ZmHMA3 enhances Zn stress tolerance and mediates Zn transport in Maize

ZmHMA3 enhances Zn stress tolerance and mediates Zn transport in Maize | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ZmHMA3 enhances Zn stress tolerance and mediates Zn transport in Maize Guihua Lv, Youqiang Li, Jianjian Chen, Zhenxing Wu, Wenmei Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4230201/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Excessive levels of Zn have the potential to be detrimental to plant health. ZmHMA3 , a member of the heavy metal ATPase (HMA) family, is responsible for the transport of Zn 2+ and Cd 2+ across cellular membranes. In order to investigate the role of the ZmHMA3 gene in response to Zn stress, ZmHMA3 knockout mutants were created using the CRISPR-Cas9 technique. Subsequently, gene specific expression, as well as agronomic traits, root morphology indicators, relative conductivity, antioxidant indicators, and Zn content in the leaf, root, and their subcellular components were assessed. The results demonstrated a significant accumulation of ZmHMA3 in both the leaf and root after 48 hours of Zn stress compared to the control group. The Zmhma3 knockout line exhibited decreased tolerance to toxic levels of Zn as compared to the wild type, resulting in a reduction in maize plant height, fresh weight, dry weight, water content, root morphology indicators (Length, SurfArea, AvgDiam, Rootvolume, Tips and Forks) and antioxidant enzyme activity (CAT, POD, SOD, and MDA), while also leading to an increase in membrane permeability and zinc accumulation. In conclusion, it can be inferred that ZmHMA3 likely functions as a crucial positive regulator in the response to Zn stress in maize. Maize (Zea mays) heavy metal ATPase (HMA) Zn stress CRISPR-Cas9 system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The overutilization of Zn fertilizer in agricultural practices, combined with the unregulated expansion of lead-zinc mining activities and the disorderly discharge of industrial wastewater, has raised concerns regarding the pollution caused by the heavy metal zinc. This pollution not only draws attention due to its environmental impact but also poses a direct threat to human health through the contamination of the food chain (Zhou et al. 2020 ). Despite being an essential trace element, necessary for the functioning of more than 1000 transcription factors and structural proteins (Brion, Heyne and Lair 2021 ), excessive levels of Zn can have a toxic effect on plants. This toxicity manifests in various ways, such as a reduction in plant biomass, leaf chlorosis, and inhibition of root growth (Tang et al. 2023 ). Zinc (Zn) uptake by plant roots occurs from the soil and is subsequently transported through the vascular system to the aboveground parts of the plant, where it is further distributed to developing tissues. A complex network in the plant that regulates zinc homeostasis to ensure its growth (Huang et al. 2020 ). The absorption and transport of Zn are primarily regulated by different transporters, mainly controlled by several gene family, including HMAs ((heavy metal ATPases), ZIPs (zinc regulatory transporters, iron regulatory transporter like proteins), YSLs (yellow stripe like proteins), CDFs (cationic diffusion promoters), NRAMPs (natural resistance associated macrophage protein), and MTPs (metal transport protein) (Kimura et al. 2023 ; Ibuot et al. 2020 ; Sinclair and Kramer 2012 ). HMAs, specifically the P-type ATPase protein known as HMA (P18 type), utilize ATP hydrolysis to provide energy for the transport of metal ions (Xu et al. 2023 ). In Arabidopsis, there are eight HMA proteins. Of those, AtHMA1 is localized in the inner membrane of chloroplasts and facilitates the detoxification of zinc (II) by reducing the zinc content within plastids (Kim et al. 2009 ); AtHMA2 and AtHMA4 are predominantly expressed in the root vasculature and play a crucial role in the translocation of Zn from roots to shoots through xylem loading. The double mutants hma2 and hma4 exhibit a significant accumulation of zinc in the underground, but severe zinc deficiency in the aboveground (Escudero et al. 2022 ); AtHMA3 is situated on the vacuolar membrane and is primarily responsible for transferring Zn ions into the vacuole (Morel et al. 2009 ). Additionally, MaHMA2 has been found to play an important role in the tolerance response of mulberry plants to Zn-induced stress (Wang et al. 2022 ). Maize ( Zea mays L.), the most cultivated crop globally, is highly susceptible to zinc (Zn) deficiency (Zhang et al. 2020 ). In areas with high Zn levels in the soil, enhancing Zn resistance and reducing grain Zn content are critical for ensuring maize yield and quality. ZmHMA3 , which exhibits homology with OsHMA2/3 and AtHMA2/3 , was found to regulate the accumulation of cadmium (Cd) in maize leaves through association analysis and linkage mapping (Cao et al. 2019 ). As HMA3 is a transporter protein for both Cd and Zn, we aimed to investigate whether the ZmHMA3 gene is also involved in maize's response to Zn stress. To address this, knockout mutant lines were generated to investigate the function of ZmHMA3 in the tolerance, absorption, and transport of excessive Zn in maize under Zn stress conditions in this study. Materials and methods Plant materials and Zn stress treatment B104 maize seeds were first washed with deionized water, then soaked in 20% H 2 O 2 and shaken for 20 minutes. After washed 4–6 times with deionized water for 3 minutes each time, the seeds were soaked overnight with saturated calcium sulfate to induce germination. The next day, they were sprouted in an artificial climate chamber using germination paper. At the second leaf (V2) stage, B104 maize seedlings were randomly allocated into two groups: the control group and the Zn stress group. The Zn stress group was exposed to a concentration of 800 µmol/L ZnSO 4 ·7H 2 O for 0 hours and 48 hours, respectively (Liao et al. 2023 ). Expression analysis of ZmHMA3 using Real-Time Quantitative PCR (qRT-PCR) Total RNA from the roots and leaves of B104 maize seedlings was extracted from each group with triplicates at 0 hours and 48 hours using TRIzol reagent (Invitrogen, Gaithersburg, MD, USA), and first-strand cDNA synthesis was performed using the primeScript TMRT 1st Strand cDNA Synthesis kit (Code Ds10A, TaKaRa, Kyoto, Japan). Subsequently, the qRT-PCR amplification program and the subcellular localization assay of ZmHMA3 were carried out according to our previous study (Liao et al. 2023 ). For the qRT-PCR assay, the relative expression level of ZmHMA3 under different treatment conditions (with three technical replicates for each sample) was analyzed using the 2 −ΔΔCT method (Livak and Schmittgen 2001 ). All primers employed in this study are listed in Supplementary Table S1 . Zmhma3 (knock-out line) constuction Zmhma3 mutants in the B104 maize line were generated using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology, following the previously described method (Ma et al. 2015 ). Briefly, single-guide RNAs (sgRNAs) were designed using the CRISPR-GE tool ( http://skl.scau.edu.cn/ ) to target the first exon of ZmHMA3 . The CRISPR/Cas9 vector was cleaved using both SacI and EcoRI enzymes, where the sgRNAs were then cloned into. These constructs were transformed into Agrobacterium tumefaciens EHA105 for inoculation with the B104 maize inbred line, as previously reported (Ishida, Hiei and Komari 2020 ). The resulting T 3 generation homozygous mutant seeds were obtained through endosperm cutting, sanger sequencing analysis, and qRT-PCR, and were subsequently used as experimental materials in the study. Physiological and antioxidant activity analysis To analyze physiological and antioxidant activities, the second leaf (V2) stage of B104 maize seedlings (wild type, WT) and Zmhma3 knock-out line (Knock-out) were subjected to treatment with 800µmol/L ZnSO 4 ·7H 2 O for 0, 24, and 48 hours, respectively, with H 2 O treatment as a mock control. Following the treatments, the agronomic traits, relative conductivity, and antioxidant indicators were determined as described below: Agronomic traits were assessed by weighing the fresh weight (Wf) of both aboveground and underground plant parts. Subsequently, the samples were dried at 80 ℃ to a constant weight and weighed as the dry weight (Wd). Plant moisture content (WC) was calculated as (Wf - Wd)/Wf * 100%. The relative reduction rate of water content was calculated as (WC0 - WC)/WC * 100%, where WC0 represents the water content on day 0. Relative conductivity was evaluated following a previously established method (Liang et al. 2023 ). Firstly, 0.3g clean fresh leaf samples were cut into sections. After soaked in a dark place for 5 hours with 30 mL deionized water, the conductivity (R1) was measured. Then 95 ℃ water bath were taken out for 1 hour. And after cooled to room temperature, the conductivity (R2) was measured. The calculation formula of relative conductivity was as followed: relative conductivity = R1/R2/W × 100%. Roots were scanned using an EPSON Expression 12000XL root scanner, and the resulting images were analyzed using TAIR high-throughput analysis software. The levels of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and malondialdehyde (MDA) were quantified using a Biochemical Reagent Kit (Solarbio, Beijing, China), following a previously described protocol (Chen et al. 2020 ). Each sampling is repeated with five plants, and 3 replicates were set. Determination of Zn ion content To measure the Zn ion content, 0.2g of dry samples were digested with 10 milliliters of nitric acid. The concentration of elements in the digestion solution was then determined using inductively coupled plasma mass spectrometry (ICP-MS 7700 X), following a previously established method (Ning et al. 2023 ). To determine the content of Zn ions in different subcellular components, a differential centrifugation technique was employed to separate these components. Specifically, 1g of fresh sample was ground into a homogenate using 10 mL of extraction buffer consisting of 250 mmol · L − 1 sucrose, 50 mmol · L − 1 Tris HCl (pH 7.5), and 1mmol · L − 1 dithio red wine. The homogenate was then subjected to centrifugation at a speed of 3000r/min for 15 minutes, resulting in the precipitation of the cell wall component (F1). Subsequently, the supernatant was subjected to a second centrifugation at a speed of 11000r/min for 45 minutes. The precipitate obtained from this second centrifugation step represented the membrane and organelle parts (F3), while the supernatant was considered the soluble component/cytosol and cytoplasm component (F2). Results Expression characteristics under Zn stress tolerance of ZmHMA3 in maize In the previous association analysis study, we obtained a candidate gene ZmHMA3 related to the accumulation of heavy metal Cd in leaves (Cao et al. 2019 ). Due to HMA3 being a Cd/Zn transporter protein, we have focused on studying the function of ZmHMA3 under Cd stress, and by the way, confirmed that overexpression of ZmHMA3 led to a higher Zn accumulation (Liao et al. 2023 ). To systematically verify the gene function of ZmHMA3 under Zn stress in maize, expression characteristics under Zn stress were firstly taken out. The examination of the expression pattern of the ZmHMA3 gene demonstrated an elevated expression level in both leaves and roots after 48 hours of exposure to Zn stress compared to the control group without Zn stress (Fig. 1 A). It suggests a potential involvement of ZmHMA3 in the response to Zn stress. CRISPR/Cas9-mediated knock-out of ZmHMA3 decreased tolerance in Zn stress To investigate the functional role of ZmHMA3 in response to Zn stress, ZmHMA3 knock-out (KO) mutants were generated using CRISPR-Cas9 technology, in which ZmHMA3 gene was rare or even not expressed (Fig. 1 B). Three guide-RNAs (gRNAs) were designed to target the first two exons of ZmHMA3 (Fig. 2 A). In the T 0 generation, 54 chimeric and heterozygous mutant maize lines harboring double-site and single-site mutations were identified. Among these, three stable lines without Cas9 were selected as ZmHMA3 homozygous mutants through sanger sequencing analysis and endosperm cutting in the T 2 generation. The mutations in these lines resulted in deletions of 11bp, 65bp, and 34bp between the gRNA1 and gRNA2 sequences, leading to frameshift mutations and subsequent alterations in the sequence and conformation of the ZmHMA3 protein (Fig. 2 B and 2 C). One of these lines, Zmhma3-2 , was chosen as the Zmhma3 knock-out (KO) line and was further analyzed in the T 3 generation. Subsequently, the relative expression levels and agronomic traits of maize seedlings under varying degrees of Zn stress were compared. Both wild-type (WT) and Zmhma3 plants were subjected to 800 µmol/L ZnSO 4 ·7H 2 O solutions for specific time periods (0 hour, 24 hours, and 48 hours). The growth phenotypes of WT and Zmhma3 indicated that both experienced wilting after 48 hours of Zn stress treatment, but the extent of wilting was more prominent in Zmhma3 compared to WT plants (Fig. 2 D). ZmHMA3 affects the seedling growth vigor and leaf cell membrane permeability of maize under Zn stress The agronomic traits of seedlings reflect their growth potential, such as the amount of water content in plants, which often reflects the strength of their life activities. Cell membrane permeability suggests the degree of lipid peroxidation in plant leaf membranes, and the relative conductivity of leaves is positively correlated with the degree of stress on the plant. We subjected the wild-type and Zmhma3 mutants to Zn stress (800µmol/L ZnSO 4 ·7H 2 O) treatment, whose groups were named Zn-WT and Zn- Zmhma3. And the control groups with H 2 0 treatment were named CK-WT and CK- Zmhma3 . In order to assess the agronomic traits of Zmhma3 and WT plants under Zn stress at different time points, various physiological indices including seedling fresh weight (SFW), seedling dry weight (SDW), seedling water content (SWC), root fresh weight (RFW), root dry weight (RDW), and root water content (RWC) were measured. After 24 hours of Zn treatment, there was no significant difference in shoots, roots, and total fresh and dry weight between WT and Zmhma3 . After 48h, differences began to appear among different treatment groups. Under 48 hours stress, Zmhma3 plants exhibited significantly lower SFW, SDW, SWC, RFW, and RWC compared to WT plants, while RDW showed no significant difference between the two genotypes (Fig. 3 A and 3 B). Similar trends were observed for total fresh weight (TFW), total dry weight (TDW), and total water content (TWC), which were consistent with RFW, RDW, and RWC (Fig. 3 C). Based on the water content, the relative reduction rate of water content for each treatment group at 24 and 48 h can be calculated compared to 0 h. Whether in the aboveground, underground, or entire plant, it can be seen that, after 48h stress, the relative reduction rate of water content in Zn- Zmhma3 was greater than that in Zn-WT, followed by CK- Zmhma3 , with CK-WT showing the smallest reduction rate (Fig. 3 D). These findings suggest that the knockout of ZmHMA3 resulted in a greater decrease in fresh weight and water content in plants under Zn stress. Furthermore, the plant height of WT and Zmhma3 showed no significant difference compared to the control during Zn stress for 24 hours. But at 48 hours under Zn stress, the plant height of Zmhma3 was significantly smaller than that of WT, indicating that the knockout of ZmHMA3 enhanced the inhibitory effect of Zn stress on maize growth (Fig. 3 E). Additionally, the relative electrical conductivity of Zmhma3 increased after Zn treatment and was significantly higher than that of WT (Fig. 3 F). This suggests that Zmhma3 plants experienced more severe damage than WT plants under Zn stress. ZmHMA3 affects the root growth and architecture of maize under Zn stress Root is the first organ to contact with heavy metals in soil in plants, which can absorb the heavy metals into root cells, thereby transferring to the above ground of the plant, and then causing the antioxidant activity change. We detected Zn heavy metal content in WT and Zmhma3 at different times of treatments. With the prolongation of stress time, For WT plants, the root length (Fig. 4 A) and surface area (Fig. 4 B) were greater than Zmhma3 before Zn stress, but there was no difference in root diameter (Fig. 4 C), root volume (Fig. 4 D), number of root tips (Fig. 4 E), and number of forks (Fig. 4 F). After Zn stress, the six root phenotype indicators were greater than those of Zmhma3 . With the prolongation of stress time, except for a decrease in root length, all other indicators of CK-WT showed an upward trend. Except for the length and forks that first increase and then decrease, all other indicators of CK- Zmhma3 also showed an upward trend. Zn stress treatment maintained the length, surface area, diameter, and volume of Zn-WT at a level similar to 0h, while the number of root tips and forks still increased. Except for tips, which first decreased and then increased, all other indicators of Zn- Zmhma3 showed a trend of first increasing and then decreasing. These results indicated that knocking out ZmHMA3 exacerbated the inhibition of root growth under Zn stress. The root antioxidant related enzyme activity was repressed in Zmhma3 Antioxidant enzymes and MDA are closely related to plant stress. CAT, SOD, and POD are effective antioxidant enzymes. And by detecting MDA, the level of membrane lipid peroxidation in plants under adverse conditions can be detected. To determine the resistance to Zn stress, we measured various antioxidant indicators of roots in WT and Zmhma3 under Zn stress. For antioxidant activity assay, the results showed that CAT content increased after 48h Zn stress in Zn-WT and Zn- Zmhma3 , and was significantly higher than that in CK-WT and CK- Zmhma3 (Fig. 5 A). The POD content of CK-WT and CK- Zmhma3 tended to stabilize during the treatment, while that of Zn-WT increased, and that of Zn- Zmhma3 decreased. Furthermore, Zn-WT was significantly greater than Zn- Zmhma3 (Fig. 5 B). At 0h, there was no significant difference between the treatment groups. At 24h, the SOD content of Zn- Zmhma3 began to significantly decrease and was significantly lower than CK-WT, CK- Zmhma3 , and Zn-WT. At 48h, the content of CK-WT, Zn-WT, and Zn- Zmhma3 decreased. The SOD content of CK- Zmhma3 was the highest, followed by CK-WT, then Zn-WT, and that of Zn- Zmhma3 was the lowest (Fig. 5 C). The MDA content of Zn-WT significantly increased after treatment, with Zn- Zmhma3 initially increasing and then decreasing, resulting in a significantly higher content of Zn-WT than Zn- Zmhma3 (Fig. 5 D). The content of CAT, POD, SOD, and MDA was higher in WT than that in Zmhma3 , which suggested that knocking out ZmHMA3 leads to a decrease in plant antioxidant enzyme activity under zinc stress. The Zn content and transport rate was increased in Zmhma3 Under heavy metal stress, the growth and development of corn are inhibited. As the duration of stress prolongs, the symptoms of victimization first manifest as wilting. The first plant organ that heavy metals come into contact with is the root, which is stored in the root and transferred to the aboveground part of the plant. The results of the study revealed that Zmhma3 plants accumulated higher Zn content compared to WT plants (Fig. 6 ), particularly in the underground parts of the plant. The Zn content in the underground parts of Zmhma3 plants was significantly higher than that in the aboveground parts after 48 hours of stress (Fig. 6 A). Furthermore, the leaf, root, and total Zn content in Zmhma3 plants were higher than those in WT plants, which corresponded to the trend observed in the transport coefficient (Fig. 6 A and 6 B). In addition, the changes in Zn content in root cells and leaf cells followed a similar trend. After Zn treatment, the Zn content in F2 (soluble part, cell sap, and cytoplasmic matrix) and F3 (cell membrane and organelle) of WT plants was significantly higher than that in Zmhma3 plants, while the Zn content in F1 (cell wall) was significantly lower in WT plants compared to Zmhma3 plants (Fig. 6 C). The Zn content in the leaves and roots of Zmhma3 plants was much higher than that in WT plants, but the Zn content within the cells was lower. Discussion With the mining of ores, industrial emissions, and the application of pesticides and fertilizers, the problem of soil heavy metal zinc pollution has become prominent in recent years(Moffat 1995 ). Zinc entering the soil is difficult to move, and natural purification time can even take about 1000 years. After the soil is polluted, it directly causes the soil to lose its natural productivity. The use of tolerance and hyperaccumulating plants for excessive zinc absorption has become a research focus for soil scientists and environmentalists (Chaney et al. 1997 ). As a zinc sensitive crop, maize is significantly affected by zinc, and the difference between concentration gradients is significant. Low concentration of zinc promotes growth, high concentration inhibits growth, and medium concentration is in a transition state (Wang 2008 ). In addition to the toxic effects on crops themselves, the most important thing is that these crops accumulate in the human body through the food chain after being consumed by humans, which can cause serious health and safety issues (Nordberg 2009 ). Although the absorption and transport mechanisms of heavy metals in plants are not yet fully understood, it is still possible to effectively reduce the concentration of heavy metals in plants by regulating the transport proteins that absorb these metals. Among numerous metal transporter families, HMAs are widely present in lower and higher plants and participate in the absorption, transportation, and accumulation of essential trace metal nutrients during plant growth and development (Morel et al. 2009 ; Wong and Cobbett 2009 ). HMA has been shown to participate in the transportation of various heavy metal cations (Williams and Mills 2005 ). In Arabidopsis, AtHMA1-4 mediate the transport of divalent heavy metal ions (Zn 2+ /Co 2+ /Cd 2+ /Pb 2+ ), while AtHMA5-8 transport Cu + and Ag + (Woeste and Kieber 2000 ; Hirayama et al. 1999 ; Axelsen and Palmgren 2001 ). In rice, OsHMA1-3 belong to Zn /Co/Cd/Pb transporters (Takahashi et al. 2012 ). Previous studies have found that ZmHMA3 protein has 72% homology with OsHMA3 protein and 62% homology with OsHMA2 protein. The homology with Arabidopsis AtHMA2 and AtHMA3 is 47% and 52% respectively. It belongs to the same Zn /Co/Cd/Pb transporter and participats in numerous heavy metal transport (Cao et al. 2019 ). However, the mechanism of action of the ZmHMA3 gene under Zn stress is still unclear. Therefore, this study conducted functional verification on ZmHMA3 gene. Analyzing the spatiotemporal expression pattern of ZmHMA3 gene is of great significance for inferring its specific function. Under normal growth conditions, ZmHMA3 demonstrates tissue specificity in nodes, anthers, and roots (Cao et al. 2019 ). In this study, the expression of ZmHMA3 was found to be upregulated in the roots and stems under Zn stress, consistent with previous research conducted on OsHMA2 and AtHMA2 (Das et al. 2021 ). This suggests that ZmHMA3 may play a positive regulatory role in enhancing resistance to Zn stress. Previous study demonstrated that Zinc stress has inhibited plant growth (Yu et al. 2023 ). Following a 48-hour Zn stress treatment on both wild-type (WT) and ZmHMA3 knockout mutants, the mutants displayed initial wilting. Subsequently, morphological characteristics such as fresh weight, dry weight, water content, plant height, and relative conductivity were measured. The results indicated that the knockout of ZmHMA3 significantly impeded maize growth under Zn stress. Previous studies primarily focused on measuring root morphological traits, such as root length and weight (Zhang et al. 2019 ; Ahmad et al. 2023 ). However, in this study, a root scanner was employed to specifically measure root diameter, root volume, and other traits, providing a more comprehensive evaluation of root morphological indicators. Overall, the WT exhibited better root growth compared to the mutant. This difference may be attributed to the disruption or alteration of ZmHMA3 protein function, which reduces or eliminates the ability of plant root cells to transport or excrete Zn. Consequently, Zn content accumulates, leading to root system toxicity and inhibition of root growth. Heavy metal stress is similar to other forms of oxidative stress, which can lead to the production of a large number of reactive oxygen species, damage major biological macromolecules, and cause membrane lipid peroxidation (Shan, Luo and Frances 1997 ). Various antioxidant mechanisms in plants can eliminate free radicals to protect cells. Among them, SOD, CAT, POD, and MDA are the most common antioxidant systems in plants. Plants employ various antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and malondialdehyde (MDA), to eliminate free radicals and protect cells. POD in plants plays a crucial role in the cross-linking of hydroxyproline-rich glycoproteins with carbolic acid, increasing cell wall strength and thereby countering Zn 2+ -induced osmotic stress (Chao et al. 2008 ). In the Zn hyperaccumulation ecotype Sedum alfredii , the activities of SOD, CAT and GPX increased under high Zn stress, surpassing those observed in the non-hyperaccumulation ecotype (Jin et al. 2008 ). In the present study, the overall antioxidant capacity of the WT was higher than that of Zmhma3 . The ZmHMA3 mutation resulted in a decrease in antioxidant enzyme activity in maize, which may also be one of the reasons for the decrease in Zn stress tolerance. Following Zn stress, the Zn content in ZmHMA3 leaves and roots, as well as the transport coefficient, were significantly higher compared to the WT. Furthermore, the Zn content in the roots was notably higher than in the leaves. Similar results were observed in Arabidopsis HMA2 knockout mutants (Eren and Argüello 2004 ). Interestingly, in contrast to plants, the Zn content in ZmHMA3 protoplasts was lower than in the WT, while the Zn content in the cell wall was higher. This means that the Zn content of Zmhma3 in leaves and roots is higher than that in WT, but the total Zn content in leaf and root cells is lower than that in WT. It is generally understood that, after heavy metals enter the plant body, the main detoxification mechanisms of plants include elimination from the body and intracellular compartmentalization. Zn precipitation on the cell wall acts as a barrier, preventing more ions from entering the cell protoplasts and causing harm (Nishizono et al. 1987 ; Küpper, Lombi and Mcgrath 2000 ). HMA2 enhances plant Zn tolerance by actively expelling excess Zn ions into the extracellular space (Eren and Argüello 2004 ). AtHMA3 is involved in the vacuolar compartmentalization of various heavy metals, such as Zn and Pb (Morel et al. 2009 ), while OsHMA3 is involved in the vacuolar compartmentalization of Cd (Miyadate et al. 2011 ). Unlike AtHMA3 , which is localized on the vacuolar membrane, ZmHMA3 is primarily located on the cell membrane, which aligns with the localization of AtHMA2 (Morel et al. 2009 ; Eren and Argüello 2004 ; Liao et al. 2023 ). Through querying the ZmHMA3 protein in the Maize GDB, it was revealed that there are amino acid motifs near the termination codon that contain phosphorylation sites. These motifs are mainly involved in providing energy for the transmembrane transport of heavy metals. This phosphorylation site is closely associated with the transmembrane domains H6 and H7 of the HMA protein. It is likely that ZmHMA3 is involved in the transport of Zn ions into and out of plant cells. Based on the findings of this study, ZmHMA3 may be involved in Zn transport into cells through the cell membrane. Although the concentration of zinc ions in the entire plant is relatively high, the concentration in protoplasts is low. This discrepancy likely leads to differences in osmotic pressure between the inside and outside of cells, resulting in cell dehydration and a decrease in ZmHMA3 water content. Zn imbalance leads to the accumulation of reactive oxygen species (ROS) and the displacement of other elements from protein active sites (Zhang et al. 2020 ). Moreover, zinc interacts with other elements in plants, and zinc toxicity typically induces steady-state changes in numerous other elements, including Fe, Ca, and P (Gupta, Ram and Kumar 2016). Therefore, measuring ion content changes related to intracellular environmental homeostasis may provide more insights into the mechanism of action of ZmHMA3 . Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Zhejiang Key R&D Program (NO. 2021C02057); Science and Technology Plan Project of Zhejiang Provincial (NO.2022C04024) Author contributions Conceptualization: Guihua Lv, Tingzhen Wang; Methodology: Youqiang Li, Jianjian Chen; Formal analysis and investigation: Youqiang Li, Zhenxing Wu; Writing - original draft preparation: Guihua Lv, Youqiang Li; Writing - review and editing: Tingzhen Wang, Wenmei Wu; Resources: Xiaohong Wu; Supervision: Haijian Lin, Guihua Lv Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Ahmad I, Rawoof A, Priyanka, Islam K, Momo J, Anju T, Kumar A, Ramchiary N (2023) Diversity and expression analysis of ZIP transporters and associated metabolites under zinc and iron stress in Capsicum. Plant Physiol Biochem 196: 415-430. https://doi.org/10.1016/j.plaphy.2023.01.060 Axelsen KB, Palmgren MG (2001) Inventory of the superfamily of p-type ion pumps in arabidopsis. Plant Physiol 126:696-706. https://doi.org/10.1104/pp.126.2.696 Brion LP, Heyne R, Lair CS (2021) Role of zinc in neonatal growth and brain growth: review and scoping review. Pediatr Res 89:1627-1640. https://doi.org/10.1038/s41390-020-01181-z Cao Y, Zhao X, Liu Y, Wang Y, Wu W, Jiang Y, Liao C, Xu X, Gao S, Shen Y, Lan H, Zou C, Pan G, Lin H (2019) Genome-wide identification of z mhmas and association of natural variation in zmhma2 and zmhma3 with leaf cadmium accumulation in maize. Peerj 7: e7877. https://doi.org/10.7717/peerj.7877 Chaney RL, Malik M, Li YM, Brown S, Brewer E, Angle J, Baker AJM (1997) Phytoremediation of soil metals. Curr Opin Biotechnol 8:279-284. https://doi.org/10.1016/s0958-1669(97)80004-3 Chao W, Song HZ, Pei FW, Hou J, Wei L, Wen JZ (2008) Metabolic adaptations to ammonia-induced oxidative stress in leaves of the submerged macrophyte Vallisneria natans (lour.) Hara. Aquat Toxicol 87:88-98. https://doi.org/10.1016/j.aquatox.2008.01.009 Chen H, Lai L, Li L, Liu L, Jakada BH, Huang Y, He Q, Chai M, Niu X, Qin Y (2020) AcoMYB4 , an Ananas comosus L. MYB Transcription Factor, Functions in Osmotic Stress through Negative Regulation of ABA Signaling. Int J Mol Sci 21(16). https://doi.org/10.3390/ijms21165727. Das U, Haque AFMM, Bari MA, Mandal A, Kabir AH (2021) Computational characterization and expression profile of MTP1 gene associated with zinc homeostasis across dicot plant species. Gene Reports 23: 101073. doi: https://doi.org/10.1016/j.genrep.2021.101073. Eren E, Argüello JM (2004) Arabidopsis hma2, a divalent heavy metal-transporting pib-type atpase, is involved in cytoplasmic Zn 2+ homeostasis. Plant Physiol 136(3): 3712-3723. https://doi.org/10.1104/pp.104.046292 Escudero V, Ferreira SD, Abreu I, Sopeña-Torres S, Makarovsky-Saavedra N, Bernal M, Krämer U, Grolimund D, González-Guerrero M, Jordá L (2022) Arabidopsis thaliana Zn 2+ -efflux ATPases HMA2 and HMA4 are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina BMM. J Exp Bot 73(1): 339-350. https://doi.org/10.1093/jxb/erab400. Wang YF (2008) Physiological and ecological characteristics of corn under different anionic forms of zinc compounds stress. Dissertation, Shandong University Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D (1998) Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci U S a 95:7220-7224. https://doi.org/10.1073/pnas.95.12.7220 Hirayama T, Kieber JJ, Hirayama N, Kogan M, Guzman P, Nourizadeh S, Alonso JM, Dailey WP, Dancis A, Ecker JR (1999) Responsive-to-antagonist1, a menkes/wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis . Cell 97:383-393. https://doi.org/10.1016/s0092-8674(00)80747-3 Huang S, Sasaki A, Yamaji N, Okada H, Mitani-Ueno N, Ma JF (2020) The zip transporter family member OsZIP9 contributes to root zinc uptake in rice under zinc-limited conditions. Plant Physiol 183:1224-1234. https://doi.org/10.1104/pp.20.00125 Ibuot A, Webster RE, Williams LE, Pittman JK (2020) Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein. Biotechnol Bioeng 117(10): 2996-3005. https://doi.org/10.1002/bit.27476. Ishida Y, Hiei Y, Komari T (2020) Tissue culture protocols for gene transfer and editing in maize ( Zea mays L.). Plant Biotechnol (Tokyo) 37(2): 121-128. https://doi.org/10.5511/plantbiotechnology.20.0113a. Jin XF, Yang XE, Islam E, Liu D, Li J (2008) Ultrastructural changes, zinc hyperaccumulation and its relation with antioxidants in two ecotypes of sedum alfredii hance. Plant Physiol Biochem 46:997-1006. https://doi.org/10.1016/j.plaphy.2008.06.012 Kim YY, Choi H, Segami S, Cho HT, Lee Y (2009) Athma1 contributes to the detoxification of excess zn(ii) in Arabidopsis . The Plant J 58:737-753. https://doi.org/10.1111/j.1365-313X.2009.03818.x Kimura S, Vaattovaara A, Ohshita T, Yokoyama K, Yoshida K, Hui A, Kaya H, Ozawa A, Kobayashi M, Mori IC, Ogata Y, Ishino Y, Sugano SS, Nagano M, Fukao Y (2023) Zinc deficiency-induced defensin-like proteins are involved in the regulation of root growth in Arabidopsis . Plant J. https://doi.org/10.1111/tpj.16281 Küpper H, Lombi E, Mcgrath Z (2000) Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212:75-84. https://doi.org/10.1007/s004250000366 Liang L, Ze M, Yang J, Xu Q, Du C, Hu X, Dong M, Zou L, Qi T (2023) Physiological and transcriptomic response reveals new insight into manganese tolerance of Celosia argentea Linn. J Hazard Mater 465: 133079. https://doi.org/ 10.1016/j.jhazmat.2023.133079. Liao C, Li Y, Wu X, Wu W, Zhang Y, Zhan P, Meng X, Hu G, Yang S, Lin H (2023) ZmHMA3 , a Member of the Heavy-Metal-Transporting ATPase Family, Regulates Cd and Zn Tolerance in Maize. Int J Mol Sci 24(17). https://doi.org/10.3390/ijms241713496. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408. https://doi.org/10.1006/meth.2001.1262. Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant 8(8): 1274-1284. https://doi.org/10.1016/j.molp.2015.04.007. Miyadate H, Adachi S, Hiraizumi A, Tezuka K, Nakazawa N, Kawamoto T, Katou K, Kodama I, Sakurai K, Takahashi H, Satoh-Nagasawa N, Watanabe A, Fujimura T, Akagi H (2011) OsHMA3 , a p1b-type of atpase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles. New Phytol 189:190-199. https://doi.org/10.1111/j.1469-8137.2010.03459.x Moffat AS (1995) Plants proving their worth in toxic metal cleanup. Science 269:302-303. https://doi.org/10.1126/science.269.5222.302 Morel M, Crouzet J, Gravot A, Auroy P, Leonhardt N, Vavasseur A, Richaud P (2009) AtHMA3, a p1b-atpase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis . Plant Physiol 149:894-904. https://doi.org/10.1104/pp.108.130294 Ning M, Liu SJ, Deng F, Huang L, Li H, Che J, Yamaji N, Hu F, Lei GJ (2023) A vacuolar transporter plays important roles in zinc and cadmium accumulation in rice grain. New Phytol 239(5): 1919-1934. https://doi.org/10.1111/nph.19070. Nishizono H, Ichikawa H, Suziki S, Ishii F (1987) The role of the root cell wall in the heavy metal tolerance ofathyrium yokoscense. Plant Soil 101:15-20. https://doi.org/10.1007/BF02371025 Nordberg GF (2009) Historical perspectives on cadmium toxicology. Toxicol Appl Pharmacol 238:192-200. https://doi.org/10.1016/j.taap.2009.03.015 Shan WY, Luo GH, Frances K (1997) Peroxidation damage of oxygen free radicals induced by cadmium to plant. Acta Botanica Sinica 39:522-526 (in Chinese) Sinclair SA, Kramer U (2012) The zinc homeostasis network of land plants. Biochim Biophys Acta 1823:1553-1567. https://doi.org/10.1016/j.bbamcr.2012.05.016 Takahashi R, Bashir K, Ishimaru Y, Nishizawa NK, Nakanishi H (2012) The role of heavy-metal atpases, hmas, in zinc and cadmium transport in rice. Plant Signal Behav 7:1605-1607. https://doi.org/10.4161/psb.22454 Tang Z, Wang HQ, Chen J, Chang JD, Zhao FJ (2023) Molecular mechanisms underlying the toxicity and detoxification of trace metals and metalloids in plants. J Integr Plant Biol 65(2): 570-593. https://doi.org/10.1111/jipb.13440. Wang L, Du Q, Shi Y, Ackah M, Guo P, Zheng D, Wu M, Jin X, Li P, Zhang Q, Li R, Yin Z, Zhao M, Zhao W (2022) Response of MaHMA2 gene expression and stress tolerance to zinc stress in mulberry (Morus alba L.). BIOCELL 46(10): 2327-2342. https://doi.org/10.32604/biocell.2022.021542 Williams LE, Mills RF (2005) P(1b)-atpases--an ancient family of transition metal pumps with diverse functions in plants. Trends Plant Sci 10:491-502. https://doi.org/10.1016/j.tplants.2005.08.008 Woeste KE, Kieber JJ (2000) A strong loss-of-function mutation in ran1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype. THE PLANT CELL ONLINE 12:443-455. https://doi.org/10.1105/tpc.12.3.443 Wong C, Cobbett CS (2009) Hma p-type atpases are the major mechanism for root-to-shoot Cd translocation in Arabidopsis thaliana. New Phytol 181:71-78. https://doi.org/10.1111/j.1469-8137.2008.02638.x Xu W, Huang H, Li X, Yang M, Chi S, Pan Y, Li N, Paterson AH, Chai Y, Lu K (2023) CaHMA1 promotes Cd accumulation in pepper fruit. J Hazard Mater 460: 132480. https://doi.org/10.1016/j.jhazmat.2023.132480. Yu L, Tang S, Kang J, Korpelainen H, Li C (2023) Responses of dioecious Populus to heavy metals: a meta-analysis. Forestry Research 3(1). https://doi.org/10.48130/FR-2023-0025. Zhang H, Yang J, Li W, Chen Y, Lu H, Zhao S, Li D, Wei M, Li C (2019) PuHSFA4a enhances tolerance to excess zinc by regulating reactive oxygen species production and root development in Populus. Plant Physiol 180(4): 2254-2271. https://doi.org/10.1104/pp.18.01495. Zhang L, Yan M, Li H, Ren Y, Zhang S (2020) Effects of zinc fertilizer on maize yield and water-use efficiency under different soil water conditions. Field Crops Res 248. https://doi.org/10.1016/j.fcr.2020.107718 Zhou M, Xiao L, Yang S, Wang B, Shi T, Tan A, Wang X, Mu G, Chen W (2020) Cross-sectional and longitudinal associations between urinary zinc and lung function among urban adults in China. Thorax 75(9): 771-779. https://doi.org/10.1136/thoraxjnl-2019-213909. Supplementary Files TableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Apr, 2024 Reviewers invited by journal 16 Apr, 2024 Editor invited by journal 09 Apr, 2024 Editor assigned by journal 09 Apr, 2024 First submitted to journal 06 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4230201","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291661769,"identity":"87bd1173-32a0-4611-9efe-4ce97077e4e9","order_by":0,"name":"Guihua Lv","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guihua","middleName":"","lastName":"Lv","suffix":""},{"id":291661770,"identity":"2726f420-b74e-47d4-bd0d-e718cc902fa2","order_by":1,"name":"Youqiang Li","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Youqiang","middleName":"","lastName":"Li","suffix":""},{"id":291661771,"identity":"5087fe03-56eb-4220-8b98-bd62071cea27","order_by":2,"name":"Jianjian Chen","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jianjian","middleName":"","lastName":"Chen","suffix":""},{"id":291661772,"identity":"b75b0c02-8d37-4c6a-89be-53093eee1901","order_by":3,"name":"Zhenxing Wu","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhenxing","middleName":"","lastName":"Wu","suffix":""},{"id":291661773,"identity":"76ad9a52-37fe-4e56-8051-16dad90e26c9","order_by":4,"name":"Wenmei Wu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenmei","middleName":"","lastName":"Wu","suffix":""},{"id":291661774,"identity":"907d3227-0705-437e-8807-7d5f086891f9","order_by":5,"name":"Xiaohong Wu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Wu","suffix":""},{"id":291661775,"identity":"01b4b6c4-904b-43f8-b5be-b81d38c0e8f3","order_by":6,"name":"Haijian Lin","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haijian","middleName":"","lastName":"Lin","suffix":""},{"id":291661776,"identity":"c3e44699-7fc9-4b84-951d-c6252ee929dd","order_by":7,"name":"Tingzhen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie3RsWrDMBCA4RMGZ7F3hdKYvIGNIHQI9FWkxVMogULxEMoFg73kBQKlj9H5zIG6mM7ZmjRDVvcNmoSMwWq3Dvo2wf0I6QA8759qumI6SgY1kS7kKHHOhyBYtrnKVq2hrr1TGbqTgOKKDW60atZVYYAcRfpZb2lY5WaJWnP8KrXAYPe16UssaJp/TFUNDXH8Jh8GECo160kmFojkU367PF5zSh4FRuFNfyKQopAFMqQcv0hzOjqSACiqeIwW0maNv0jubQ6XT440dVaqrHS8ZVi+77/Pq0wO3OnF83Gn5W7fl1wR/G3c8zzPu+IHX4FYngxdeHgAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Tingzhen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-04-07 07:46:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4230201/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4230201/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54900535,"identity":"44816f18-2914-4cf8-9bd5-79d72d104c56","added_by":"auto","created_at":"2024-04-18 10:12:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":204714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression pattern of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmHMA3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder Zn stress\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003e(A) Expression analysis of \u003cem\u003eZmHMA3\u003c/em\u003e using qRT-PCR. (B) Relative expression of \u003cem\u003eZmhma3-2\u003c/em\u003ehomozygous mutant line and WT in leaf and root of maize. Standard error is indicated. The ** represent significant differences as determined using Student’s \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/6e7411ff58298d3d78140f75.png"},{"id":54900532,"identity":"138c597f-da88-4dee-a0eb-0eb27ed50021","added_by":"auto","created_at":"2024-04-18 10:12:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":663110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9 system and knock-out phenotype of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmHMA3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in maize.\u003c/strong\u003e (A) Schematic diagram of CRISPR/cas9 target site in \u003cem\u003eZmHMA3\u003c/em\u003e gene. Fold lines represent introns and orange boxes represent exons and PAMS (NGGs or CCNs) are marked in red. (B) Mutation site in \u003cem\u003eZmhma3\u003c/em\u003e homozygous mutant lines. (C) Amino acid sequence and conformational mutations caused by base frameshift mutation. (D) The WT and KO plants under Zn stress for 0, 24 and 48hours. Bar = 2 cm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/0cc286fe687990b5334c5260.png"},{"id":54900923,"identity":"a51c1436-503c-4fbc-a1ff-617dcc59708b","added_by":"auto","created_at":"2024-04-18 10:20:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":659991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe seedling growth vigor and leaf cell membrane permeability of WT and KO plants under Zn stress for 0, 24 and 48h.\u003c/strong\u003e The SFW, SDW, SWC (A), RFW, RDW, RWC (B), TEW, TDW, TWC (C), relative reduction rate of water content (D), plant height (E) and relative conductivity (F) of WT and KO plants treated with (Zn-WT and Zn-\u003cem\u003eZmhma3\u003c/em\u003e) or without (CK-WT and CK-\u003cem\u003eZmhma3\u003c/em\u003e) Zn stress. Standard error is indicated. Different letters represent significant differences as determined using one-way ANOVA followed by Duncan’s test. \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/c7bc0de310b8361f87c1b4f5.png"},{"id":54900534,"identity":"023b0b53-a2d4-4f4b-9d8c-433f6451d618","added_by":"auto","created_at":"2024-04-18 10:12:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":671813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe root architectures of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmhma3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under Zn stress for 0, 24 and 48h.\u003c/strong\u003e (A) Total root length. (B) Root surface area (SurfArea). (C) Root diameter (AvgDiam). (D) Root volume. (E) Tips. (F) Forks. Standard error is indicated.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/c64687b5de4fdb9749fee86b.png"},{"id":54901577,"identity":"86758c62-a4d8-43f6-95ee-4e551e796db6","added_by":"auto","created_at":"2024-04-18 10:28:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":630266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe root antioxidant properties of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmhma3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder Zn stress maize.\u003c/strong\u003e CAT (\u003cstrong\u003eA\u003c/strong\u003e), POD (\u003cstrong\u003eB\u003c/strong\u003e), SOD (\u003cstrong\u003eC\u003c/strong\u003e) and MDA (\u003cstrong\u003eD\u003c/strong\u003e) of WT and \u003cem\u003eZmhma3\u003c/em\u003e under Zn stress for 0, 24 and 48h. Standard error is indicated.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/e73d980a0d3dfeea8f8ff2d0.png"},{"id":54900537,"identity":"d9de4b76-8f5c-4c35-9591-a7025a0b00b4","added_by":"auto","created_at":"2024-04-18 10:12:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":233950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Zn content and transport rate of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eZmhma3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under Zn stress maize.\u003c/strong\u003e The Zn content (A), Zn transport coefficient (B), and Zn subcellular content (C) under Zn stress. F1: cell wall. F2: soluble part, cell sap and cytoplasmic matrix. F3: cell membrane and organelle. Standard error is indicated. The * (\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05) and the **(\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01) represent significant differences as determined using Student’ s \u003cem\u003et\u003c/em\u003e-test followed by Duncan’ s test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/10d0f672e1caf206136dde0d.png"},{"id":54902094,"identity":"8b43efd5-fd28-4b81-807b-ce4799d3f1ea","added_by":"auto","created_at":"2024-04-18 10:36:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3435904,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/346f90cd-b16b-4793-918d-25b62672aecf.pdf"},{"id":54900924,"identity":"988a17bb-af3b-4a23-a817-c49fb57381b3","added_by":"auto","created_at":"2024-04-18 10:20:01","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":10389,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4230201/v1/7efba00c4b236bc920801bc3.xlsx"}],"financialInterests":"","formattedTitle":"ZmHMA3 enhances Zn stress tolerance and mediates Zn transport in Maize","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe overutilization of Zn fertilizer in agricultural practices, combined with the unregulated expansion of lead-zinc mining activities and the disorderly discharge of industrial wastewater, has raised concerns regarding the pollution caused by the heavy metal zinc. This pollution not only draws attention due to its environmental impact but also poses a direct threat to human health through the contamination of the food chain (Zhou et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite being an essential trace element, necessary for the functioning of more than 1000 transcription factors and structural proteins (Brion, Heyne and Lair \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), excessive levels of Zn can have a toxic effect on plants. This toxicity manifests in various ways, such as a reduction in plant biomass, leaf chlorosis, and inhibition of root growth (Tang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZinc (Zn) uptake by plant roots occurs from the soil and is subsequently transported through the vascular system to the aboveground parts of the plant, where it is further distributed to developing tissues. A complex network in the plant that regulates zinc homeostasis to ensure its growth (Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The absorption and transport of Zn are primarily regulated by different transporters, mainly controlled by several gene family, including HMAs ((heavy metal ATPases), ZIPs (zinc regulatory transporters, iron regulatory transporter like proteins), YSLs (yellow stripe like proteins), CDFs (cationic diffusion promoters), NRAMPs (natural resistance associated macrophage protein), and MTPs (metal transport protein) (Kimura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ibuot et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sinclair and Kramer \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). HMAs, specifically the P-type ATPase protein known as HMA (P18 type), utilize ATP hydrolysis to provide energy for the transport of metal ions (Xu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In Arabidopsis, there are eight HMA proteins. Of those, \u003cem\u003eAtHMA1\u003c/em\u003e is localized in the inner membrane of chloroplasts and facilitates the detoxification of zinc (II) by reducing the zinc content within plastids (Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e); \u003cem\u003eAtHMA2\u003c/em\u003e and \u003cem\u003eAtHMA4\u003c/em\u003e are predominantly expressed in the root vasculature and play a crucial role in the translocation of Zn from roots to shoots through xylem loading. The double mutants \u003cem\u003ehma2\u003c/em\u003e and \u003cem\u003ehma4\u003c/em\u003e exhibit a significant accumulation of zinc in the underground, but severe zinc deficiency in the aboveground (Escudero et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); \u003cem\u003eAtHMA3\u003c/em\u003e is situated on the vacuolar membrane and is primarily responsible for transferring Zn ions into the vacuole (Morel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, \u003cem\u003eMaHMA2\u003c/em\u003e has been found to play an important role in the tolerance response of mulberry plants to Zn-induced stress (Wang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMaize (\u003cem\u003eZea mays\u003c/em\u003e L.), the most cultivated crop globally, is highly susceptible to zinc (Zn) deficiency (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In areas with high Zn levels in the soil, enhancing Zn resistance and reducing grain Zn content are critical for ensuring maize yield and quality. \u003cem\u003eZmHMA3\u003c/em\u003e, which exhibits homology with \u003cem\u003eOsHMA2/3\u003c/em\u003e and \u003cem\u003eAtHMA2/3\u003c/em\u003e, was found to regulate the accumulation of cadmium (Cd) in maize leaves through association analysis and linkage mapping (Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As HMA3 is a transporter protein for both Cd and Zn, we aimed to investigate whether the \u003cem\u003eZmHMA3\u003c/em\u003e gene is also involved in maize's response to Zn stress. To address this, knockout mutant lines were generated to investigate the function of \u003cem\u003eZmHMA3\u003c/em\u003e in the tolerance, absorption, and transport of excessive Zn in maize under Zn stress conditions in this study.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and Zn stress treatment\u003c/h2\u003e \u003cp\u003eB104 maize seeds were first washed with deionized water, then soaked in 20% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and shaken for 20 minutes. After washed 4\u0026ndash;6 times with deionized water for 3 minutes each time, the seeds were soaked overnight with saturated calcium sulfate to induce germination. The next day, they were sprouted in an artificial climate chamber using germination paper. At the second leaf (V2) stage, B104 maize seedlings were randomly allocated into two groups: the control group and the Zn stress group. The Zn stress group was exposed to a concentration of 800 \u0026micro;mol/L ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO for 0 hours and 48 hours, respectively (Liao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of\u003c/b\u003e \u003cb\u003eZmHMA3\u003c/b\u003e \u003cb\u003eusing Real-Time Quantitative PCR (qRT-PCR)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTotal RNA from the roots and leaves of B104 maize seedlings was extracted from each group with triplicates at 0 hours and 48 hours using TRIzol reagent (Invitrogen, Gaithersburg, MD, USA), and first-strand cDNA synthesis was performed using the primeScript TMRT 1st Strand cDNA Synthesis kit (Code Ds10A, TaKaRa, Kyoto, Japan). Subsequently, the qRT-PCR amplification program and the subcellular localization assay of \u003cem\u003eZmHMA3\u003c/em\u003e were carried out according to our previous study (Liao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For the qRT-PCR assay, the relative expression level of \u003cem\u003eZmHMA3\u003c/em\u003e under different treatment conditions (with three technical replicates for each sample) was analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method (Livak and Schmittgen \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). All primers employed in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eZmhma3\u003c/b\u003e \u003cb\u003e(knock-out line) constuction\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eZmhma3\u003c/em\u003e mutants in the B104 maize line were generated using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology, following the previously described method (Ma et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Briefly, single-guide RNAs (sgRNAs) were designed using the CRISPR-GE tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://skl.scau.edu.cn/\u003c/span\u003e\u003cspan address=\"http://skl.scau.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to target the first exon of \u003cem\u003eZmHMA3\u003c/em\u003e. The CRISPR/Cas9 vector was cleaved using both SacI and EcoRI enzymes, where the sgRNAs were then cloned into. These constructs were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105 for inoculation with the B104 maize inbred line, as previously reported (Ishida, Hiei and Komari \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The resulting T\u003csub\u003e3\u003c/sub\u003e generation homozygous mutant seeds were obtained through endosperm cutting, sanger sequencing analysis, and qRT-PCR, and were subsequently used as experimental materials in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological and antioxidant activity analysis\u003c/h2\u003e \u003cp\u003eTo analyze physiological and antioxidant activities, the second leaf (V2) stage of B104 maize seedlings (wild type, WT) and \u003cem\u003eZmhma3\u003c/em\u003e knock-out line (Knock-out) were subjected to treatment with 800\u0026micro;mol/L ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO for 0, 24, and 48 hours, respectively, with H\u003csub\u003e2\u003c/sub\u003eO treatment as a mock control. Following the treatments, the agronomic traits, relative conductivity, and antioxidant indicators were determined as described below:\u003c/p\u003e \u003cp\u003eAgronomic traits were assessed by weighing the fresh weight (Wf) of both aboveground and underground plant parts. Subsequently, the samples were dried at 80 ℃ to a constant weight and weighed as the dry weight (Wd). Plant moisture content (WC) was calculated as (Wf - Wd)/Wf * 100%. The relative reduction rate of water content was calculated as (WC0 - WC)/WC * 100%, where WC0 represents the water content on day 0.\u003c/p\u003e \u003cp\u003eRelative conductivity was evaluated following a previously established method (Liang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Firstly, 0.3g clean fresh leaf samples were cut into sections. After soaked in a dark place for 5 hours with 30 mL deionized water, the conductivity (R1) was measured. Then 95 ℃ water bath were taken out for 1 hour. And after cooled to room temperature, the conductivity (R2) was measured. The calculation formula of relative conductivity was as followed: relative conductivity\u0026thinsp;=\u0026thinsp;R1/R2/W \u0026times; 100%.\u003c/p\u003e \u003cp\u003eRoots were scanned using an EPSON Expression 12000XL root scanner, and the resulting images were analyzed using TAIR high-throughput analysis software. The levels of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and malondialdehyde (MDA) were quantified using a Biochemical Reagent Kit (Solarbio, Beijing, China), following a previously described protocol (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Each sampling is repeated with five plants, and 3 replicates were set.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Zn ion content\u003c/h2\u003e \u003cp\u003eTo measure the Zn ion content, 0.2g of dry samples were digested with 10 milliliters of nitric acid. The concentration of elements in the digestion solution was then determined using inductively coupled plasma mass spectrometry (ICP-MS 7700 X), following a previously established method (Ning et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To determine the content of Zn ions in different subcellular components, a differential centrifugation technique was employed to separate these components. Specifically, 1g of fresh sample was ground into a homogenate using 10 mL of extraction buffer consisting of 250 mmol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose, 50 mmol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Tris HCl (pH 7.5), and 1mmol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dithio red wine. The homogenate was then subjected to centrifugation at a speed of 3000r/min for 15 minutes, resulting in the precipitation of the cell wall component (F1). Subsequently, the supernatant was subjected to a second centrifugation at a speed of 11000r/min for 45 minutes. The precipitate obtained from this second centrifugation step represented the membrane and organelle parts (F3), while the supernatant was considered the soluble component/cytosol and cytoplasm component (F2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExpression characteristics under Zn stress tolerance of\u003c/b\u003e \u003cb\u003eZmHMA3\u003c/b\u003e \u003cb\u003ein maize\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the previous association analysis study, we obtained a candidate gene \u003cem\u003eZmHMA3\u003c/em\u003e related to the accumulation of heavy metal Cd in leaves (Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Due to HMA3 being a Cd/Zn transporter protein, we have focused on studying the function of \u003cem\u003eZmHMA3\u003c/em\u003e under Cd stress, and by the way, confirmed that overexpression of \u003cem\u003eZmHMA3\u003c/em\u003e led to a higher Zn accumulation (Liao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To systematically verify the gene function of \u003cem\u003eZmHMA3\u003c/em\u003e under Zn stress in maize, expression characteristics under Zn stress were firstly taken out. The examination of the expression pattern of the \u003cem\u003eZmHMA3\u003c/em\u003e gene demonstrated an elevated expression level in both leaves and roots after 48 hours of exposure to Zn stress compared to the control group without Zn stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It suggests a potential involvement of \u003cem\u003eZmHMA3\u003c/em\u003e in the response to Zn stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCRISPR/Cas9-mediated knock-out of\u003c/b\u003e \u003cb\u003eZmHMA3\u003c/b\u003e \u003cb\u003edecreased tolerance in Zn stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the functional role of \u003cem\u003eZmHMA3\u003c/em\u003e in response to Zn stress, \u003cem\u003eZmHMA3\u003c/em\u003e knock-out (KO) mutants were generated using CRISPR-Cas9 technology, in which \u003cem\u003eZmHMA3\u003c/em\u003e gene was rare or even not expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Three guide-RNAs (gRNAs) were designed to target the first two exons of \u003cem\u003eZmHMA3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the T\u003csub\u003e0\u003c/sub\u003e generation, 54 chimeric and heterozygous mutant maize lines harboring double-site and single-site mutations were identified. Among these, three stable lines without Cas9 were selected as \u003cem\u003eZmHMA3\u003c/em\u003e homozygous mutants through sanger sequencing analysis and endosperm cutting in the T\u003csub\u003e2\u003c/sub\u003e generation. The mutations in these lines resulted in deletions of 11bp, 65bp, and 34bp between the gRNA1 and gRNA2 sequences, leading to frameshift mutations and subsequent alterations in the sequence and conformation of the ZmHMA3 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). One of these lines, \u003cem\u003eZmhma3-2\u003c/em\u003e, was chosen as the \u003cem\u003eZmhma3\u003c/em\u003e knock-out (KO) line and was further analyzed in the T\u003csub\u003e3\u003c/sub\u003e generation. Subsequently, the relative expression levels and agronomic traits of maize seedlings under varying degrees of Zn stress were compared. Both wild-type (WT) and \u003cem\u003eZmhma3\u003c/em\u003e plants were subjected to 800 \u0026micro;mol/L ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO solutions for specific time periods (0 hour, 24 hours, and 48 hours). The growth phenotypes of WT and \u003cem\u003eZmhma3\u003c/em\u003e indicated that both experienced wilting after 48 hours of Zn stress treatment, but the extent of wilting was more prominent in \u003cem\u003eZmhma3\u003c/em\u003e compared to WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eZmHMA3\u003c/b\u003e \u003cb\u003eaffects the seedling growth vigor and leaf cell membrane permeability of maize under Zn stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe agronomic traits of seedlings reflect their growth potential, such as the amount of water content in plants, which often reflects the strength of their life activities. Cell membrane permeability suggests the degree of lipid peroxidation in plant leaf membranes, and the relative conductivity of leaves is positively correlated with the degree of stress on the plant.\u003c/p\u003e \u003cp\u003eWe subjected the wild-type and \u003cem\u003eZmhma3\u003c/em\u003e mutants to Zn stress (800\u0026micro;mol/L ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO) treatment, whose groups were named Zn-WT and Zn-\u003cem\u003eZmhma3.\u003c/em\u003e And the control groups with H\u003csub\u003e2\u003c/sub\u003e0 treatment were named CK-WT and CK-\u003cem\u003eZmhma3\u003c/em\u003e. In order to assess the agronomic traits of \u003cem\u003eZmhma3\u003c/em\u003e and WT plants under Zn stress at different time points, various physiological indices including seedling fresh weight (SFW), seedling dry weight (SDW), seedling water content (SWC), root fresh weight (RFW), root dry weight (RDW), and root water content (RWC) were measured.\u003c/p\u003e \u003cp\u003eAfter 24 hours of Zn treatment, there was no significant difference in shoots, roots, and total fresh and dry weight between WT and \u003cem\u003eZmhma3\u003c/em\u003e. After 48h, differences began to appear among different treatment groups. Under 48 hours stress, \u003cem\u003eZmhma3\u003c/em\u003e plants exhibited significantly lower SFW, SDW, SWC, RFW, and RWC compared to WT plants, while RDW showed no significant difference between the two genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar trends were observed for total fresh weight (TFW), total dry weight (TDW), and total water content (TWC), which were consistent with RFW, RDW, and RWC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Based on the water content, the relative reduction rate of water content for each treatment group at 24 and 48 h can be calculated compared to 0 h. Whether in the aboveground, underground, or entire plant, it can be seen that, after 48h stress, the relative reduction rate of water content in Zn-\u003cem\u003eZmhma3\u003c/em\u003e was greater than that in Zn-WT, followed by CK-\u003cem\u003eZmhma3\u003c/em\u003e, with CK-WT showing the smallest reduction rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings suggest that the knockout of \u003cem\u003eZmHMA3\u003c/em\u003e resulted in a greater decrease in fresh weight and water content in plants under Zn stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the plant height of WT and Zmhma3 showed no significant difference compared to the control during Zn stress for 24 hours. But at 48 hours under Zn stress, the plant height of \u003cem\u003eZmhma3\u003c/em\u003e was significantly smaller than that of WT, indicating that the knockout of \u003cem\u003eZmHMA3\u003c/em\u003e enhanced the inhibitory effect of Zn stress on maize growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Additionally, the relative electrical conductivity of \u003cem\u003eZmhma3\u003c/em\u003e increased after Zn treatment and was significantly higher than that of WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This suggests that \u003cem\u003eZmhma3\u003c/em\u003e plants experienced more severe damage than WT plants under Zn stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eZmHMA3\u003c/b\u003e \u003cb\u003eaffects the root growth and architecture of maize under Zn stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRoot is the first organ to contact with heavy metals in soil in plants, which can absorb the heavy metals into root cells, thereby transferring to the above ground of the plant, and then causing the antioxidant activity change. We detected Zn heavy metal content in WT and \u003cem\u003eZmhma3\u003c/em\u003e at different times of treatments.\u003c/p\u003e \u003cp\u003eWith the prolongation of stress time, For WT plants, the root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and surface area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) were greater than \u003cem\u003eZmhma3\u003c/em\u003e before Zn stress, but there was no difference in root diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), root volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), number of root tips (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and number of forks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). After Zn stress, the six root phenotype indicators were greater than those of \u003cem\u003eZmhma3\u003c/em\u003e. With the prolongation of stress time, except for a decrease in root length, all other indicators of CK-WT showed an upward trend. Except for the length and forks that first increase and then decrease, all other indicators of CK-\u003cem\u003eZmhma3\u003c/em\u003e also showed an upward trend. Zn stress treatment maintained the length, surface area, diameter, and volume of Zn-WT at a level similar to 0h, while the number of root tips and forks still increased. Except for tips, which first decreased and then increased, all other indicators of Zn-\u003cem\u003eZmhma3\u003c/em\u003e showed a trend of first increasing and then decreasing. These results indicated that knocking out \u003cem\u003eZmHMA3\u003c/em\u003e exacerbated the inhibition of root growth under Zn stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe root antioxidant related enzyme activity was repressed in\u003c/b\u003e \u003cb\u003eZmhma3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAntioxidant enzymes and MDA are closely related to plant stress. CAT, SOD, and POD are effective antioxidant enzymes. And by detecting MDA, the level of membrane lipid peroxidation in plants under adverse conditions can be detected. To determine the resistance to Zn stress, we measured various antioxidant indicators of roots in WT and \u003cem\u003eZmhma3\u003c/em\u003e under Zn stress.\u003c/p\u003e \u003cp\u003eFor antioxidant activity assay, the results showed that CAT content increased after 48h Zn stress in Zn-WT and Zn-\u003cem\u003eZmhma3\u003c/em\u003e, and was significantly higher than that in CK-WT and CK-\u003cem\u003eZmhma3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The POD content of CK-WT and CK-\u003cem\u003eZmhma3\u003c/em\u003e tended to stabilize during the treatment, while that of Zn-WT increased, and that of Zn-\u003cem\u003eZmhma3\u003c/em\u003e decreased. Furthermore, Zn-WT was significantly greater than Zn-\u003cem\u003eZmhma3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At 0h, there was no significant difference between the treatment groups. At 24h, the SOD content of Zn-\u003cem\u003eZmhma3\u003c/em\u003e began to significantly decrease and was significantly lower than CK-WT, CK-\u003cem\u003eZmhma3\u003c/em\u003e, and Zn-WT. At 48h, the content of CK-WT, Zn-WT, and Zn-\u003cem\u003eZmhma3\u003c/em\u003e decreased. The SOD content of CK-\u003cem\u003eZmhma3\u003c/em\u003e was the highest, followed by CK-WT, then Zn-WT, and that of Zn-\u003cem\u003eZmhma3\u003c/em\u003e was the lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The MDA content of Zn-WT significantly increased after treatment, with Zn-\u003cem\u003eZmhma3\u003c/em\u003e initially increasing and then decreasing, resulting in a significantly higher content of Zn-WT than Zn-\u003cem\u003eZmhma3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The content of CAT, POD, SOD, and MDA was higher in WT than that in \u003cem\u003eZmhma3\u003c/em\u003e, which suggested that knocking out \u003cem\u003eZmHMA3\u003c/em\u003e leads to a decrease in plant antioxidant enzyme activity under zinc stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Zn content and transport rate was increased in\u003c/b\u003e \u003cb\u003eZmhma3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUnder heavy metal stress, the growth and development of corn are inhibited. As the duration of stress prolongs, the symptoms of victimization first manifest as wilting. The first plant organ that heavy metals come into contact with is the root, which is stored in the root and transferred to the aboveground part of the plant. The results of the study revealed that \u003cem\u003eZmhma3\u003c/em\u003e plants accumulated higher Zn content compared to WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), particularly in the underground parts of the plant. The Zn content in the underground parts of \u003cem\u003eZmhma3\u003c/em\u003e plants was significantly higher than that in the aboveground parts after 48 hours of stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Furthermore, the leaf, root, and total Zn content in \u003cem\u003eZmhma3\u003c/em\u003e plants were higher than those in WT plants, which corresponded to the trend observed in the transport coefficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, the changes in Zn content in root cells and leaf cells followed a similar trend. After Zn treatment, the Zn content in F2 (soluble part, cell sap, and cytoplasmic matrix) and F3 (cell membrane and organelle) of WT plants was significantly higher than that in \u003cem\u003eZmhma3\u003c/em\u003e plants, while the Zn content in F1 (cell wall) was significantly lower in WT plants compared to \u003cem\u003eZmhma3\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The Zn content in the leaves and roots of \u003cem\u003eZmhma3\u003c/em\u003e plants was much higher than that in WT plants, but the Zn content within the cells was lower.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith the mining of ores, industrial emissions, and the application of pesticides and fertilizers, the problem of soil heavy metal zinc pollution has become prominent in recent years(Moffat \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Zinc entering the soil is difficult to move, and natural purification time can even take about 1000 years. After the soil is polluted, it directly causes the soil to lose its natural productivity. The use of tolerance and hyperaccumulating plants for excessive zinc absorption has become a research focus for soil scientists and environmentalists (Chaney et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). As a zinc sensitive crop, maize is significantly affected by zinc, and the difference between concentration gradients is significant. Low concentration of zinc promotes growth, high concentration inhibits growth, and medium concentration is in a transition state (Wang \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In addition to the toxic effects on crops themselves, the most important thing is that these crops accumulate in the human body through the food chain after being consumed by humans, which can cause serious health and safety issues (Nordberg \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although the absorption and transport mechanisms of heavy metals in plants are not yet fully understood, it is still possible to effectively reduce the concentration of heavy metals in plants by regulating the transport proteins that absorb these metals. Among numerous metal transporter families, HMAs are widely present in lower and higher plants and participate in the absorption, transportation, and accumulation of essential trace metal nutrients during plant growth and development (Morel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wong and Cobbett \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). HMA has been shown to participate in the transportation of various heavy metal cations (Williams and Mills \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In Arabidopsis, AtHMA1-4 mediate the transport of divalent heavy metal ions (Zn\u003csup\u003e2+\u003c/sup\u003e/Co\u003csup\u003e2+\u003c/sup\u003e/Cd\u003csup\u003e2+\u003c/sup\u003e/Pb\u003csup\u003e2+\u003c/sup\u003e), while AtHMA5-8 transport Cu\u003csup\u003e+\u003c/sup\u003e and Ag\u003csup\u003e+\u003c/sup\u003e (Woeste and Kieber \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hirayama et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Axelsen and Palmgren \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In rice, OsHMA1-3 belong to Zn /Co/Cd/Pb transporters (Takahashi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Previous studies have found that ZmHMA3 protein has 72% homology with OsHMA3 protein and 62% homology with OsHMA2 protein. The homology with Arabidopsis AtHMA2 and AtHMA3 is 47% and 52% respectively. It belongs to the same Zn /Co/Cd/Pb transporter and participats in numerous heavy metal transport (Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the mechanism of action of the \u003cem\u003eZmHMA3\u003c/em\u003e gene under Zn stress is still unclear. Therefore, this study conducted functional verification on \u003cem\u003eZmHMA3\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eAnalyzing the spatiotemporal expression pattern of \u003cem\u003eZmHMA3\u003c/em\u003e gene is of great significance for inferring its specific function. Under normal growth conditions, \u003cem\u003eZmHMA3\u003c/em\u003e demonstrates tissue specificity in nodes, anthers, and roots (Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the expression of \u003cem\u003eZmHMA3\u003c/em\u003e was found to be upregulated in the roots and stems under Zn stress, consistent with previous research conducted on \u003cem\u003eOsHMA2\u003c/em\u003e and \u003cem\u003eAtHMA2\u003c/em\u003e (Das et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This suggests that \u003cem\u003eZmHMA3\u003c/em\u003e may play a positive regulatory role in enhancing resistance to Zn stress. Previous study demonstrated that Zinc stress has inhibited plant growth (Yu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Following a 48-hour Zn stress treatment on both wild-type (WT) and \u003cem\u003eZmHMA3\u003c/em\u003e knockout mutants, the mutants displayed initial wilting. Subsequently, morphological characteristics such as fresh weight, dry weight, water content, plant height, and relative conductivity were measured. The results indicated that the knockout of \u003cem\u003eZmHMA3\u003c/em\u003e significantly impeded maize growth under Zn stress. Previous studies primarily focused on measuring root morphological traits, such as root length and weight (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, in this study, a root scanner was employed to specifically measure root diameter, root volume, and other traits, providing a more comprehensive evaluation of root morphological indicators. Overall, the WT exhibited better root growth compared to the mutant. This difference may be attributed to the disruption or alteration of \u003cem\u003eZmHMA3\u003c/em\u003e protein function, which reduces or eliminates the ability of plant root cells to transport or excrete Zn. Consequently, Zn content accumulates, leading to root system toxicity and inhibition of root growth.\u003c/p\u003e \u003cp\u003eHeavy metal stress is similar to other forms of oxidative stress, which can lead to the production of a large number of reactive oxygen species, damage major biological macromolecules, and cause membrane lipid peroxidation (Shan, Luo and Frances \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Various antioxidant mechanisms in plants can eliminate free radicals to protect cells. Among them, SOD, CAT, POD, and MDA are the most common antioxidant systems in plants. Plants employ various antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and malondialdehyde (MDA), to eliminate free radicals and protect cells. POD in plants plays a crucial role in the cross-linking of hydroxyproline-rich glycoproteins with carbolic acid, increasing cell wall strength and thereby countering Zn\u003csup\u003e2+\u003c/sup\u003e-induced osmotic stress (Chao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In the Zn hyperaccumulation ecotype \u003cem\u003eSedum alfredii\u003c/em\u003e, the activities of SOD, CAT and GPX increased under high Zn stress, surpassing those observed in the non-hyperaccumulation ecotype (Jin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In the present study, the overall antioxidant capacity of the WT was higher than that of \u003cem\u003eZmhma3\u003c/em\u003e. The \u003cem\u003eZmHMA3\u003c/em\u003e mutation resulted in a decrease in antioxidant enzyme activity in maize, which may also be one of the reasons for the decrease in Zn stress tolerance.\u003c/p\u003e \u003cp\u003eFollowing Zn stress, the Zn content in \u003cem\u003eZmHMA3\u003c/em\u003e leaves and roots, as well as the transport coefficient, were significantly higher compared to the WT. Furthermore, the Zn content in the roots was notably higher than in the leaves. Similar results were observed in \u003cem\u003eArabidopsis HMA2\u003c/em\u003e knockout mutants (Eren and Arg\u0026uuml;ello \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Interestingly, in contrast to plants, the Zn content in \u003cem\u003eZmHMA3\u003c/em\u003e protoplasts was lower than in the WT, while the Zn content in the cell wall was higher. This means that the Zn content of \u003cem\u003eZmhma3\u003c/em\u003e in leaves and roots is higher than that in WT, but the total Zn content in leaf and root cells is lower than that in WT. It is generally understood that, after heavy metals enter the plant body, the main detoxification mechanisms of plants include elimination from the body and intracellular compartmentalization. Zn precipitation on the cell wall acts as a barrier, preventing more ions from entering the cell protoplasts and causing harm (Nishizono et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; K\u0026uuml;pper, Lombi and Mcgrath \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). HMA2 enhances plant Zn tolerance by actively expelling excess Zn ions into the extracellular space (Eren and Arg\u0026uuml;ello \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cem\u003eAtHMA3\u003c/em\u003e is involved in the vacuolar compartmentalization of various heavy metals, such as Zn and Pb (Morel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), while \u003cem\u003eOsHMA3\u003c/em\u003e is involved in the vacuolar compartmentalization of Cd (Miyadate et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Unlike \u003cem\u003eAtHMA3\u003c/em\u003e, which is localized on the vacuolar membrane, \u003cem\u003eZmHMA3\u003c/em\u003e is primarily located on the cell membrane, which aligns with the localization of \u003cem\u003eAtHMA2\u003c/em\u003e (Morel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Eren and Arg\u0026uuml;ello \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Liao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Through querying the ZmHMA3 protein in the Maize GDB, it was revealed that there are amino acid motifs near the termination codon that contain phosphorylation sites. These motifs are mainly involved in providing energy for the transmembrane transport of heavy metals. This phosphorylation site is closely associated with the transmembrane domains H6 and H7 of the HMA protein. It is likely that ZmHMA3 is involved in the transport of Zn ions into and out of plant cells. Based on the findings of this study, \u003cem\u003eZmHMA3\u003c/em\u003e may be involved in Zn transport into cells through the cell membrane. Although the concentration of zinc ions in the entire plant is relatively high, the concentration in protoplasts is low. This discrepancy likely leads to differences in osmotic pressure between the inside and outside of cells, resulting in cell dehydration and a decrease in \u003cem\u003eZmHMA3\u003c/em\u003e water content. Zn imbalance leads to the accumulation of reactive oxygen species (ROS) and the displacement of other elements from protein active sites (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, zinc interacts with other elements in plants, and zinc toxicity typically induces steady-state changes in numerous other elements, including Fe, Ca, and P (Gupta, Ram and Kumar 2016). Therefore, measuring ion content changes related to intracellular environmental homeostasis may provide more insights into the mechanism of action of \u003cem\u003eZmHMA3\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Zhejiang Key R\u0026amp;D Program (NO. 2021C02057); Science and Technology Plan Project of Zhejiang Provincial (NO.2022C04024)\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eConceptualization: Guihua Lv, Tingzhen Wang; Methodology: Youqiang Li, Jianjian Chen; Formal analysis and investigation: Youqiang Li, Zhenxing Wu; Writing - original draft preparation: Guihua Lv, Youqiang Li; Writing - review and editing: Tingzhen Wang, Wenmei Wu; Resources: Xiaohong Wu; Supervision: Haijian Lin, Guihua Lv\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad I, Rawoof A, Priyanka, Islam K, Momo J, Anju T, Kumar A, Ramchiary N (2023) Diversity and expression analysis of ZIP transporters and associated metabolites under zinc and iron stress in Capsicum. Plant Physiol Biochem 196: 415-430. https://doi.org/10.1016/j.plaphy.2023.01.060\u003c/li\u003e\n\u003cli\u003eAxelsen KB, Palmgren MG (2001) Inventory of the superfamily of p-type ion pumps in arabidopsis. Plant Physiol 126:696-706. https://doi.org/10.1104/pp.126.2.696\u003c/li\u003e\n\u003cli\u003eBrion LP, Heyne R, Lair CS (2021) Role of zinc in neonatal growth and brain growth: review and scoping review. Pediatr Res 89:1627-1640. https://doi.org/10.1038/s41390-020-01181-z\u003c/li\u003e\n\u003cli\u003eCao Y, Zhao X, Liu Y, Wang Y, Wu W, Jiang Y, Liao C, Xu X, Gao S, Shen Y, Lan H, Zou C, Pan G, Lin H (2019) Genome-wide identification of z\u003cem\u003emhmas\u003c/em\u003e and association of natural variation in \u003cem\u003ezmhma2\u003c/em\u003e and \u003cem\u003ezmhma3\u003c/em\u003e with leaf cadmium accumulation in maize. Peerj 7: e7877. https://doi.org/10.7717/peerj.7877\u003c/li\u003e\n\u003cli\u003eChaney RL, Malik M, Li YM, Brown S, Brewer E, Angle J, Baker AJM (1997) Phytoremediation of soil metals. Curr Opin Biotechnol 8:279-284. https://doi.org/10.1016/s0958-1669(97)80004-3\u003c/li\u003e\n\u003cli\u003eChao W, Song HZ, Pei FW, Hou J, Wei L, Wen JZ (2008) Metabolic adaptations to ammonia-induced oxidative stress in leaves of the submerged macrophyte \u003cem\u003eVallisneria natans\u003c/em\u003e (lour.) Hara. Aquat Toxicol 87:88-98. https://doi.org/10.1016/j.aquatox.2008.01.009\u003c/li\u003e\n\u003cli\u003eChen H, Lai L, Li L, Liu L, Jakada BH, Huang Y, He Q, Chai M, Niu X, Qin Y (2020) \u003cem\u003eAcoMYB4\u003c/em\u003e, an \u003cem\u003eAnanas comosus\u003c/em\u003e L. MYB Transcription Factor, Functions in Osmotic Stress through Negative Regulation of ABA Signaling. Int J Mol Sci 21(16). https://doi.org/10.3390/ijms21165727.\u003c/li\u003e\n\u003cli\u003eDas U, Haque AFMM, Bari MA, Mandal A, Kabir AH (2021) Computational characterization and expression profile of MTP1 gene associated with zinc homeostasis across dicot plant species. Gene Reports 23: 101073. doi: https://doi.org/10.1016/j.genrep.2021.101073.\u003c/li\u003e\n\u003cli\u003eEren E, Arg\u0026uuml;ello JM (2004) Arabidopsis hma2, a divalent heavy metal-transporting pib-type atpase, is involved in cytoplasmic Zn\u003csup\u003e2+\u003c/sup\u003e homeostasis. Plant Physiol 136(3): 3712-3723. https://doi.org/10.1104/pp.104.046292\u003c/li\u003e\n\u003cli\u003eEscudero V, Ferreira SD, Abreu I, Sope\u0026ntilde;a-Torres S, Makarovsky-Saavedra N, Bernal M, Kr\u0026auml;mer U, Grolimund D, Gonz\u0026aacute;lez-Guerrero M, Jord\u0026aacute; L (2022) Arabidopsis thaliana Zn\u003csup\u003e2+\u003c/sup\u003e-efflux ATPases HMA2 and HMA4 are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina BMM. J Exp Bot 73(1): 339-350. https://doi.org/10.1093/jxb/erab400.\u003c/li\u003e\n\u003cli\u003eWang YF (2008) Physiological and ecological characteristics of corn under different anionic forms of zinc compounds stress. Dissertation, Shandong University\u003c/li\u003e\n\u003cli\u003eGrotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D (1998) Identification of a family of zinc transporter genes from \u003cem\u003eArabidopsis\u003c/em\u003e that respond to zinc deficiency. Proc Natl Acad Sci U S a 95:7220-7224. https://doi.org/10.1073/pnas.95.12.7220\u003c/li\u003e\n\u003cli\u003eHirayama T, Kieber JJ, Hirayama N, Kogan M, Guzman P, Nourizadeh S, Alonso JM, Dailey WP, Dancis A, Ecker JR (1999) Responsive-to-antagonist1, a menkes/wilson disease-related copper transporter, is required for ethylene signaling in \u003cem\u003eArabidopsis\u003c/em\u003e. Cell 97:383-393. https://doi.org/10.1016/s0092-8674(00)80747-3\u003c/li\u003e\n\u003cli\u003eHuang S, Sasaki A, Yamaji N, Okada H, Mitani-Ueno N, Ma JF (2020) The zip transporter family member\u003cem\u003e OsZIP9\u003c/em\u003e contributes to root zinc uptake in rice under zinc-limited conditions. Plant Physiol 183:1224-1234. https://doi.org/10.1104/pp.20.00125\u003c/li\u003e\n\u003cli\u003eIbuot A, Webster RE, Williams LE, Pittman JK (2020) Increased metal tolerance and bioaccumulation of zinc and cadmium in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e expressing a AtHMA4 C-terminal domain protein. Biotechnol Bioeng 117(10): 2996-3005. https://doi.org/10.1002/bit.27476.\u003c/li\u003e\n\u003cli\u003eIshida Y, Hiei Y, Komari T (2020) Tissue culture protocols for gene transfer and editing in maize (\u003cem\u003eZea mays\u003c/em\u003e L.). Plant Biotechnol (Tokyo) 37(2): 121-128. https://doi.org/10.5511/plantbiotechnology.20.0113a.\u003c/li\u003e\n\u003cli\u003eJin XF, Yang XE, Islam E, Liu D, Li J (2008) Ultrastructural changes, zinc hyperaccumulation and its relation with antioxidants in two ecotypes of sedum alfredii hance. Plant Physiol Biochem 46:997-1006. https://doi.org/10.1016/j.plaphy.2008.06.012\u003c/li\u003e\n\u003cli\u003eKim YY, Choi H, Segami S, Cho HT, Lee Y (2009) Athma1 contributes to the detoxification of excess zn(ii) in \u003cem\u003eArabidopsis\u003c/em\u003e. The Plant J 58:737-753. https://doi.org/10.1111/j.1365-313X.2009.03818.x\u003c/li\u003e\n\u003cli\u003eKimura S, Vaattovaara A, Ohshita T, Yokoyama K, Yoshida K, Hui A, Kaya H, Ozawa A, Kobayashi M, Mori IC, Ogata Y, Ishino Y, Sugano SS, Nagano M, Fukao Y (2023) Zinc deficiency-induced defensin-like proteins are involved in the regulation of root growth in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant J. https://doi.org/10.1111/tpj.16281\u003c/li\u003e\n\u003cli\u003eK\u0026uuml;pper H, Lombi E, Mcgrath Z (2000) Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator \u003cem\u003eArabidopsis\u003c/em\u003e halleri. Planta 212:75-84. https://doi.org/10.1007/s004250000366\u003c/li\u003e\n\u003cli\u003eLiang L, Ze M, Yang J, Xu Q, Du C, Hu X, Dong M, Zou L, Qi T (2023) Physiological and transcriptomic response reveals new insight into manganese tolerance of Celosia argentea Linn. J Hazard Mater 465: 133079. https://doi.org/ 10.1016/j.jhazmat.2023.133079.\u003c/li\u003e\n\u003cli\u003eLiao C, Li Y, Wu X, Wu W, Zhang Y, Zhan P, Meng X, Hu G, Yang S, Lin H (2023) \u003cem\u003eZmHMA3\u003c/em\u003e, a Member of the Heavy-Metal-Transporting ATPase Family, Regulates Cd and Zn Tolerance in Maize. Int J Mol Sci 24(17). https://doi.org/10.3390/ijms241713496.\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408. https://doi.org/10.1006/meth.2001.1262.\u003c/li\u003e\n\u003cli\u003eMa X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant 8(8): 1274-1284. https://doi.org/10.1016/j.molp.2015.04.007.\u003c/li\u003e\n\u003cli\u003eMiyadate H, Adachi S, Hiraizumi A, Tezuka K, Nakazawa N, Kawamoto T, Katou K, Kodama I, Sakurai K, Takahashi H, Satoh-Nagasawa N, Watanabe A, Fujimura T, Akagi H (2011) \u003cem\u003eOsHMA3\u003c/em\u003e, a p1b-type of atpase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles. New Phytol 189:190-199. https://doi.org/10.1111/j.1469-8137.2010.03459.x\u003c/li\u003e\n\u003cli\u003eMoffat AS (1995) Plants proving their worth in toxic metal cleanup. Science 269:302-303. https://doi.org/10.1126/science.269.5222.302\u003c/li\u003e\n\u003cli\u003eMorel M, Crouzet J, Gravot A, Auroy P, Leonhardt N, Vavasseur A, Richaud P (2009) AtHMA3, a p1b-atpase allowing Cd/Zn/Co/Pb vacuolar storage in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Physiol 149:894-904. https://doi.org/10.1104/pp.108.130294\u003c/li\u003e\n\u003cli\u003eNing M, Liu SJ, Deng F, Huang L, Li H, Che J, Yamaji N, Hu F, Lei GJ (2023) A vacuolar transporter plays important roles in zinc and cadmium accumulation in rice grain. New Phytol 239(5): 1919-1934. https://doi.org/10.1111/nph.19070.\u003c/li\u003e\n\u003cli\u003eNishizono H, Ichikawa H, Suziki S, Ishii F (1987) The role of the root cell wall in the heavy metal tolerance ofathyrium yokoscense. Plant Soil 101:15-20. https://doi.org/10.1007/BF02371025\u003c/li\u003e\n\u003cli\u003eNordberg GF (2009) Historical perspectives on cadmium toxicology. Toxicol Appl Pharmacol 238:192-200. https://doi.org/10.1016/j.taap.2009.03.015\u003c/li\u003e\n\u003cli\u003eShan WY, Luo GH, Frances K (1997) Peroxidation damage of oxygen free radicals induced by cadmium to plant. Acta Botanica Sinica 39:522-526 (in Chinese)\u003c/li\u003e\n\u003cli\u003eSinclair SA, Kramer U (2012) The zinc homeostasis network of land plants. Biochim Biophys Acta 1823:1553-1567. https://doi.org/10.1016/j.bbamcr.2012.05.016\u003c/li\u003e\n\u003cli\u003eTakahashi R, Bashir K, Ishimaru Y, Nishizawa NK, Nakanishi H (2012) The role of heavy-metal atpases, hmas, in zinc and cadmium transport in rice. Plant Signal Behav 7:1605-1607. https://doi.org/10.4161/psb.22454\u003c/li\u003e\n\u003cli\u003eTang Z, Wang HQ, Chen J, Chang JD, Zhao FJ (2023) Molecular mechanisms underlying the toxicity and detoxification of trace metals and metalloids in plants. J Integr Plant Biol 65(2): 570-593. https://doi.org/10.1111/jipb.13440. \u003c/li\u003e\n\u003cli\u003eWang L, Du Q, Shi Y, Ackah M, Guo P, Zheng D, Wu M, Jin X, Li P, Zhang Q, Li R, Yin Z, Zhao M, Zhao W (2022) Response of \u003cem\u003eMaHMA2\u003c/em\u003e gene expression and stress tolerance to zinc stress in mulberry (Morus alba L.). BIOCELL 46(10): 2327-2342. https://doi.org/10.32604/biocell.2022.021542\u003c/li\u003e\n\u003cli\u003eWilliams LE, Mills RF (2005) P(1b)-atpases--an ancient family of transition metal pumps with diverse functions in plants. Trends Plant Sci 10:491-502. https://doi.org/10.1016/j.tplants.2005.08.008\u003c/li\u003e\n\u003cli\u003eWoeste KE, Kieber JJ (2000) A strong loss-of-function mutation in ran1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype. THE PLANT CELL ONLINE 12:443-455. https://doi.org/10.1105/tpc.12.3.443\u003c/li\u003e\n\u003cli\u003eWong C, Cobbett CS (2009) Hma p-type atpases are the major mechanism for root-to-shoot Cd translocation in Arabidopsis thaliana. New Phytol 181:71-78. https://doi.org/10.1111/j.1469-8137.2008.02638.x\u003c/li\u003e\n\u003cli\u003eXu W, Huang H, Li X, Yang M, Chi S, Pan Y, Li N, Paterson AH, Chai Y, Lu K (2023) \u003cem\u003eCaHMA1\u003c/em\u003e promotes Cd accumulation in pepper fruit. J Hazard Mater 460: 132480. https://doi.org/10.1016/j.jhazmat.2023.132480.\u003c/li\u003e\n\u003cli\u003eYu L, Tang S, Kang J, Korpelainen H, Li C (2023) Responses of dioecious Populus to heavy metals: a meta-analysis. Forestry Research 3(1). https://doi.org/10.48130/FR-2023-0025.\u003c/li\u003e\n\u003cli\u003eZhang H, Yang J, Li W, Chen Y, Lu H, Zhao S, Li D, Wei M, Li C (2019) PuHSFA4a enhances tolerance to excess zinc by regulating reactive oxygen species production and root development in Populus. Plant Physiol 180(4): 2254-2271. https://doi.org/10.1104/pp.18.01495.\u003c/li\u003e\n\u003cli\u003eZhang L, Yan M, Li H, Ren Y, Zhang S (2020) Effects of zinc fertilizer on maize yield and water-use efficiency under different soil water conditions. Field Crops Res 248. https://doi.org/10.1016/j.fcr.2020.107718\u003c/li\u003e\n\u003cli\u003eZhou M, Xiao L, Yang S, Wang B, Shi T, Tan A, Wang X, Mu G, Chen W (2020) Cross-sectional and longitudinal associations between urinary zinc and lung function among urban adults in China. Thorax 75(9): 771-779. https://doi.org/10.1136/thoraxjnl-2019-213909.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Maize (Zea mays), heavy metal ATPase (HMA), Zn stress, CRISPR-Cas9 system","lastPublishedDoi":"10.21203/rs.3.rs-4230201/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4230201/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExcessive levels of Zn have the potential to be detrimental to plant health. \u003cem\u003eZmHMA3\u003c/em\u003e, a member of the heavy metal ATPase (HMA) family, is responsible for the transport of Zn\u003csup\u003e2+\u003c/sup\u003e and Cd\u003csup\u003e2+\u003c/sup\u003e across cellular membranes. In order to investigate the role of the \u003cem\u003eZmHMA3\u003c/em\u003e gene in response to Zn stress, \u003cem\u003eZmHMA3\u003c/em\u003e knockout mutants were created using the CRISPR-Cas9 technique. Subsequently, gene specific expression, as well as agronomic traits, root morphology indicators, relative conductivity, antioxidant indicators, and Zn content in the leaf, root, and their subcellular components were assessed. The results demonstrated a significant accumulation of \u003cem\u003eZmHMA3\u003c/em\u003e in both the leaf and root after 48 hours of Zn stress compared to the control group. The \u003cem\u003eZmhma3\u003c/em\u003e knockout line exhibited decreased tolerance to toxic levels of Zn as compared to the wild type, resulting in a reduction in maize plant height, fresh weight, dry weight, water content, root morphology indicators (Length, SurfArea, AvgDiam, Rootvolume, Tips and Forks) and antioxidant enzyme activity (CAT, POD, SOD, and MDA), while also leading to an increase in membrane permeability and zinc accumulation. In conclusion, it can be inferred that ZmHMA3 likely functions as a crucial positive regulator in the response to Zn stress in maize.\u003c/p\u003e","manuscriptTitle":"ZmHMA3 enhances Zn stress tolerance and mediates Zn transport in Maize","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-18 10:11:56","doi":"10.21203/rs.3.rs-4230201/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-20T19:49:12+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-16T04:09:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2024-04-10T03:28:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-09T17:32:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2024-04-07T03:45:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"be090ec3-5508-47be-ab79-ee8e75fab116","owner":[],"postedDate":"April 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-04-18T10:11:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-18 10:11:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4230201","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4230201","identity":"rs-4230201","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}