized if sufficient Zn is not available in soil . The capThe unavailability and unaffordability of a healthy diet are largely responsible for the prevalence of malnutrition across the globe. As per an estimate, over 3 billion people worldwide are far from easy access to a healthy diet. Thus, both accessibility and affordability of a healthy diet must be ensured. By 2030, diet-related health cost linked to death and non-communicable diseases are expected to exceed USD 1.3 trillion while, the diet-related social cost of GHG (greenhouse gas) emission is projected to be greater than USD 1.7 trillion. Though these costs are often ignored, a dietary shift to a healthy diet can reduce the cost related to health and climate change. In fact, adoption of a healthy diet can reduce the direct and indirect health costs up to 97% and a 41–74% reduction in social cost in GHG emission by 2030 [As the application of Zn may not necessarily increase the yield, farmers may be sceptical to apply Zn as it incurs an additional cost. However, the health benefits obtained from Zn application are usually overlooked in such conditions. Zn and other micronutrient deficiency cause huge economic losses in developing countries and have a huge impact on gross domestic product (GDP) and costs related to health care ,130. MicWang et al.  calculatThe suboptimal utilization of the human resource potential is expected to reduce the work productivity and thus will have an undesirable impact on economic output at all levels, starting from the individual to the national level. Intervention is required to reduce micronutrient malnutrition so that human resource potential can be fully utilized to their potential and cost of health can be reduced. Biofortification may be seen as an investment in human health, which will also reduce the cost of health. Unless we evaluate this health benefit of biofortified grains and see grain yield as the only criteria for evaluating the economic output of crop production; then the benefits of biofortification can be hardly realized. Social awareness on the importance of micronutrient nutrition, policies to promote micronutrient application in crops to realize the benefits of agronomic and/or genetic biofortification, and investment in the research and development on biofortification can help in sustainably alleviating micronutrient malnutrition.A comprehensive 4R  approach of Zn application can be developed for different crops at the regional level and the best combination can be found for achieving high grain Zn concentration.Physiological constraints of grain Zn accumulation must be identified for different crops under different conditions and agronomic and genetic approaches for ameliorating these constraints may be found to further improve the grain Zn density.Biofortification options must be studied under stressed environments and their effects must be evaluated under such conditions. As climate change is expected to bring more weather anomalies, a stress-proof biofortification approach must be developed.The bioavailability of Zn obtained through foliar application can be compared with other application methods. Agronomic management that improves grain Zn bioavailability should be studied.The environmental implications of continuous Zn application should be studied. Continuous application of Zn over a long period may cause Zn toxicity and therefore should be regularly monitored.The performance of Zn-efficient genotypes under different soil Zn availability should be evaluated. The beneficial effects of combining the agronomic and genetic biofortification approach should be explored.Though research has advanced in biofortification, some key areas need to be addressed or improved further. Some have been highlighted below:From this comprehensive review, it can be concluded that the biofortification approach has outstanding potential for ameliorating the problem of micronutrient malnutrition. The cost-effectiveness of this approach makes it a suitable option for low-income countries. As biofortification improves the micronutrient concentration of the staple food grains that is predominantly consumed by people it does not involve any dietary change and can be adapted by people quickly. Agronomic biofortification not only improves the grain Zn concentration providing health benefits, but it can also help in reducing the extent of Zn deficiency especially in regions where intensive cropping is practiced and micronutrient application is overlooked. With the advent of genetic engineering and molecular tools, the breeding approach of biofortification can also be fastened and developing a superior Zn-efficient and Zn-rich varieties will be comparatively easier to find. Policy initiative and government support will also help in further research and dissemination of biofortification technologies and practices.


ZmZIP3, ZmZIP4, ZmZIP5, ZmZIP7, and ZmZIP8 in the roots of two inbred lines. These results indicate that extended time length of low-Zn stress will enlarge the difference of multiple physiological traits, especially biomass, between Zn-sensitive and Zn-tolerant inbred lines. There were significant genotypic differences of root morphology in response to heterogeneous Zn supply. Compared with split-supply with +Zn/+Zn, the difference of above-ground biomass between Zn-sensitive and Zn-tolerant inbred lines under split-supply with –Zn/+Zn was higher. Under the condition of heterogeneous Zn supply, several ZmZIP genes may play important roles in tolerance to low Zn stress, which can provide a basis for further functional characterization.All over the world, a common problem in the soil is the low content of available zinc (Zn), which is unevenly distributed and difficult to move. However, information on the foraging strategies of roots in response to heterogeneous Zn supply is still very limited. Few studies have analyzed the adaptability of maize inbred lines with different Zn efficiencies to different low Zn stress time lengths in maize. This study analyzed the effects of different time lengths of low Zn stress on various related traits in different inbred lines. In addition, morphological plasticity of roots and the response of Zn-related important gene iron-regulated transporter-like proteins (ZIPs) were studied via simulating the heterogeneity of Zn nutrition in the soil. In this report, when Zn deficiency stress duration was extended (from 14 to 21 days), under Zn-deficient supply (0.5 μM), Zn efficiency (ZE) based on shoot dry weight of Wu312 displayed no significant difference, and ZE for Ye478 was increased by 92.9%. Under longer-term Zn deficiency, shoot, and root dry weights of Ye478 were 6.5 and 2.1-fold higher than those of Wu312, respectively. Uneven Zn supply strongly inhibited the development of some root traits in the -Zn region. Difference in shoot dry weights between Wu312 and Ye478 was larger in T1 (1.97 times) than in T2 (1.53 times). Under heterogeneous condition of Zn supply, both the –Zn region and the +Zn region upregulated the expressions of  Zinc (Zn) is an essential micronutrient in plant growth and development. It plays an important role in various enzymatic reactions, metabolic processes, redox reactions, plant hormone metabolism, promoting the development of plant reproductive organs, resistance to infection by certain pathogens, and improving plant resistance to stress , followed by rice (up to 27%) and maize (9%)  transcription factor gene family, bZIP19 and bZIP23, contributes to the upregulation of ZIPs and improves the adaptation to low Zn supply  are highly induced in roots of Zn-deficient plants. Tissue-specific expression in roots supports the roles of these genes in uptake and root-to-shoot translocation of Zn under Zn starvation conditions : 0.5 NH4NO3, 0.5 CaCl2, 1.5 Ca(NO3)2, 0.75 K2SO4, 0.65 MgSO4, 0.1 KCl, 0.25 KH2PO4, 1.0 × 10−3 H3BO3, 0.35 Fe(II)-EDTA, 8.0 × 10−3 CuSO4, 1.2 × 10−2 MnSO4, 4.0 × 10−5 (NH4)Mo7O24, and 4.0 × 10−3 NiCl. Growth chamber condition was set as a 14-h light period from 8:00 to 22:00 with 28°C and a 10 h dark period with 22°C. The average light intensity measured at canopy was 350 μmol m−2 s−1. The pH of solution was adjusted to 5.5–6.0.Maize seeds of inbred lines Wu312 and Ye478 were sterilized for 30 min in a 10% solution of H4·7H2O. However, a study showed that compared with ZnSO4, ethylene diamine tetra acetic acid-Zn (EDTA-Zn) was found to be better for growth and yield of rice  and +Zn (8 μM EDTA-Zn) in Treatment 1 (T1), and with +Zn (8 μM EDTA-Zn) and +Zn (8 μM EDTA-Zn) in Treatment 2 (T2), respectively . After 4In Experiment 1 and 2, shoots and roots for plants were separately collected in an envelope. All samples were heat-treated at 105°C for 30 min and dried at 75°C until constant weight. Zn concentrations in shoots and roots were analyzed by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). Zn uptake efficiency, Zn transport efficiency, and Zn use efficiency were calculated using the following equations from (1) to (3), respectively.−1) = Zn uptake efficiency (μg root dry weight gZn transport efficiency (%) = −1) = Zn use efficiency .Total RNA was isolated from each root sample for Wu312 and Ye478 with TRIzol (Takara). We used 1.5 μg of total RNA to synthesize cDNA. Quantitative real-time polymerase chain reaction (PCR) was performed using SYBR Green Real-time RT-PCR (Applied Biosystems) and an ABI7500 Fast Real-Time PCR System (Applied Biosystems). The primers used for real-time PCR are shown in t-test. A probability level of p < 0.05 was required for statistical significance.Each experiment was arranged in accordance with a randomized complete design. Means among different treatments were compared using the least significant difference (LSD) test. Means between inbred lines Wu312 and Ye478 were compared using In Experiment 1, when the Zn supply concentration was 0.5 μM, shoot and root dry weights of Ye478 were 154 and 109% higher than those of Wu312, respectively. Compared with Zn-sufficient supply, shoot dry weights of Wu312 and Ye478 under Zn deficient condition (0.5 μM) were decreased by 33.6 and 18.8%, respectively , while rIn Experiment 2, at the nil Zn supply (0 μM), plant of Wu312 was tended to be dead and Ye478 showed serious Zn-deficient symptoms . When thIn Experiment 2, when the Zn supply level was 0.5 μM, compared with Zn-sufficient treatment (8 μM), leaf soil plant analysis development (SPAD) of Wu312 was decreased by 38.7% and SPAD of Ye478 was increased by 14.9%. When the Zn supply concentration was increased to 16 and 32 μM, leaf SPAD values of two inbred lines were significantly reduced . Under dBased on visible phenotypic variation of plants, shoot and root dry weight, and leaf SPAD of Wu312 and Ye478 in Experiments 1 and 2, Zn nutritional status of 8 μM was determined to be the Zn-sufficient condition (control treatment). When Zn concentration was 0.5 μM, compared with 21-day Experiment 2, a 14-day Experiment 1 was designed as a short-term zinc deficiency experiment. In addition, shoot dry weights of Ye478 under Zn-deficient (0.5 μM EDTA-Zn), and Zn-sufficient condition (8 μM EDTA-Zn) under short-term stress were 1.5- and 1.3-fold higher than that of Wu312 in Experiment 1, respectively. Under longer-term Zn deficiency stress in Experiment 2, these multiple values were reduced to 6.5 and 2.1, respectively.Relative values of shoot dry weight, which were estimated by the ratio of shoot dry weight under 0.5 μM low Zn stress to the shoot dry weight under Zn-sufficient condition (8 μM EDTA-Zn), were considered as Zn efficiency (ZE) based on shoot dry weight. ZEs for Zn-sensitive inbred line Wu312 showed no significant difference between Experiment 1 and 2. However, ZE for Zn-tolerant inbred line Ye478 in Experiment 2 was 192.9% higher than that in Experiment 1 . This fiIn Experiments 1 and 2, shoot and root Zn concentration of Wu312 and Ye478 gradually enhanced with the increase of Zn supply , 4C,D. EIn Experiment 1, compared with Zn sufficient condition (8 μM), Zn uptake and transport efficiency of Wu312 and Ye478 under Zn deficient condition (0.5 μM) was decreased by 59.5 and 67.8%, and 42.9 and 38.8%, respectively . And Zn Under the condition of heterogeneous Zn supply, total root length, root surface area, and lateral root number of the Wu312 and Ye478 under Zn deficiency were significantly reduced  comparedSpecific root length of two inbred lines differed greatly, but the difference in main root length was relatively small . In the ZmZIP1, ZmZIP2, and ZmZIP6 showed no significant difference . Also, the expression levels of these genes in Zn-efficient and Zn-inefficient genotypes were also different.Under the condition of heterogeneous Zn supply, compared with homogeneous Zn-sufficient supply, the expression levels of fference . Howeverifferent  while Zmfference . Uneven of Wu312 . Heterogn region . CompareZmZIP1-7 . These rIn Experiment 1, shoot dry weight of Ye478 under Zn-deficient condition (0.5 μM) was 1.5 times higher than that of Wu312 despite there being no significant difference in root dry weight between two inbred lines . HoweverApart from extending stress duration, we also investigated the effects of excess Zn on maize via increasing the Zn supply to 16 and 32 μM. Previous studies have shown that excess Zn inhibited the production of biomass, chlorophyll, total soluble protein, and strongly increased accumulation of Zn in both root and shoot mainly because Zn-induced oxidative stress can cause loss of plasma membrane integrity, electrolyte leakage, increase in malondialdehyde content, and damage photosynthesis to produce reactive oxygen species  indicated that the root surface of Wu312 have begun to rot and become grayish brown since the 21st day after transfer, which would probably lead to necrosis in the whole plant . But theThe R/S ratio of genotypes increased as Zn application decreased , Ye478 could continue to grow healthily, while the development of Wu312 was significantly inhibited. This may be because genotypes with different tolerance to Zn stress have different minimum Zn requirements  in ZIP genes are crucial in plant adaptation to low and fluctuating Zn availability in soil . The expression levels of each gene in the two lines are basically consistent with the results of this study. According to the results of this study, the expression of ZmZIP1 in two inbred lines under the condition of non-uniform Zn supply did not show a significant difference compared with the control  of the nodes and responsible for the preferential distribution of Zn to developing tissues  mainly accumulate Zn in the roots rather than in shoots. OsZIP4 is a Zn transporter that localizes to the phloem cells of stems, vascular bundles of leaves, and roots. Constitutive expression of it changes the Zn distribution within rice plants . However, low levels are found in the ZmZIP5i (RNAi line) plants. ZmZIP5 may play a key role in Zn uptake and root-to-shoot translocation , ZmZIP7 may display characteristics of low-affinity Zn transport in the plant  demonstrated a much stronger activity than OsZIP5  and other rice ZIP proteins .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The high-areal-capacity full cells demonstrate hundreds of cycles under high Zn utilization conditions.Rechargeable aqueous zinc-ion batteries (RZIBs) provide a promising complementarity to the existing lithium-ion batteries due to their low cost, non-toxicity and intrinsic safety. However, Zn anodes suffer from zinc dendrite growth and electrolyte corrosion, resulting in poor reversibility. Here, we develop an ultrathin, fluorinated two-dimensional porous covalent organic framework (FCOF) film as a protective layer on the Zn surface. The strong interaction between fluorine (F) in FCOF and Zn reduces the surface energy of the Zn (002) crystal plane, enabling the preferred growth of (002) planes during the electrodeposition process. As a result, Zn deposits show horizontally arranged platelet morphology with (002) orientations preferred. Furthermore, F-containing nanochannels facilitate ion transport and prevent electrolyte penetration for improving corrosion resistance. The FCOF@Zn symmetric cells achieve stability for over 750 h at an ultrahigh current density of 40 mA cm Rechargeable aqueous zinc-ion batteries are promising but the zinc anode suffers from dendrite growth and electrolyte corrosion. Here, the authors develop a fluorinated covalent organic framework film as a protective layer for aqueous zinc anode battery. Unfortunately, previous Zn anodes showed poor reversibility in aqueous electrolytes5. Issues including Zn dendrite formation, continuous parasitic hydrogen evolution reaction (HER), and irreversible by-products, resulting in low Coulombic efficiency (CE), and shortened battery life9. To stabilize the Zn anode, various strategies including electrolyte optimization , surface coating materials  and Zn bulk structure engineering  have been proposed to achieve higher performance Zn anodes. However, there are still some unsolved issues with these strategies, which restricts them to subdued performance levels in Zn batteries. For instance, manipulating previous electrolyte compositions leads to an increase in overall costs15, sacrifices rate performance of the batteries owing to their slow ionic conductivity and the HER is only lowered, not eliminated2. Interfacial modification layers are effective for suppressing HER16, however, the huge volume change during repeated Zn plating/stripping can damage protective layers, even peeling them completely off the Zn matrix17. Employing conductive 3D hosts could help to realize high-rate Zn deposition18, but adds additional porosity and weight, thereby reducing the volumetric/gravimetric energy density of the batteries. Therefore, developing alternative techniques to achieve dendrite-free Zn anodes while maintaining fast Zn deposition is urgently needed.Zinc (Zn) anode-based aqueous batteries are attracting tremendous interest owing to Zn’s high theoretical capacity  and (103) planes, which mitigates dendrite formation and reduces the later corrosion rates. Substrate such as stainless steel modified with an aligned graphene layer5, shows good lattice matching with Zn, which induces epitaxial deposition of Zn along the (002) planes, achieving ultra-long cycling life. Recently, fields generated by rotating disc electrodes19 are reported to promote the crystallographic reorientation of Zn to be grow parallel to the substrate, and the reversibility of Zn deposition/stripping is greatly increased. Therefore, correlating the crystallography and morphology to deeply understand and regulate the electrodeposition behavior of Zn is of great significance for developing long-life Zn batteries. However, there is an obvious lacks of fundamental elucidation of the mechanism controlling planar Zn deposition. Furthermore, the surface stability of inorganic crystals has long been thought to be dominated by their surface energy33. From the perspective of crystal growth, controlling the surface energy of Zn crystal planes offers exciting opportunities to realize planar zinc deposition.The crystallinity and morphology of Zn electrodeposits dominates the reversibility of Zn plating/stripping−2). The assembled full cells paired with manganese dioxide (MnO2) cathodes show a stable cycle life for over 250 cycles under practical condition of lean electrolyte, high areal capacity cathode and limited Zn excess.Here, by using two-dimensional (2D) covalent organic frameworks (COF) as a multi-functional platform Fig. , we deveThe imine-linked FCOF thin films are prepared through a solvothermal procedure Fig. . In a tyq = 0.20, 0.35, 0.40, 0.53 A−1 correspond to the planes (100), (110), (200) and (210), respectively, consistent with a previous report34, confirming the high degree of crystallinity of the as-prepared film. Findings from theoretical simulation and Pawley refinement confirm that the 2D stacked structure in FCOF is an AA stacking mode  of the N1s spectra . The high intensity of the peak at 398.96 eV in the N1s spectra  measurement Fig. . From thter Fig. , which itra Fig.  is assigtra Fig.  shows thtra Fig.  further 2 and polyvinylidene difluoride (PVDF) hybrid matrix (2.67 GPa)36. The good mechanical strength is greatly beneficial to buffer volume expansion and retard dendrite propagation during the dissolution/deposition of Zn anodes.The F atoms are the crucial elements within the FCOF films for achieving high-performance Zn anodes. The element mapping obtained from energy dispersive X-ray spectroscopy (EDX) indicates that F is evenly distributed in the film  results  than bare Zn. In particular, the Rct of Zn anodes coated by the 100 nm thick FCOF film is lowest (90 Ω), about half that of bare Zn (180 Ω). Apparently, the Zn2+ transport is increased by the fluorinated 1D nanochannels. This is mainly due to the F atoms surrounded within the nanopores that endow the film a strong hydrophobic effect. When hydrated Zn2+ transport through fluorinated nanochannels is driven by electric field force, water molecules coordinated with Zn2+ will be repelled because of the strong hydrophobicity of F element in the FCOF film covering the Zn metal. Consequently, a large portion of water molecules is retarded and not able to penetrate the fluorinated nanochannels. The de-solvation effect of Zn2+ is therefore significantly promoted by F element. To confirm the ability of FCOF film to promote de-solvation, a study of the activation energy (Ea) is made, Ea represents the de-solvation barrier to Zn2+ transport. A computation for Ea based on temperature-dependent EIS is performed  for FCOF@Zn is 14.5 kJ mol−1, whilst that for bare Zn is significantly greater at 32.2 kJ mol−1. The lower activation energy for FCOF@Zn over bare Zn strongly suggests that the FCOF layer ensures fast de-solvation of Zn2+ and facilitates fast ion-transference. Studies have demonstrated that COFs are an excellent ionic conductor39. The inherent ordered structures provide regular ionic coordination sites to facilitate ions to hop along the 1D aligned nanochannels. This highly boosts ionic conductivity. To study Zn2+ transport in FCOF, the transport sites for Zn2+ on the COF skeleton are determined theoretically. The charge density distribution of the chemical structural unit in FCOF is computed. As is shown in Supplementary Fig. 39. Because of electrostatic attraction, the positively charged Zn2+ hops around the F atom sites and transports along the 1D aligned channels during charge/discharge . The impedance of the FCOF@Zn symmetric cells increases from 180 to 600 Ω within 8 h (h) after cell assembly 6·5H2O (JCPDS# 39-0688)11. When plain Zn anodes are immersed in aqueous electrolyte for 48 h, the peak intensity of by-products increases sharply. Much less irreversible by-product is accumulated on the surface of FCOF@Zn anodes during the same time duration. To evaluate corrosion resistance of the FCOF film, potentiodynamic and open-circuit, tests are conducted using a home-made test apparatus  decreased from 2.5 × 10−3 A cm−2 for bare Zn to 4.1 × 10−5 A cm−2 for FCOF@Zn. Despite 8 days of electrolyte immersion, FCOF@Zn continued to exhibit a greater corrosion potential (Ecorr) of −1.063 V and a lower corrosion current (Icorr) of 4.8 × 10−4 A cm−2, compared with bare Zn of, respectively, −1.068 V and 1.2 × 10−3 A cm−2. The open circuit potential (OCP) for both electrodes is shown in Supplementary Fig. 4 remained stable at ~ −1.042 V vs. SCE, whilst that for FCOF@Zn is ~ −1.020 V in the initial stage, and remained greater at −1.032 V than that for bare Zn of −1.041 V, despite 5000 s. It is reliably concluded therefore that the corrosion resistance for Zn anode is significantly improved by FCOF film surface protection.The corrosion resistance of the FCOF film on the Zn surface is investigated in aqueous electrolyte 6 •5H2O (JCPDS NO: 39-0688) by-product. In contrast, these by-product signals are significantly weaker for Zn deposits under the FCOF film, strongly evidencing that the FCOF film provides protection for Zn, and inhibits accumulation of by-product. The orientation of the Zn deposits can also be quantified by calculating the texture coefficient41  of Zn deposits underneath the FCOF film is 19.2, much higher than that of the deposits on bare Zn (11.5), verifying the preferential growth on the (002) plane of Zn modulated by an FCOF film. XRD patterns for Zn deposits following the 30th and 80th cycles are shown in Supplementary Fig. Tc for the (002) plane is stable with increase in cycle number , however is higher than that for bare Zn . These evidences validate that the preferred (002) plane orientation is maintained, despite long cycling.In addition to the fast ion conduction and suppression of side reactions features, the morphology and texture of Zn deposits has been proven to have large impact on the cycling life of Zn batteries. Attaining an even planar deposition can ensure the batteries running for a prolonged time without short circuiting. An electronic resistance property is important in Zn deposition underneath the FCOF film. From the polarization I–V curve, the 100 nm FCOF film exhibits an electronic resistance of 3 × 10ion Fig. , disordelms Fig. , while tlms Fig. . This chlms Fig. , and are42 . As shown in Fig. d-spacings of 0.230 and 0.133 nm with an interfacial angle of 90°, corresponding to the (100) and (1–20) planes, respectively. The HRTEM result is in accord with the indexed SAED diffraction spots. To further verify the indexing results, an atomic arrangement model of Zn along the (001) direction is simulated, as shown in Fig. X-ray diffraction pole figures are used to further identify the texture information of Zn deposits. The (002) pole figure Fig.  of Zn un42 Fig. . In cont42 Fig.  shows al42 Fig. , while f42 Fig. . This in2+ transport, and corrosion resistance properties enabled by the FCOF film are expected to greatly improve the electrochemical performance of Zn anodes. The reversibility of Zn anodes can be measured by a procedure that wherein a specific amount of Zn is plated on the substrate and then stripped away. Coulombic efficiency (CE) is an important index to evaluate such reversibility. The CE using the half cells in FCOF@Ti/Zn and Ti/Zn configurations is measured. At a moderate current density . Remarkably, as evidenced from the AFM height and phase imaging . The average height difference  is only 170 nm 2 or Zn4SO4(OH)6·nH2O on Zn surface16. Raman spectroscopy is carried out to reveal the components on Zn deposits surfaces after cycling . Sharp peaks at 1152, 1110, 1011, and 967 cm−1 are observed on the Zn deposits on bare Ti 6·5H2O43. In contrast, the peaks of the Zn deposits underneath FCOF are not obvious and their intensity is much lower  is also far superior to most previous reported values  manganese dioxide (MnO2) cathodes. For the FCOF@Zn/MnO2cells, cyclic voltammetry curve (CV) curves demonstrate a larger current density at 0.1 mV s−1 and a smaller voltage gap between typical redox peaks than in Zn/MnO2 cells  is lower than that of Zn/MnO2 cells . Reducing the capacity ratio of the negative electrode to the positive electrode (N/P) during full cell operation is a key parameter to achieve high energy density45. In previous studies, many systems chose to use thick zinc foil (≥100 µm) paired with low mass loading cathodes to assemble full cells, the N/P reported in these studies is typically higher than 50, which is not beneficial for achieving high energy density. In our case, the excellent performance of the FCOF@Zn anodes allows us to further evaluate the cycle performance of full cells under harsh conditions. Using thin FCOF film-protected Zn plates as anodes (the thin Zn plates is rolled to desired thickness to satisfy the required N/P condition), FCOF@Zn/MnO2 cells with N/P = 10:1 and N/P = 5:1 show stable specific capacity at current density of 4 mA cm−2 for over 300 and 200 cycles, respectively  and controlled electrolyte-to-capacity ratio  is assembled. When the mass loading of MnO2 increases from 8 to 16 mg cm−2, the charge and discharge curves for FCOF@Zn-MnO2 full cell exhibit low polarization as shown in Supplementary Fig. 2 full cell  is cycled at a current density of 3 mA cm−2. The charge/discharge curves determined from different cycles almost overlap , which is significantly increased (by approx. 6.5 times) compared with many reported Zn/MnO2 cells using low mass loading cathodes and thick Zn foils30  indicator  plane with lowest the surface energy tends to stay exposed instead of its intrinsic thermodynamically stable plane. In our case, the uniformly distributed F atoms within FCOF film have a strong binding interaction with the Zn atoms, thereby regulating the relative surface energy value of the (101) and (002) planes and influencing the deposited morphology. To verify this, the surface energy and adsorption energy of the two crystal planes terminated by F atoms are studied by first-principles calculations  and lower conduction band (CB). With the formation of Zn–F interaction, however, localized states of Zn 4s are observed, indicating that F atoms are prone to stabilize Zn atoms. To qualitatively determine the strength of the interaction between F atoms and Zn atoms and its impact on different crystal planes, the electron states of the Zn 3d and F 2p are analyzed. Due to the strong electron-withdrawing property of F atoms, the F 2p could accept electrons from the Zn 4s. As a result, the Zn 4s planes would partially lose electrons, forming quasi-stable bonds with the F species. Thereafter, the electrons from the Zn 3d may be excited toward the Fermi level. The displacement of Zn 3d electrons from the FT-(002) planes is more energetically-favorable than from FT-(101) planes. On the other hand, compared with the F 2p electron states of the in FT-(101) planes, the F 2p electron distribution in a FT-(002) plane is more localized. These results clearly suggest that F atoms interact more strongly with the (002) planes than with the (101) plane. The XPS data for the F 1s and Zn 2p at the FCOF-Zn interface also indicate the existence of strong F–Zn interaction50  crystal planes of Zn. The growth of (002) planes is most stable during the deposition process, and anisotropic growth of Zn along other planes is done in such a way that a (002) plane is formed, resulting in platelet-like Zn deposits. Given that the F atoms bonded within the FCOF film are arranged in parallel along the surfaces of the current collectors, which induces each Zn platelet to also be arranged in parallel. Furthermore, the F-containing porous nano channels endow good hydrophobic effects on the film, which is beneficial for de-solvation and facilitates fast transport of hydrated Zn2 bath) and degassed by three freeze-pump-thaw cycles and finally sealed under vacuum conditions. The sealed tube is heated at 120 °C for 3 days. For the postprocessing, the tube is opened, washed with acetone several times and then the tube is charged with deionized water overnight. The free-standing FCOF films easily detach from the tube wall by gently shaking the tube. The obtained FCOF films is immersed in acetone for 2 days to wash out any impurities. Finally, the as-prepared FCOF films is transferred to various substrates via a pulling method in acetone solvent. The thickness of the membrane/film can be also easily tuned by controlling the concentration of monomers. The MnO2 cathodes are synthesized using an aging method according to a previous report51. First, 5.07 g manganese sulfate monohydrate (MnSO4·H2O) is put in 100 mL deionized water, followed by ultrasound and stir to ensure dissolution (named Solution 1). Then 4.32 g sodium hydroxide (NaOH) and 20 mL hydrogen peroxide  are dissolved in 180 mL deionized water (named Solution 2). Solution 2 is then dropwise added into Solution 1 under vigorous stirring conditions. The mixed solution is stirred for 1 h and aged at 25 °C for 24 h. The precipitated product is centrifuged for three times, washed with deionized water, and dried to obtain MnO2.For the typical synthesis of FCOF film, a pyrex tube is charged with 3.75 mg (0.0182 mmol) 2, 3, 5, 6-tetrafluoroterephthalaldehyde (TFA), 4.40 mg (0.0125 mmol) 1,3,5-Tris(4-aminophenyl) benzene (TAPB), 1.8 mL dioxane (Dio) and 0.2 mL 1, 3, 5-trimethylbenzene (TMB). After sonication for 10 min, 0.1 mL HOAc (1.5 M) solution is added. After this, the tube is frozen at 77 K (liquid Nλ = 0.154056 nm), using an operating voltage of 40 kV and a 40 mA current. Fourier transform infrared (FTIR) spectra are collected on a ThermoFisher Nicolet 6700 spectrometer. N2 adsorption–desorption isotherms are measured at 77 K on a Micromeritics TriStar II 3020 volumetric adsorption analyzer after degassing in a vacuum at 120 °C overnight. Atomic force microscope (AFM) is performed on a NT-MDT NTEGRA Spectra II microscope. Field-emission scanning electron microscopy (FESEM) images are acquired from a Zeiss Gemini SEM500, equipped with an Aztec X-Max Extreme energy dispersive spectrometer (EDS). High-resolution transmission electron microscope (HRTEM) images are collected from a JEM-2010F transmission electron microscope. XPS measurements are carried out with a Thermo Scientific K-Alpha+ spectrometer under vacuum. Nano-indentation surface hardness measurements are conducted on a TI-950, NHT.Wide-angle X-ray scattering (WAXS) measurement is conducted on a XenocsXeuss2.0 with 8 KeV Cu Kα radiation. X-ray diffraction (XRD) data is measured by a Bruker D8 Advance with Cu-Kα X-ray radiation  in a mass ratio of 7:2:1. The mixture is compressed onto a Ti grid. The electrodes are dried at 80 °C under vacuum for 12 h and then punched into disks. All the electrochemical properties are tested by assembling 2016-coin cells with glass fiber separators. All Galvanostatic charge-discharge measurements are carried out on a battery testing instrument  at different current densities. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) are performed by a CHI 660E electrochemical workstation. CV curves of full cells are recorded over the voltage range of 1–1.85 V. EIS is measured in a frequency range of 100 kHz to 0.1 Hz at open circuit potential and an amplitude of 5 mV. The fitting parameters of the equivalent circuit are analyzed by ZSimpWin software.Cycling tests for symmetric cells and Zn/Ti half cells of bare Zn or FCOF@Zn are conducted with 2 M ZnSOThe DFT calculations are carried out using the Vienna Ab-initio Simulation Package (VASP) with the frozen-core all-electron projector-augment-wave (PAW) method. The Perdew–Burke–Ernzerhof (PBE) form of the generalized gradient approximation (GGA) is adopted to describe the exchange and correlation potential. The cutoff energy for the plane-wave basis set is set to 500 eV. The Monkhorst-Pack k-point6 sampling is set to 3 × 3 × 1. The geometry optimizations are performed until the forces on each ion is reduced below 0.05 eV/Å. The vacuum slab models are used to calculate the adsorption of F atom on Zn (002) and (101) surfaces. These Zn surface slabs comprise four layers of Zn atoms, and a vacuum region of 20 Å above them is used to ensure the decoupling between neighboring systems. For the geometry optimization, the atoms in the 2-bottom layers of slab are fixed to their bulk positions.The adsorption energy, 31:2.The surface free energy (Supplementary Information


This review addresses the physiological and biochemical connections between steroidal implants and Zn and their interaction to influence growth in beef cattle. Steroidal implants have been widely accepted as a growth-promoting technology that provides an unmatched economic return to the producer and improves beef production’s environmental sustainability. Likewise, decades of research have indicated Zn is vital for skeletal muscle growth. Considering Zn is an essential trace mineral, strategic Zn supplementation to implanted cattle may optimize beef production. Although many interrelationships between steroidal implants and Zn are new and forthcoming, the literature reviewed hereafter indicates roles for Zn in a multitude of growth processes pertinent to steroidal implant-induced growth and uncover changes in Zn metabolism due to steroidal implant use.Growth-promoting technologies such as steroidal implants have been utilized in the beef industry for over 60 years and remain an indispensable tool for improving economic returns through consistently improved average daily gain via increased skeletal muscle hypertrophy. Zinc has been implicated in skeletal muscle growth through protein synthesis, satellite cell function, and many other growth processes. Therefore, the objective of this review was to present the available literature linking Zn to steroidal implant-induced protein synthesis and other metabolic processes. Herein, steroidal implants and their mode of action, the biological importance of Zn, and several connections between steroidal implants and Zn related to growth processes are discussed. These include the influence of Zn on hormone receptor signaling, circulating insulin-like growth factor-1 concentrations, glucose metabolism, protein synthesis via mTOR, and satellite cell proliferation and differentiation. Supplemental Zn has also been implicated in improved growth rates of cattle utilizing growth-promoting technologies, and steroidal implants appear to alter liver and circulating Zn concentrations. Therefore, this review provides evidence of the role of Zn in steroidal implant-induced growth yet reveals gaps in the current knowledge base related to optimizing Zn supplementation strategies to best capture growth performance improvements offered through steroidal implants. Increasing supplementation of trace minerals (TM)  and moreSteroidal implants have been used since the 1950s in the U.S. beef industry although they are no longer an accepted growth-promoting technology in several countries. This technology routinely improves cattle gains by 16–20%  and is a2) or the synthetic prodrug of E2, estradiol benzoate (EB). The estrogenic component can be included alone, but most often, it is included in combination with either progesterone, testosterone propionate, or trenbolone acetate (TBA) thymidine, which functions as a labeled DNA precursor, linearly decreased in the liver, kidney, and spleen, reflective of the importance of Zn in DNA synthesis. Additionally, protein synthesis was markedly decreased in the kidney and spleen of rats, measured by [14C]lysine incorporation in these tissues. These studies were instrumental in demonstrating a vital role for Zn in protein utilization in the body.Zinc functions in mediating whole body growth and protein synthesis. It was demonstrated that in piglets fed Zn deficient diets, growth retardation was evident as early as 1 week following dietary treatment administration . FurtherOfficial beef cattle TM requirements are established to prevent deficiency and allow for growth . The curAlthough Zn supplementation is greater across the beef industry  than NASYet, Zn absorption may be influenced by additional factors that alter whole body use of Zn, such as growth. Skeletal muscle and bone comprise a large percentage of Zn storage. However, classified as slow Zn metabolizers, release of Zn from muscle and bone is less likely . Seeing 2 receptor’s signaling in some manner.Zinc has been implicated in many pathways downstream of hormone receptors often with a role in affecting the phosphorylation state of the cell, these responses typically begin at the receptor. Similar to the non-genomic pathways by which hormone signaling occurs, Zn treatment can also directly influence GPER signaling. Interestingly, Pisano et al.  found th2+ for 2 h resulted in increased release of hbEGF from EGFR, resulting in EGFR phosphorylation [Treatment of normal human bronchial epithelial cells with 100 µM Znrylation . Phosphorylation . Zinc surylation . Furtherrylation  observedrylation ,68. As arylation ,70, MMP rylation ,72. Furtrylation . Althougrylation ,26, thes2 and androgen hormone receptor signaling.Although Zn has not been directly related to androgen receptor function like the previously discussed estrogen receptor, a role for Zn in androgen signaling is emerging. Interestingly, the Zn transporter ZIP9 was discovered as a membrane androgen receptor that facilitates non-genomic signaling in fish  and huma2 implant [Steroidal implants have consistently been shown to increase both hepatic and/or local skeletal muscle IGF-I production in steers implanted with a combination TBA + E implant ,77,78. I implant . Additio implant . The imp implant , who repAlthough Zn supplementation has been shown to improve growth rates in rats , links bThe majority of circulating IGF-1 is bound by a binding protein that acts to prolong the half-life of IGF-1 . Due to Circulating concentrations of IGF-1 and/or IGFBP-3 have been observed to increase with Zn supplementation ,8,87,88.As previously suggested, the literature strongly indicates a role for Zn concerning glucose metabolism. While ruminants derive a substantial amount of energy from volatile fatty acids, gluconeogenesis plays an important role in ruminant energy metabolism . GlucoseThe behavior of proinsulin and insulin in the presence of Zn suggests Zn is important in β-cell production of insulin for most animal species. Zinc is an essential TM that plays a vital role in the formation of insulin, stimulation of phosphorylation of the β-subunit of this hormone receptor, activation of PI3K, and the translocation of glucose transporter 4 (GLUT4) ,93. The Wu et al.  showed tAdditionally, Ohashi et al.  reported2 resulted in a dose-dependent increase in protein synthesis rate coupled with a decrease in protein degradation rate [Downstream of GH and IGF-1 signaling, a considerable body of the literature reveals additional interactions for Zn and steroidal implants in protein synthesis exist . Steroidion rate .Zinc appears to be relevant to many of these same protein synthesis pathways. Nimmanon et al.  reportedAs it pertains to cattle, BSC provide the additional nuclei source necessary to support postnatal skeletal muscle fiber hypertrophy and are critical in determining the extent of muscle growth . Satelli2 influence satellite cell function. While it is clear Zn plays a role in satellite cell function, past evidence provides controversy about the exact relationship of Zn to proliferation and differentiation of myoblasts. Differential expression of Zn transporters were analyzed by Paskavitz et al. [2+ indicates cellular Zn concentration is dynamic during the myogenesis process. Petrie et al. [In addition to playing a structural role, Zn appears to have an important regulatory function related to cell proliferation and differentiation in satellite cells that likely interacts or overlaps with the mechanisms by which TBA and Ez et al.  to deterz et al.  demonstre et al.  found in2 on myogenic proliferation and differentiation and found that exogenous Zn treatment increased both myoblast proliferation and differentiation. Mnatsakanyan et al. [−1·d−1 of Zn as Zn-methionine. Skeletal muscle SC, extracted and cultured from these steers, possessed increased Pax7 gene expression. This indicates animals treated with Zn-methionine had greater capacity to increase total nuclei present in skeletal muscle through proliferation and differentiation of the Pax7 SC. Although some of the previous literature is in disagreement with regard to how Zn specifically impacts different aspects of SC lineage, these data do indicate that Zn does in fact play a role in SC proliferation and differentiation and require further investigation.Additionally, Petrie et al.  reportedn et al.  also repn et al.  found Znn et al.  reportedn et al.  and Ohasn et al. , contrasn et al.  conducteAlthough TM recommendations for beef cattle were recently updated , few cha4 was fed to heifers and steers, steroidal implant administration improved average daily gain (ADG) by more than 17% [4 (100 mg Zn/kg DM) resulted in a 24% improvement in ADG over non-implanted steers fed the same Zn treatment [4 resulted in a linear improvement in performance of steers administered a high potency implant during the first 18 d post-implant administration. In contrast, no differences in performance were observed due to Zn supplementation within non-implanted steers [Indeed, Zn supplementation influences steroidal implant-induced performance of cattle. When 200 mg Zn/kg DM from ZnSOthan 17% . Howeverthan 17% . Interesreatment . Both ofd steers . These d4 (30 vs. 100 mg Zn/kg DM) [Although supplementation of Zn to cattle utilizing growth-promoting technologies has been proven advantageous, timing of Zn supplementation may be vital to capturing this growth response. Peak payout of hormone from steroidal implants occurs within 40 d of implant administration , resultin/kg DM) . These dAlthough effects of Zn supplementation on cattle performance clearly indicate the importance of Zn in growth, changes in liver and plasma Zn concentrations provide compelling evidence of modified physiological Zn requirements due to steroidal implants. In lambs implanted with zeranol (12 mg), Zn retention was increased by 60% . FurtherRecent studies have found increasing evidence of implants altering Zn requirements by assessing plasma and liver Zn concentrations . It is i2 cannot be quantified and accounted for in a controlled study. Therefore, the effects of steroidal implants on Zn metabolism in heifers may not be as reliable as in steers or spayed heifers.In contrast, Huerta et al.  observed4 or Zn-methionine [In addition to circulating Zn concentrations, steroidal implants have been found to influence concentrations of Zn in the liver. As previously mentioned, liver Zn concentrations are more difficult to change than plasma due to the less labile nature of Zn stored in the liver. Interestingly, Messersmith et al.  observedthionine . In compthionine . This rethionine  observedthionine  suggeststhionine  observedthionine  Zn recomThese data indicate changes in plasma Zn concentrations may be more consistent than liver Zn concentrations due to steroidal implant administration. Although the detected differences in Zn concentrations remained within the wide reference ranges  for plasStrategic supplementation of TM, including Zn, to cattle based upon expected performance and management practices such as steroidal implant use is critical to a future that features precision cattle feeding. This review details the proposed multi-faceted interactions between Zn and steroidal implants on growth ranging from effects on hormone receptors to protein synthesis. The literature reveals supporting roles for additional dietary Zn in implanted cattle and many avenues for future research. With more than 20 million cattle on feed in the U.S. each year and Zn being a low-cost input, strategic supplementation of Zn could potentially better capture the growth performance advantages offered by steroidal implants while improving producer return on investment and the efficiencies of beef production. However, the optimal concentration of supplemental Zn to capitalize on the genetic potential and implant-induced growth rates of cattle is unclear. Similar to the energy and protein demands of implanted cattle, Zn requirements of the beef animal are likely increased to accommodate these higher rates of growth. An association between Zn metabolism and steroidal implant-induced growth, as outlined in this review, provide evidence of Zn as a potential limiting micronutrient in beef cattle growth.Furthermore, optimal timing of Zn supplementation is unknown. Pinpointing a Zn supplementation window may prove useful in best accommodating the micronutrient demands of fed beef cattle. Phase feeding of greater concentrations of Zn during peak hormonal payout or other periods of high growth rates may optimize performance and best utilize TM resources. Supplementation of Zn at high concentrations in cattle poses minimal risk for toxicity, and supplemented concentrations are well below pharmacological concentrations fed in other species . Therefo


This allowed us to calculate the fraction of Zn in the shoots that was derived from fertilizer, soil, and seed over 4 successive cuts. In addition, we measured the 67Zn:66Zn isotope ratio with the diffusive gradients in thin films technique (DGT) on soils labeled with 67Zn and incubated with the same fertilizers. After 48 days of growth, the largest fraction of Zn in the ryegrass shoots was derived from the soil (79–88%), followed by the Zn-containing fertilizer (11–20%); the least (<2.3%) came from the seed. Only a minor fraction of the Zn applied with the fertilizer was transferred to the shoots (4.7–12%), which indicates that most of the freshly added Zn remained in the soil after one crop cycle and may thereby contribute to a residual Zn pool in the soil. The 67Zn:66Zn isotope ratios in the DGT extracts and the shoots measured at cut 4 were identical, suggesting that the DGT and plant took up Zn from the same pool. The proportion of Zn derived from the fertilizers in the DGT extracts was also identical to that measured in ryegrass shoots at cut 4. In conclusion, this work shows that stable Zn isotope labeling of the soil available Zn can be used to precisely quantify the impact of complex organic fertilizers on the Zn nutrition of crops. It also demonstrates that DGT extractions on labeled soils could be used to estimate the contribution of Zn fertilizers to plant nutrition.Manure and sewage sludge are known to add significant amounts of zinc (Zn) and other metals to soils. However, there is a paucity of information on the fate of Zn that derives from complex organic fertilizers in soil–plant systems and the contribution of these fertilizers to the Zn nutrition of crops. To answer these questions, we grew Italian ryegrass in the presence of ZnSO Tella et al. (65Zn strongly enhanced the uptake of Zn derived from the soil (non-labeled) by wheat. An increased solubility of soil Zn may be caused by nitrification of ammonium added with the organic fertilizer, which leads to acidification, and also by the decomposition of organic compounds leading to the release of water-soluble organic compounds that can form complexes with soil Zn  in agroecosystems . AUE is the difference between the amount of Zn taken up by a plant that has been fertilized with Zn and the amount of Zn taken by the same plant grown in the absence of Zn fertilizer, divided by the amount of Zn added as fertilizer. Using this mass balance approach, Berenguer et al.  estimate67Zn can be used to assess the plant uptake of Zn derived from wheat straw compost ICP-MS. Hence, labeling the pool of soil exchangeable Zn with 67Zn could be used to quantify the plant uptake of Zn derived from the soil and from sewage sludge or animal manure. Moreover, the isotope label can be used to calculate the recovery of the Zn applied with the fertilizer in the plant. Such recovery calculations can provide valuable insights about the potential fate of Zn in soil–plant systems after harvest  is a well-established tool to predict the plant available pool of Zn in the soil. A strong correlation between the amount of Zn extracted by the DGT sampler and the concentration of Zn in plant tissues has been shown in several studies  was grown as a model plant that takes up most of its Zn from the soil isotopically exchangeable pool  precisely quantify the amount of Zn in plants derived from fertilizer, soil, and seeds in soils amended with distinct complex organic fertilizers, (2) determine the fate of Zn input with complex organic fertilizer in soil–plant systems, and (3) test whether DGT can be employed to predict the relative contribution of soil and fertilizer sources to the plant uptake of Zn. To this end, two cropped soils, an acidic soil from Heitenried, Switzerland, and an alkaline soil from Strickhof, Switzerland, were labeled with 67Zn-enriched isotope (67Zn abundance = 32%) used in a wheat growth trial . Poultry manure and cattle manure in the form of dried pellets were bought from the Landi farmers' shop (https://www.landi.ch). These organic fertilizers were finely ground before their application. Mineral Zn fertilizer was supplied as a ZnSO4 solution. These Zn-containing fertilizers were thoroughly mixed with each labeled soil. The application rate was based on the Zn concentration of the fertilizer, leading to similar amounts of Zn added per kilogram of soil  but different N and C inputs among these treatments  was sown in each pot. As observed in our previous isotope labeling studies on phosphorus and Zn, the root system of ryegrass can colonize well in a pot with a relatively small volume of soil over a short growth time, which is a prerequisite so that the plant shoots can provide a proper estimation of the isotopic signature of the available P and Zn , 55 mg P kg−1 soil (phosphate), 138 mg K kg−1 soil (K+), 31 mg S kg−1 soil (sulfate), 23 mg Mg kg−1 soil (Mg2+), 5 mg Fe kg−1 soil (Fe-EDTA), 232 μg B kg−1 soil (boric acid), 127 μg Mn kg−1 soil (Mn2+), 31 μg Cu kg−1 soil (Cu2+), and 54 μg Mo kg−1 soil (molybdic acid). Distilled water was supplied regularly to the pot by weighing to maintain soil moisture between 40 and 80% water-holding capacity. No drought symptoms were observed on the ryegrass plants throughout the experiment. Each treatment had 4 replicates with 1 pot per replicate. These pots were randomly rearranged after each watering. The shoots were harvested 4 times by cutting at 21, 30, 38, and 48 days after sowing, to minimize the influence of seed Zn on the tracing of fertilizer Zn. After each cut, the nutrient solution was resupplied at a rate of 20% of the first load.The plant growing conditions were similar to those described in Dürr-Auster et al. . BrieflyA soil incubation experiment was carried out in parallel with the growth trial for deploying the DGT sampler to extract Zn from the soil available Zn pool. To this end, one pot per treatment was prepared for each type of soil, but no ryegrass was grown. The pot received only the first load of the abovementioned nutrient solution and was then placed in the same growth chamber as the growth trial. Distilled water was supplied weekly to adjust the soil moisture at 80% water-holding capacity to keep the soil moisture in the same range as for the plant trial. The soil incubation lasted until the fourth cut of the ryegrass .3 for 24 h .After the incubation, 4 beakers, each containing about 50 g of incubated soils , were prepared for each incubated pot, to make 4 replicates. Following the protocol described in Hooda et al. , the soi3 in a high-pressure single-reaction microwave chamber . The same digestion was applied to the dried fertilizers. An aliquot of the digest was purified to extract Zn from the sample matrix. Sample purification was shown to be necessary for Zn isotope analysis with quadrupole single-collector (Q-)ICP-MS in stable Zn isotope soil labeling experiments ICP-MS  for the purified samples and corrected for mass bias using standard sample bracketing, as explained in Dürr-Auster et al. , with i varying from 1 to 4] was obtained from the Zn concentration [CZni (μg g−1 DW)] and the dry weight [DWi (g kg−1 soil)] of the corresponding cut:For each cut of each pot, the Zn uptake in the ryegrass shoots at cut QZni was normalized to 1 kg of soil to describe the transfer of Zn from the soil. The values of DWi, CZni, and QZni in the ryegrass shoot at each cut are shown in QZni was composed of the uptake of Zn derived from the fertilizer [QZndfferti (μg kg−1 soil)], soil [QZndfsoili (μg kg−1 soil)], and seeds [QZndfseedi (μg kg−1 soil)]:Zndfferti (%)], soil [Zndfsoili (%)], and seed [Zndfseedi (%)]add up to 100%:In the ryegrass shoot, the relative proportion of Zn derived from the fertilizer 2−4:Zndfferti and Zndfsoili in the ryegrass shoot at each cut and Zndfseed at cut 1 and cuts 2–4 are shown in The values of QZndfferti and QZndfsoili in the ryegrass shoots at cut i were then calculated:QZndfseed, QZndfferti, and QZndfsoili in the ryegrass shoot at each cut are shown in The values of ZnReci (%)] in the ryegrass shoot at cut i was calculated using:The recovery of Zn from the fertilizer [QZninput is the input of Zn into the soil with the Zn-containing fertilizers ], total Zn uptake [QZnsum (μg kg−1 soil)], and averaged Zn concentration [CZnavg (μg g−1 DW)] in the ryegrass shoots of the 4 cuts are discussed. They were calculated for each pot, as follows:In this paper, the total dry weight  and the soil  of the 4 cuts were calculated:Then, the total uptake and averaged proportion of Zn derived from the fertilizer [ZnRecsum (%)] in the ryegrass shoot at cuts 1–4 was calculated:The total recovery of Zn from the fertilizer [AUE (%)] was also calculated to compare the results to ZnRecsum, which relies only on the relative increase of QZnsum resulting from the addition of Zn with the fertilizer:The apparent fertilizer use efficiency  was calculated following the same principles as for the plant (Equation 5). In the equation, 67Zn:66Zn ratios measured in the DGT extracts were used instead of those in the plant, whereas the soil Zn isotope abundances of 67Zn and 66Zn were measured in the DGT extracts in the no Zn treatment instead of the abundances measured in the ryegrass shoots. In ZndffertDGT values were compared with Zndffert4 values measured in the ryegrass shoots at cut 4 . Meanwhile, ZndffertDGT and Zndffert4 were highly positively correlated in each of the soils (p < 0.05) and in both soils , compared with the no Zn treatment . For the higher shoot dry weight in the Strickhof soil, one explanation would be that the growth of Italian ryegrass was favored under neutral to alkaline soil conditions  may have caused the overall higher shoot dry weight, lower Zn concentration and uptake, and lower hof soil . These rhof soil . The highof soil . The lowZndffertavg and QZndffertsum were significantly higher with the application of the animal manures than with ZnSO4 , followed by the Zn-containing fertilizer (11–20%), and the seed (<2.3%) after 48 days of growth. The plant recovery of Zn derived from freshly applied complex organic fertilizers was low (4.7–12%), whereas the major fraction of fertilizer Zn remained in the soil (>88%). These findings illustrate that complex organic fertilizer may contribute to the buildup of a Zn residual pool in the soil. In both the acidic Heitenried soil and the alkaline Strickhof, animal manures increased the plant uptake of Zn more effectively than the water-soluble ZnSOThe original contributions presented in the study are included in the article/B-FY wrote original draft, formal analyses, data curation, and visualization. TD-A involved in methodology, resources, validation, experiments, and data collection. EF conducted conceptualization, methodology, validation, data curation, writing review and editing, and funding acquisition. MW contributed to review and editing the manuscript and to investigations. All authors contributed to the article and approved the submitted version.This study was funded by the Swiss State Secretariat for Education and Research within the framework of the COST (European Cooperation for Science and Technology) action Mineral-Improved Crop Production for Healthy Food and Feed (FA0905) with the Project Number C10.0085 and by the Group of Plant Nutrition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.