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Key Message: ZmWRKY64 positively regulates Arabidopsis and maize Cd stress through modulating Cd uptake, translocation, and ROS scavenging genes expression. Cadmium (Cd) is a highly toxic heavy metal with severe impacts on crops growth and development. The WRKY transcription factor is a significant regulator influencing plant stress response. Nevertheless, the function of the WRKY protein in maize Cd stress response remains unclear. Here, we identified a maize WRKY gene, ZmWRKY64, the expression of which was enhanced in maize roots and leaves under Cd stress. ZmWRKY64 was localized in the nucleus and displayed transcriptional activity in yeast. Heterologous expression of ZmWRKY64 in Arabidopsis diminished Cd accumulation in plants by negatively regulating the expression of AtIRT1, AtZIP1, AtHMA2, AtNRAMP3, and AtNRAMP4, which are involved in Cd uptake and transport, resulting in Cd stress tolerance. Knockdown of ZmWRKY64 in maize led to excessive Cd accumulation in leaf cells and in the cytosol of the root cells, resulting in a Cd hypersensitive phenotype. Further analysis confirmed that ZmWRKY64 positively regulated ZmABCC4, ZmHMA3, ZmNRAMP5, ZmPIN2, ZmABCG51, ZmABCB13/32, and ZmABCB10, which may influence Cd translocation and auxin transport, thus mitigating Cd toxicity in maize. Moreover, ZmWRKY64 could directly enhance the transcription of ZmSRG7, a reported key gene regulating reactive oxygen species homeostasis under abiotic stress. Our results indicate that ZmWRKY64 is important in maize Cd stress response. This work provides new insights into the WRKY transcription factor regulatory mechanism under a Cd-polluted environment and may lead to the genetic improvement of Cd tolerance in maize., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cadmium (Cd), which is a nonessential heavy metal element for organisms, can have a severe impact on the growth and development of organisms that absorb excessive Cd. Studies have shown that Brassica carinata, a semiwild oil crop, has strong tolerance to various abiotic stresses, and RNA-seq has revealed that the B. carinata superoxide dismutase gene (BcaSOD1) likely responds to Cd stress. To elucidate the BcaSOD1 function involved in tolerance of Cd stress, we cloned the coding sequences of BcaSOD1 from a purple B. carinata accession and successfully transferred it into Arabidopsis thaliana. The subcellular localization results demonstrated that BcaSOD1 was primarily located in the plasma membrane, mitochondria and nucleus. Overexpression of BcaSOD1 in transgenic Arabidopsis (OE) effectively decreased the toxicity caused by Cd stress. Compared to the WT (wild type lines), the OE lines exhibited significantly increased activities of antioxidant enzymes (APX, CAT, POD, and SOD) after exposure to 2.5 mM CdCl 2 . The Cd content of underground (root) in the OE line was dominantly higher than that in the WT; however, the Cd content of aboveground (shoot) was comparable between the OE and WT types. Moreover, the qRT‒PCR results showed that several heavy metal detoxification-related genes (AtIREG2, AtMTP3, AtHMA3, and AtNAS4) were significantly upregulated in the roots of OE lines under Cd treatment, suggesting that these genes are likely involved in Cd absorption in the roots of OE lines. In addition, both comparable transcriptome and qRT-PCR analyses revealed that exogenous BcaSOD1 noticeably facilitates detoxification by stimulating the expression of two arginine (Arg) biosynthesis genes (AtGDH1 and AtGDH2) while inhibiting the expression of AtARGAH1, a negative regulator in biosynthesis of Arg. The Arg content was subsequently confirmed to be significantly enhanced in OE lines under Cd treatment, indicating that BcaSOD1 likely strengthened Cd tolerance by regulating the expression of Arg-related genes. This study demonstrates that BcaSOD1 can enhance Cd tolerance and reveals the molecular mechanism of this gene, providing valuable insights into the molecular mechanism of Cd tolerance in plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Submergence stress represents a serious threat to the yield and quality of maize because it can lead to oxygen deficiency and the accumulation of toxic metabolites. However, the mechanisms by which maize resists the adverse effects of submergence stress have yet to be fully elucidated. Here, we cloned a gene from maize Balem (Barley aleurone and embryo), ZmB12D, which was expressed at significant levels in seed embryos during imbibition and in leaves under submergence stress. Subcellular localization analysis indicated that the ZmB12D protein was localized in the mitochondria. The overexpression of ZmB12D in increased the tolerance of Arabidopsis to submergence stress, probably due to a reduction in the levels of malonaldehyde (MDA), the increased activity of antioxidant enzymes (SOD, POD and CAT), enhanced electron transport by coordinating the expression of non-symbiotic hemoglobin-2 (AHb2) and Fe transport-related (AtNAS3) genes (mediating Fe and oxygen availability) and also modulated the anaerobic respiration rates through upregulated the AtPDC1, AtADH1, AtSUS4 genes under submergence. Yeast one-hybrid (Y1H) and transient transactivation assays demonstrated that ZmWRKY70 bound to the ZmB12D promoter and activated ZmB12D. Collectively, out findings indicate that ZmB12D plays an important role in the tolerance of maize to submergence stress. This research provides new insights into the genetic improvement of maize with regards to submergence tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Cadmium (Cd) is a toxic heavy metal that seriously affects metabolism after accumulation in plants, and it also causes adverse effects on humans through the food chain. The HIPP gene family has been shown to be highly tolerant to Cd stress due to its special domain and molecular structure. This study described the Cd-induced gene ApHIPP26 from the hyperaccumulator Arabis paniculata . Its subcellular localization showed that ApHIPP26 was located in the nucleus. Transgenic Arabidopsis overexpressing ApHIPP26 exhibited a significant increase in main root length and fresh weight under Cd stress. Compared with wild-type lines, Cd accumulated much more in transgenic Arabidopsis both aboveground and underground. Under Cd stress, the expression of genes related to the absorption and transport of heavy metals underwent different changes in parallel, which were involved in the accumulation and distribution of Cd in plants, such as AtNRAMP6 and AtNRAMP3 . Under Cd stress, the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) in the transgenic lines were higher than those in the wild type. The physiological and biochemical indices showed that the proline and chlorophyll contents in the transgenic lines increased significantly after Cd treatment, while the malondialdehyde (MDA) content decreased. In addition, the gene expression profile analysis showed that ApHIPP26 improved the tolerance of Arabidopsis to Cd by regulating the changes of related genes in plant hormone signal transduction pathway. In conclusion, ApHIPP26 plays an important role in cadmium tolerance by alleviating oxidative stress and regulating plant hormones, which provides a basis for understanding the molecular mechanism of cadmium tolerance in plants and provides new insights for phytoremediation in Cd-contaminated areas.

Maize is often subjected to various environmental stresses. The strictosidine synthase-like (SSL) family is thought to catalyze the key step in the monoterpene alkaloids synthesis pathway in response to environmental stresses. However, the role of ZmSSL genes in maize growth and development and its response to stresses is unknown. Herein, we undertook the systematic identification and analysis of maize SSL genes. Twenty SSL genes were identified in the maize genome. Except for chromosomes 3, 5, 6, and 10, they were unevenly distributed on the remaining 6 chromosomes. A total of 105 SSL genes from maize, sorghum, rice, Aegilops tauschii , and Arabidopsis were divided into five evolutionary groups, and ZmSSL gene structures and conserved protein motifs in the same group were similar. A collinearity analysis showed that tandem duplication plays an important role in the evolution of the SSL family in maize, and ZmSSL genes share more collinear genes in crops (maize, sorghum, rice, and Ae. tauschii ) than in Arabidopsis. Cis -element analysis in the ZmSSL gene promoter region revealed that most genes contained many development and stress response elements. We evaluated the expression levels of ZmSSL genes under normal conditions and stress treatments. ZmSSL4-9 were widely expressed in different tissues and were positively or negatively regulated by heat, cold, and infection stress from Colletotrichum graminicola and Cercospora zeina . Moreover, ZmSSL4 and ZmSSL5 were localized in the chloroplast. Taken together, we provide insight into the evolutionary relationships of the ZmSSL genes, which would be useful to further identify the potential functions of ZmSSLs in maize.

Hypoxic stress due to submergence is a serious threat to the growth and development of maize. WRKY transcription factors are significant regulators of plant responses to various abiotic and biotic stresses. Nevertheless, their function and regulatory mechanisms in the resistance of maize to submergence stress remain unclear. Here we report the cloning of a maize WRKY transcription factor gene, ZmWRKY70, transcripts of which accumulate under submergence stress in maize seedlings. Subcellular localization analysis and yeast transcriptional activation assay indicated that ZmWRKY70 was localized in the nucleus and had transcriptional activation activity. Heterologous overexpression of ZmWRKY70 in Arabidopsis increased the tolerance of seeds and seedlings to submergence stress by upregulating the transcripts of several key genes involved in anaerobic respiration, such as group VII ethylene-responsive factor (ERFVII) (AtRAP2.2), alcohol dehydrogenase (AtADH1), pyruvate decarboxylase (AtPDC1/2), and sucrose synthase (AtSUS4), under submergence conditions. Moreover, the overexpression of ZmWRKY70 in maize mesophyll protoplasts enhanced the expression of ZmERFVII members (ZmERF148, ZmERF179, and ZmERF193), ZmADH1, ZmPDC2/3, and ZmSUS1. Yeast one-hybrid and dual-luciferase activity assays further confirmed that ZmWRKY70 enhanced the expression of ZmERF148 by binding to the W box motif located in the promoter region of ZmERF148. Together, these results indicate that ZmWRKY70 plays a significant role in tolerance of submergence stress. This work provides a theoretical basis, and suggests excellent genes, for biotechnological breeding to improve the tolerance of maize to submergence through the regulation of ZmWRKY genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Key Message: Cadmium-induced TaWAK20 regulates the cadmium stress response by phosphorylating TaSPL5 in wheat. Receptor-like kinases (RLKs) are thought to play important roles in responses to abiotic stresses in plants. In this study, we identified a cadmium (Cd)-induced RLK in wheat, TaWAK20, which is a positive regulator of the Cd stress response. TaWAK20 is specifically expressed in root tissue. Overexpression of TaWAK20 significantly improved the tolerance of Cd stress in wheat and decreased Cd accumulation in wheat plants by regulating reactive oxygen species production and scavenging. Yeast one-hybrid assays, electrophoretic mobility shift assays, and firefly luciferase activity analyses demonstrated that the TaWAK20 promoter was bound by the TabHLH35 transcription factor. TaWAK20 interacted with and phosphorylated squamosa promoter binding protein-like 5 (TaSPL5). Furthermore, phosphorylation of TaSPL5 increased its DNA-binding activity. In addition, Arabidopsis-expressing phosphorylated TaSPL5 exhibited greater Cd tolerance than Arabidopsis-expressing unphosphorylated TaSPL5. Taken together, these data identify a TabHLH35-TaWAK20-TaSPL5 module that regulates Cd stress., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Arabis paniculata has been reported as a hyperaccumulator and functions in cadmium (Cd) tolerance and accumulation. However, the genes involved in Cd stress resistance in A. paniculata are still unknown. In this work, genes of the natural resistanceassociated macrophage proteins ( NRAMPs ) were characterized in A. paniculata , and their evolutionary relationship and expression patterns were analysed. Expression profiles indicated that ApNRAMPs showed large differences in response to Cd stress. It was highly induced by Cd in root and shoot tissues. To investigate the function of ApNRAMP4 under Cd stress, ApNRAMP4 was cloned and expressed in yeast and Arabidopsis . The results indicated that yeast and Arabidopsis expressing ApNRAMP4 showed normal growth under Cd stress. In addition, transgenic yeast and Arabidopsis showed the ability to concentrate Cd. Under 20 μM CdCl 2 , Cd concentrations in wild type (WT) and transgenic yeast were 3.11 and 5.92 mg/kg, respectively. Cd concentrations in root tissues of WTand transgenic Arabidopsis were 0.18 and 0.54 mg/kg, respectively. In shoot tissues of WT and transgenic Arabidopsis , Cd concentrations were 0.13 and 0.49 mg/kg, respectively. This report provides genomic information on hyperaccumulator A. paniculata . In addition, the present work identified key NRAMP genes that may serve as resources for heavy metal phytoremediation.

AT14A has a small domain that has sequence similarities to integrins from animals. Integrins serve as a transmembrane linker between the extracellular matrix and the cytoskeleton, which play critical roles in a variety of biological processes. Because the function of AT14A is unknown, Arabidopsis thaliana AT14A, which is a transmembrane receptor for cell adhesion molecules and a middle member of the cell wall-plasma membrane-cytoskeleton continuum in plants, has been described. AT14A, co-expressed with green fluorescent protein (GFP), was found to localize mainly to the plasma membrane. The mutant Arabidopsis at14a-1 cells exhibit various phenotypes with cell shape, cell cluster size, thickness, and cellulose content of cell wall, the adhesion between cells, and the adhesion of plasma membrane to cell wall varied by plasmolysis. Using direct staining of filamentous actin and indirect immunofluorescence staining of microtubules, cortical actin filaments and microtubules arrays were significantly altered in cells, either where AT14A was absent or over-expressed. It is concluded that AT14A may be a substantial middle member of the cell wall-plasma membrane-cytoskeleton continuum and play an important role in the continuum by regulating cell wall and cortical cytoskeleton organization.

• TaWRKY70 increased Cd tolerance of transgenic Arabidopsis. • Expression of TaWRKY70 in Arabidopsis suppress Cd transfer coefficient. • TaWRKY70 binds to the promoter of TaCAT5 as well as regulating the transcription of TaCAT5. The WRKY transcription factors (TFs) are involved in plant responses to multiple biotic and abiotic stressors; however, the role of WRKY TFs in the regulation of Cd stress in wheat remains unknown. In this study, we investigated the mechanism of the wheat TaWRKY70 TF in Cd stress. TaWRKY70 expression in Arabidopsis was used for the functional analysis. TaWRKY70 regulated Cd tolerance with Cd accumulating in roots but not in the leaf tissues of transgenic Arabidopsis. The net Cd2+ influx into Arabidopsis roots decreased when TaWRKY70 was expressed. The quantitative real-time-polymerase chain reaction (qRT-PCR) results showed that the expression levels of heavy metal ATPase (AtHMA3), natural resistance-associated macrophage protein (AtNRAMP5) , yellow stripe1-like (AtYSL3), and iron transport protein (AtIRT1) decreased under Cd-stress condition. Electrolyte leakage, as well as malondialdehyde and hydrogen peroxide contents, were lower in transgenic Arabidopsis than those in the wild-type (WT), whereas antioxidant enzyme activities were higher. Electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay, and transient transactivation assay confrmed that TaWRKY70 could directly bind to the TaCAT5 promoter. Our findings provide a novel understanding of the WRKY TFs involved in the response to heavy metal stress. [ABSTRACT FROM AUTHOR]