Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis.

BACKGROUND: Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles. CONCLUSIONS: Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

Comparative transcriptome and metabolome analysis reveal glutathione metabolic network and functional genes underlying blue and red-light mediation in maize seedling leaf.

BACKGROUND: Light quality severely affects biosynthesis and metabolism-associated process of glutathione. However, the role of specific light is still unclear on the glutathione metabolism. In this article, comparatively transcriptome and metabolome methods are used to fully understand the blue and red-light conditions working on the glutathione metabolism in maize seedling leaf. RESULTS: There are 20 differently expressed genes and 4 differently expressed metabolites in KEGG pathway of glutathione metabolism. Among them, 12 genes belong to the glutathione S-transferase family, 3 genes belong to the ascorbate peroxidase gene family and 2 genes belong to the ribonucleoside-diphosphate reductase gene family. Three genes, G6PD, SPDS1, and GPX1 belong to the gene family of glucose 6-phosphate dehydrogenase, spermidine synthase, and glutathione peroxidase, respectively. Four differently expressed metabolites are identified. Three of them, Glutathione disulfide, Glutathione, and l-γ-Glutamyl-L-amino acid are decreased while L-Glutamate is increased. In addition, Through PPI analysis, two annotated genes gst16 and DAAT, and 3 unidentified genes 100381533, pco105094 and umc2770, identified as RPP13-like3, BCAT-like1and GMPS, were obtained. By the analysis of protein sequence and PPI network, we predict that pco105094 and umc2770 were involved in the GSSG-GSH and AsA-GSH cycle in the network of glutathione metabolism. CONCLUSIONS: Compared to red light, blue light remarkably changed the transcription signal transduction and metabolism of glutathione metabolism. Differently expressed genes and metabolic mapped to the glutathione metabolism signaling pathways. In total, we obtained three unidentified genes, and two of them were predicted in current glutathione metabolism network. This result will contribute to the research of glutathione metabolism of maize.

ZmXTH, a xyloglucan endotransglucosylase/hydrolase gene of maize, conferred aluminum tolerance in Arabidopsis.

Aluminum (Al) toxicity is one of the primary factors limiting crop production in acid soils worldwide. The cell wall is the major target of Al toxicity owing to the presence of many Al binding sites. Previous studies have found that XTH, encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), could participate in cell wall extension and affect the binding ability of the cell wall to Al by impeding the activities of these two enzymes. In this study, we found that ZmXTH, an XTH gene in maize, was involved in Al detoxification. The Al-induced up-regulation of ZmXTH occurred in the roots, prominently in the root tips. Additionally, the expression of ZmXTH was specifically induced by Al3+ but no other divalent or trivalent cations. Compared with the wild-type Arabidopsis, ZmXTH overexpressing plants grew more healthy and had decreased Al content in their root and root cell wall after Al stress. Overall, the results suggest that ZmXTH could confer the Al tolerance of transgenic Arabidopsis plants by reducing the Al accumulation in their roots and cell walls.

Biochemical Characterization of Recombinant UDPG-Dependent IAA Glucosyltransferase from Maize (Zea mays).

Here, we report a biochemical characterization of recombinant maize indole-3-acetyl-ß-d-glucose (IAGlc) synthase which glucosylates indole-3-acetic acid (IAA) and thus abolishes its auxinic activity affecting plant hormonal homeostasis. Substrate specificity analysis revealed that IAA is a preferred substrate of IAGlc synthase; however, the enzyme can also glucosylate indole-3-butyric acid and indole-3-propionic acid with the relative activity of 66% and 49.7%, respectively. KM values determined for IAA and UDP glucose are 0.8 and 0.7 mM, respectively. 2,4-Dichlorophenoxyacetic acid is a competitive inhibitor of the synthase and causes a 1.5-fold decrease in the enzyme affinity towards IAA, with the Ki value determined as 117 µM, while IAA-Asp acts as an activator of the synthase. Two sugar-phosphate compounds, ATP and glucose-1-phosphate, have a unique effect on the enzyme by acting as activators at low concentrations and showing inhibitory effect at higher concentrations (above 0.6 and 4 mM for ATP and glucose-1-phosphate, respectively). Results of molecular docking revealed that both compounds can bind to the PSPG (plant secondary product glycosyltransferase) motif of IAGlc synthase; however, there are also different potential binding sites present in the enzyme. We postulate that IAGlc synthase may contain more than one binding site for ATP and glucose-1-phosphate as reflected in its activity modulation.

Genetic Variation of the Serine Acetyltransferase Gene Family for Sulfur Assimilation in Maize.

Improving sulfur assimilation in maize kernels is essential due to humans and animals' inability to synthesize methionine. Serine acetyltransferase (SAT) is a critical enzyme that controls cystine biosynthesis in plants. In this study, all SAT gene members were genome-wide characterized by using a sequence homology search. The RNA-seq quantification indicates that they are highly expressed in leaves, other than root and seeds, consistent with their biological functions in sulfur assimilation. With the recently released 25 genomes of nested association mapping (NAM) founders representing the diverse maize stock, we had the opportunity to investigate the SAT genetic variation comprehensively. The abundant transposon insertions into SAT genes indicate their driving power in terms of gene structure and genome evolution. We found that the transposon insertion into exons could change SAT gene transcription, whereas there was no significant correlation between transposable element (TE) insertion into introns and their gene expression, indicating that other regulatory elements such as promoters could also be involved. Understanding the SAT gene structure, gene expression and genetic variation involved in natural selection and species adaption could precisely guide genetic engineering to manipulate sulfur assimilation in maize and to improve nutritional quality.

The maize ZmMIEL1 E3 ligase and ZmMYB83 transcription factor proteins interact and regulate the hypersensitive defence response.

The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide-binding site, leucine-rich repeat (NLR) resistance proteins (R-proteins) that recognize the presence of specific pathogen-derived proteins. Rp1-D21 is an autoactive maize NLR R-protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1-D21. Here we identify the E3 ligase ZmMIEL1 as the causal gene at a chromosome 10 modifier locus. Transient ZmMIEL1 expression in Nicotiana benthamiana reduced HR induced by Rp1-D21, while suppression of ZmMIEL1 expression in maize carrying Rp1-D21 increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, the Arabidopsis R-protein RPM1D505V, in N. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1-D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in the N. benthamiana system. By comparison to a similar system in Arabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression of ZmMYB83 expression in maize lines carrying Rp1-D21 suppressed HR. Suppression of ZmMIEL1 expression caused an increase in ZmMYB83 transcript and protein levels in N. benthamiana and maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally, ZmMYB83 and ZmMIEL1 regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR.

Induction of defense-related enzymes and enhanced disease resistance in maize against Fusarium verticillioides by seed treatment with Jacaranda mimosifolia formulations.

Fusarium verticillioides is an important fungal pathogen of maize, causing stalk rot and severely affecting crop production. The aim of this study was to characterize the protective effects of formulations based on Jacaranda mimosifolia leaf extracts against F. verticillioides in maize. We compared different seed treatments comprising J. mimosifolia extracts, chemical fungicide (mefenoxam) and salicylic acid to modulate the defense system of maize host plants. Both aqueous and methanolic leaf extracts of J. mimosifolia (1.2% w/v) resulted in 96-97% inhibition of mycelial growth of F. verticillioides. While a full-dose (1.2%) extract of J. mimosifolia provided significant protective effects on maize plants compared to the inoculated control, a half-dose (0.6% w/v) application of J. mimosifolia in combination with half-strength mefenoxam was the most effective treatment in reducing stalk rot disease in pot and field experiments. The same seed treatment significantly upregulated the expression of genes in the leaves encoding chitinase, glucanase, lipid transfer protein, and pathogenesis-related proteins PR-1, PR-5 and PR-10, 72 h after inoculation. This treatment also induced the activities of peroxidase, polyphenol oxidase, protease, acid invertase, chitinase and phenylalanine ammonia lyase. We conclude that seed pre-treatment with J. mimosifolia extract with half-strength chemical mefenoxam is a promising approach for the management of stalk rot in maize.

Maize metacaspases modulate the defense response mediated by the NLR protein Rp1-D21 likely by affecting its subcellular localization.

Plants usually employ resistance (R) genes to defend against the infection of pathogens, and most R genes encode intracellular nucleotide-binding, leucine-rich repeat (NLR) proteins. The recognition between R proteins and their cognate pathogens often triggers a rapid localized cell death at the pathogen infection sites, termed the hypersensitive response (HR). Metacaspases (MCs) belong to a cysteine protease family, structurally related to metazoan caspases. MCs play crucial roles in plant immunity. However, the underlying molecular mechanism and the link between MCs and NLR-mediated HR are not clear. In this study, we systematically investigated the MC gene family in maize and identified 11 ZmMCs belonging to two types. Further functional analysis showed that the type I ZmMC1 and ZmMC2, but not the type II ZmMC9, suppress the HR-inducing activity of the autoactive NLR protein Rp1-D21 and of its N-terminal coiled-coil (CCD21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CCD21 in vivo. We further showed that ZmMC1 and ZmMC2, but not ZmMC9, are predominantly localized in a punctate distribution in both N. benthamiana and maize (Zea mays) protoplasts. Furthermore, the co-expression of ZmMC1 and ZmMC2 with Rp1-D21 and CCD21 causes their re-distribution from being uniformly distributed in the nucleocytoplasm to a punctate distribution co-localizing with ZmMC1 and ZmMC2. We reveal a novel role of plant MCs in modulating the NLR-mediated defense response and derive a model to explain it.

An embedded gene selection method using knockoffs optimizing neural network.

BACKGROUND: Gene selection refers to find a small subset of discriminant genes from the gene expression profiles. How to select genes that affect specific phenotypic traits effectively is an important research work in the field of biology. The neural network has better fitting ability when dealing with nonlinear data, and it can capture features automatically and flexibly. In this work, we propose an embedded gene selection method using neural network. The important genes can be obtained by calculating the weight coefficient after the training is completed. In order to solve the problem of black box of neural network and further make the training results interpretable in neural network, we use the idea of knockoffs to construct the knockoff feature genes of the original feature genes. This method not only make each feature gene to compete with each other, but also make each feature gene compete with its knockoff feature gene. This approach can help to select the key genes that affect the decision-making of neural networks. RESULTS: We use maize carotenoids, tocopherol methyltransferase, raffinose family oligosaccharides and human breast cancer dataset to do verification and analysis. CONCLUSIONS: The experiment results demonstrate that the knockoffs optimizing neural network method has better detection effect than the other existing algorithms, and specially for processing the nonlinear gene expression and phenotype data.

Crystal structure of DRIK1, a stress-responsive receptor-like pseudokinase, reveals the molecular basis for the absence of ATP binding.

BACKGROUND: Plants reprogram metabolism and development to rapidly adapt to biotic and abiotic stress. Protein kinases play a significant role in this process by phosphorylating protein substrates that activate or inactivate signaling cascades that regulate cellular and metabolic adaptations. Despite their importance in plant biology, a notably small fraction of the plant kinomes has been studied to date. RESULTS: In this report, we describe ZmDRIK1, a stress-responsive receptor-like pseudokinase whose expression is downregulated under water restriction. We show the structural features and molecular basis of the absence of ATP binding exhibited by ZmDRIK1. The ZmDRIK1 kinase domain lacks conserved amino acids that are essential for phosphorylation activity. The crystal structure of the ZmDRIK1 kinase domain revealed the presence of a spine formed by the side chain of the triad Leu240, Tyr363, and Leu375 that occludes the ATP binding pocket. Although ZmDRIK1 is unable to bind nucleotides, it does bind the small molecule ENMD-2076 which, in a cocrystal structure, revealed the potential to serve as a ZmDRIK1 inhibitor. CONCLUSION: ZmDRIK1 is a novel receptor-like pseudokinase responsive to biotic and abiotic stress. The absence of ATP binding and consequently, the absence of phosphorylation activity, was proven by the crystal structure of the apo form of the protein kinase domain. The expression profiling of the gene encoding ZmDRIK1 suggests this kinase may play a role in downregulating the expression of stress responsive genes that are not necessary under normal conditions. Under biotic and abiotic stress, ZmDRIK1 is down-regulated to release the expression of these stress-responsive genes.

Structural basis for monolignol oxidation by a maize laccase.

Plant laccases catalyse the oxidation of monolignols in lignification, a process reinforcing the cell wall of many different cell types that provide mechanical support, nutrient transportation and defence against pathogens in plants1. The isozymes display a broad range of substrate preferences. Here, the substrate preference of a laccase (ZmLac3) from Zea mays (maize) was characterized. The crystal structure of ZmLac3 revealed a compact and deep substrate-binding pocket, and the binding modes of sinapyl alcohol (SinA) and coniferyl alcohol (ConA) were solved. On the basis of structural data and kinetics analysis, we propose that the regionalization of polar and hydrophobic surfaces in the binding pocket of ZmLac3 is vital for defining the orientation of SinA/ConA binding. The extra methoxyl group in SinA makes substantial contributions to interactions between SinA and ZmLac3, which are absent in the ZmLac3-ConA complex. In summary, the polar and hydrophobic interactions between SinA/ConA and ZmLac3 determine the binding positions of the monolignols in ZmLac3. These results provide valuable insight about ZmLac3 catalysis and should aid industrial processes that use plant laccases.

Thr420 and Ser454 of ZmCCaMK play a crucial role in brassinosteroid-induced antioxidant defense in maize.

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play important roles in brassinosteroid (BR)-induced antioxidant defense and enhancing the tolerance of plants to drought stress. The autophosphorylation of CCaMK is a key step for the activation of CCaMK, thus promoting substrate phosphorylation. However, how CCaMK autophosphorylation function in BR-induced antioxidant defense is not known yet. Here, seven potential autophosphorylation sites of ZmCCaMK were identified using mass spectroscopy (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) analysis. The transient gene expression analysis in maize protoplasts showed that Thr420 and Ser454 of ZmCCaMK were important for BR-induced antioxidant defense. Furthermore, Thr420 and Ser454 of ZmCCaMK were crucial for improving drought tolerance and alleviating drought induced oxidative damage of plants via overexpressing various mutant versions of ZmCCaMK in tobacco (Nicotiana tabacum). Mutations of Thr420 and Ser454 in ZmCCaMK substantially blocked the autophosphorylation and substrate phosphorylation of ZmCCaMK in vitro. Taken together, our results demonstrate that Thr420 and Ser454 of ZmCCaMK are crucial for BR-induced antioxidant defense and drought tolerance through modulating the autophosphorylation and substrate phosphorylation activities of ZmCCaMK.

Maize roots and shoots show distinct profiles of oxidative stress and antioxidant defense under heavy metal toxicity.

Heavy metal accumulation in agricultural land causes crop production losses worldwide. Metal homeostasis within cells is tightly regulated. However, homeostasis breakdown leads to accumulation of reactive oxygen species (ROS). Overall plant fitness under stressful environment is determined by coordination between roots and shoots. But little is known about organ specific responses to heavy metals, whether it depends on the metal category (redox or non-redox reactive) and if these responses are associated with heavy metal accumulation in each organ or there are driven by other signals. Maize seedlings were subjected to sub-lethal concentrations of four metals (Zn, Ni, Cd and Cu) individually, and were quantified for growth, ABA level, and redox alterations in roots, mature leaves (L1,2) and young leaves (L3,4) at 14 and 21 days after sowing (DAS). The treatments caused significant increase in endogenous metal levels in all organs but to different degrees, where roots showed the highest levels. Biomass was significantly reduced under heavy metal stress. Although old leaves accumulated less heavy metal content than root, the reduction in their biomass (FW) was more pronounced. Metal exposure triggered ABA accumulation and stomatal closure mainly in older leaves, which consequently reduced photosynthesis. Heavy metals induced oxidative stress in the maize organs, but to different degrees. Tocopherols, polyphenols and flavonoids increased specifically in the shoot under Zn, Ni and Cu, while under Cd treatment they played a minor role. Under Cu and Cd stress, superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were induced in the roots, however ascorbate peroxidase (APX) activity was only increased in the older leaves. Overall, it can be concluded that root and shoot organs specific responses to heavy metal toxicity are not only associated with heavy metal accumulation and they are specialized at the level of antioxidants to cope with.

Biosynthesis pathway of indole-3-acetyl-myo-inositol during development of maize (Zea mays L.) seeds.

Indole-3-acetic acid (IAA) conjugation is one of the mechanisms responsible for auxin homeostasis. IAA ester conjugates biosynthesis has been studied during development of maize seeds where IAA-inositol (IAInos) and its glycosidic forms make up about 50 % of its ester conjugates pool. 1-O-indole-3-acetyl-ß-d-glucose (IAGlc) synthase and indole-3-acetyl transferase (IAInos synthase) are key enzymes in a two-step pathway of IAInos synthesis. In the first reaction, IAA is glucosylated to a high energy acetal, 1-O-indole-3-acetyl-ß-d-glucose by IAGlc synthase, whereas in the second step, IAInos synthase transfers IAA moiety to myo-inositol forming a stable auxin ester, indole-3-acetyl-myo-inositol (IAInos). It should be mentioned that IAGlc synthase catalyzes a reversible reaction with unfavourable equilibrium that delivers IAGlc for favourable transacylation to IAInos. This is the first study where IAGlc synthase and IAInos synthase are simultaneously analyzed by enzymatic activity assay and quantitative RT-PCR in maize seeds at four stages of development (13, 26, 39 and 52 Days After Flowering). Activity of IAGlc/IAInos synthases as well as their expression profiles during seed development were different. While both enzymatic activities and ZmIAIn expression were the highest in seeds at 26 DAF, the highest expression of ZmIAGlc was observed at 13 DAF. Protein gel blot analysis showed that IAInos synthase exists as a mixture of several isoforms at a similar protein level at particular stages of seed development. Neither of other ester conjugates of IAA (IAA-mannose) nor IAA-amino acids were detected at the stages studied. Catalytic activity of l-tryptophan aminotransferase involved in IAA biosynthesis as well as UDPG pyrophosphorylase, synthesizing UDPG as a substrate for IAGlc synthase, were also analyzed. l-tryptophan aminotransferase activity was the highest at 26 DAF. Changes in enzyme activity of UDPG pyrophosphorylase are difficult to interpret. Expression levels of ZmIPS and ZmIPP encoding two enzymes of myo-inositol biosynthesis pathway: inositol-x-phosphate synthase (IPS) and inositol-x-phosphate phosphatase (IPP), respectively, were analyzed. 26 DAF seeds displayed the highest expression level of ZmIPS, whereas transcription of ZmIPP was the highest at 13 DAF.

A 6&1-FEH Encodes an Enzyme for Fructan Degradation and Interact with Invertase Inhibitor Protein in Maize (Zea mays L.).

About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.

Identification of a 119-bp Promoter of the Maize Sulfite Oxidase Gene (ZmSO) That Confers High-Level Gene Expression and ABA or Drought Inducibility in Transgenic Plants.

Drought adversely affects crop growth and yields. The cloning and characterization of drought- or abscisic acid (ABA)-inducible promoters is of great significance for their utilization in the genetic improvement of crop resistance. Our previous studies have shown that maize sulfite oxidase (SO) has a sulfite-oxidizing function and is involved in the drought stress response. However, the promoter of the maize SO gene has not yet been characterized. In this study, the promoter (ZmSOPro, 1194 bp upstream region of the translation initiation site) was isolated from the maize genome. The in-silico analysis of the ZmSOPro promoter identified several cis-elements responsive to the phytohormone ABA and drought stress such as ABA-responsive element (ABRE) and MYB binding site (MBS), besides a number of core cis-acting elements, such as TATA-box and CAAT-box. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmSO. The ZmSOPro activity was detected by ß-glucuronidase (GUS) staining at nearly all developmental stages and in most plant organs, except for the roots in transgenic Arabidopsis. Moreover, its activity was significantly induced by ABA and drought stress. The 5'-deletion mutant analysis of the ZmSOPro in tobacco plants revealed that a 119-bp fragment in the ZmSOPro (upstream of the transcription start site) is a minimal region, which is required for its high-level expression. Moreover, the minimal ZmSOPro was significantly activated by ABA or drought stress in transgenic plants. Further mutant analysis indicated that the MBS element in the minimal ZmSOPro region (119 bp upstream of the transcription start site) is responsible for ABA and drought-stress induced expression. These results improve our understanding of the transcriptional regulation mechanism of the ZmSO gene, and the characterized 119-bp promoter fragment could be an ideal candidate for drought-tolerant gene engineering in both monocot and dicot crops.

A maize polygalacturonase functions as a suppressor of programmed cell death in plants.

BACKGROUND: The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7. RESULTS: Expression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field. CONCLUSIONS: ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.

Rational design, synthesis and structure-activity relationship of novel substituted oxazole isoxazole carboxamides as herbicide safener.

A series of novel substituted oxazole isoxazole carboxamides derivatives were designed on the basis of active subunit combination. Forty-four novel compounds were synthesized by an efficient one-pot procedure under microwave irradiation. The bioactivity was evaluated as herbicide safener against the injury of chlorsulfuron. It was found that most of the synthesized compounds displayed remarkable protection against chlorsulfuron via enhanced glutathione content and glutathione S transferase activity. Especially compound I-11 exhibited better bioactivity than the safeners isoxadifen-ethyl and R-28725. Molecular docking simulations suggested that the target compounds could compete with chlorsulfuron in the active site of acetolactate synthase, which could explain the protective effects of safeners. The present work demonstrates that the target compounds containing oxazole isoxazole groups could be considered as potential candidates for developing novel safeners in the future.

Maize polyamine oxidase in the presence of spermine/spermidine induces the apoptosis of LoVo human colon adenocarcinoma cells.

Amine oxidases, which contribute to the regulation of polyamine levels, catalyze the oxidative deamination of polyamines to generate H2O2 and aldehyde(s). In this study, and at least to the best of our knowledge, maize polyamine oxidase (ZmPAO) was used for the first time with the aim of identifying a novel strategy for cancer therapy. The cytotoxicity and the mechanisms of cell death induced by the enzymatic oxidation products of polyamine generated by ZmPAO were investigated. Exogenous spermine and ZmPAO treatment decreased cell viability in a spermine dose­ and time­dependent manner, particularly, the viability of the multidrug­resistant (MDR) colon adenocarcinoma cells, LoVo DX, when compared with drug­sensitive ones (LoVo WT). Further analyses revealed that H2O2 derived from spermine was mainly responsible for the cytotoxicity. Flow cytometric analysis revealed that treatment with ZmPAO and spermine increased the apoptotic population of LoVo WT and LoVo DX cells. In addition, we found that treatment with ZmPAO and spermine markedly reduced mitochondrial membrane potential in the LoVo DX cells, in agreement with the results of cell viability and apoptosis assays. Transmission electron microscopic observations supported the involvement of mitochondrial depolarization in the apoptotic process. Therefore, the dysregulation of polyamine metabolism in tumor cells may be a potential therapeutic target. In addition, the development of MDR tumor cells is recognized as a major obstacle in cancer therapy. Therefore, the design of a novel therapeutic strategy based on the use of this combination may be taken into account, making this approach attractive mainly in treating MDR cancer patients.

Functional analysis of Arabidopsis and maize transgenic lines overexpressing the ADP-ribose/NADH pyrophosphohydrolase, AtNUDX7.

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.