The Effect of Granulocyte Colony-Stimulating Factor on Endometrial Receptivity of Implantation Failure Mouse.

The purpose of this study was to investigate the effect of G-CSF on the endometrial receptivity of implantation failure mice. Sixty female mice were treated mifepristone to establish an implant failure model. The treatment groups received different doses of G-CSF. Endometrial tissue and serum were collected on day 5 after mating. The abundance of pinopodes on the endometrium was observed by scanning electron microscopy. The expressions of LPAR3, COX2, and HOXA10 were detected by RT-qPCR and Western blotting. Serum levels of E2, P, VEGF, LIF, TNF-α and IL-10 were measured by ELISA. The expressions of VEGF, CD34, CD57, TNF-α, and IL-10 were assessed by immunohistochemistry. Immunofluorescence analysis was performed to determine the number of CD57, Treg, and Th17 cells. G-CSF increased implantation and pregnancy rates of mifepristone-induced implantation failure mice, with the most significant effect seen at the intermediate dose. G-CSF increased the serum levels of E2 and P, the abundance of endometrial pinopodes, and the level of LIF in the endometrium. It also promoted the expression of VEGF, HOXA10, LPAR3, and COX2. Moreover, G-CSF reduced the level of CD57 cells and the ratio of Th17/Treg cells in endometrium. G-CSF reduced the inflammatory factor TNF-α, but IL-10 did not change significantly. G-CSF can enhance embryo implantation rate and pregnancy rate and improve endometrial receptivity by attenuating degeneration of pinopodes, upregulating estrogen and progesterone, facilitating angiogenesis, maintaining immune cell homeostasis, and reducing the production of inflammatory cytokines in implantation failure mouse.

Overexpression of ZmSTOP1-A Enhances Aluminum Tolerance in Arabidopsis by Stimulating Organic Acid Secretion and Reactive Oxygen Species Scavenging.

Aluminum (Al) toxicity and low pH are major factors limiting plant growth in acidic soils. Sensitive to Proton Rhizotoxicity 1 (STOP1) transcription factors respond to these stresses by regulating the expression of multiple Al- or low pH-responsive genes. ZmSTOP1-A, a STOP1-like protein from maize (Zea mays), was localized to the nucleus and showed transactivation activity. ZmSTOP1-A was expressed moderately in both roots and shoots of maize seedlings, but was not induced by Al stress or low pH. Overexpression of ZmSTOP1-A in Arabidopsis Atstop1 mutant partially restored Al tolerance and improved low pH tolerance with respect to root growth. Regarding Al tolerance, ZmSTOP1-A/Atstop1 plants showed clear upregulation of organic acid transporter genes, leading to increased organic acid secretion and reduced Al accumulation in roots. In addition, the antioxidant enzyme activity in roots and shoots of ZmSTOP1-A/Atstop1 plants was significantly enhanced, ultimately alleviating Al toxicity via scavenging reactive oxygen species. Similarly, ZmSTOP1-A could directly activate ZmMATE1 expression in maize, positively correlated with the number of Al-responsive GGNVS cis-elements in the ZmMATE1 promoter. Our results reveal that ZmSTOP1-A is an important transcription factor conferring Al tolerance by enhancing organic acid secretion and reactive oxygen species scavenging in Arabidopsis.

GWAS across multiple environments and WGCNA suggest the involvement of ZmARF23 in embryonic callus induction from immature maize embryos.

KEY MESSAGE: Combined GWAS, WGCNA, and gene-based association studies identified the co-expression network and hub genes for maize EC induction. ZmARF23 bound to ZmSAUR15 promoter and regulated its expression, affecting EC induction. Embryonic callus (EC) induction in immature maize embryos shows high genotype dependence, which limits the application of genetic transformation in transgenic breeding and gene function elucidation in maize. Herein, we conducted a genome-wide association mapping (GWAS) for four EC induction-related traits, namely rate of embryonic callus induction (REC), increased callus diameter (ICD), ratio of shoot formation (RSF), and length of shoot (LS) across different environments. A total of 77 SNPs were significantly associated these traits under three environments and using the averages (across environments). Among these significant SNPs, five were simultaneously detected under multiple environments and 11 had respective phenotypic variation explained > 10%. A total of 257 genes were located in the linkage disequilibrium decay of these REC- and ICD-associated SNPs, of which 178 were responsive to EC induction. According to the expression values of the 178 genes, we performed a weighted gene co-expression network analysis (WGCNA) and revealed an EC induction-associated module and five hub genes. Hub gene-based association studies uncovered that the intragenic variations in GRMZM2G105473 and ZmARF23 influenced EC induction efficiency among different maize lines. Dual-luciferase reporter assay indicated that ZmARF23 bound to the promoter of a known causal gene (ZmSAUR15) for EC induction and positively regulated its expression on the transcription level. Our study will deepen the understanding of genetic and molecular mechanisms underlying EC induction and contribute to the use of genetic transformation in maize.

Analgesics can affect the sensitivity of temozolomide to glioma chemotherapy through gap junction.

This study investigated the effect of frequently used analgesics in cancer pain management (flurbiprofen (FLU), tramadol (TRA), and morphine (MOR)) and a novel α2-adrenergic agonist (dexmedetomidine, DEX) on temozolomide (TMZ) sensitivity in glioma cells. Cell counting kit-8 and colony-formation assays were performed to analyze the viability of U87 and SHG-44 cell lines. A high and low cell density of colony method, pharmacological methods, and connexin43 mimetic peptide GAP27 were used to manipulate the function of gap junctions; "Parachute" dye coupling and western blot were employed to determine junctional channel transfer ability and connexin expression. The results showed that DEX (in the concentration range of 0.1 to 5.0 ng/ml) and TRA (in the concentration range of 1.0 to 10.0 µg/ml) reduced the TMZ cytotoxicity in a concentration-dependent manner but was only observed with high cell density (having formed gap junction). The cell viability percentage was 71.3 to 86.8% when DEX was applied at 5.0 ng/ml, while tramadol showed 69.6 to 83.7% viability at 5.0 µg/ml in U87 cells. Similarly, 5.0 ng/ml of DEX resulted in 62.6 to 80.5%, and 5.0 µg/ml TRA showed 63.5 to 77.3% viability in SHG-44 cells. Further investigating the impact of analgesics on gap junctions, only DEX and TRA were found to decrease channel dye transfer through connexin phosphorylation and ERK pathway, while no such effect was observed for FLU and MOR. Analgesics that can affect junctional communication may compromise the effectiveness of TMZ when used simultaneously.

The protective effect of total glucosides of white paeony capsules on experimental autoimmune encephalomyelitis.

AIMS: To learn about the effect and mechanism of total glucosides of white peony capsule (TGP), on experimental autoimmune encephalomyelitis (EAE), an acknowledged animal model of multiple sclerosis (MS). METHODS: The rat model of EAE was induced by subcutaneous injection with guinea pig spinal cord homogenate. The severity of the disease model was assessed by clinical score, hematoxylin and eosin (H&E) and luxol fast blue (LFB). Immunohistochemical assay was used to observe the types of inflammatory cells and adhesive molecule expression. Enzyme-linked immunosorbent assay (ELISA) was applied to detect content of the stem cell growth factor / mast cell growth factor (scf/MGF), interleukin-6 (IL-6) and IL-2. Immunofluorescence assay was applied to observe the expression of connexin43 (Cx43), glial fibrillary acidic protein (GFAP), connexin47 (Cx47) and the monoclonal antibody anti-adenomatous polyposis coli (APC) clone CC1. RESULTS: Compare with the animals in EAE model group, TGP treated rats (particularly those treated with high doses) showed a significant decrease in morbidity, clinical scores, CNS infiltration of inflammatory cells (including mononuclear macrophages, CD4+ and CD8+ T cells) and demyelination. The key adhesion molecule ICAM-1, cytokines IL-2、IL-6 and scf/MGF were significantly decreased with TGP treatment. Oppositely, PD-1, connexin47 in oligodendrocytes and connexin43 in astrocytes were elevated with TGP treatment. CONCLUSION: To sum up, TGP exhibited a significantly prevention and treatment effect on EAE rat model, and this improvement was achieved through a combination way composed of glial and inflammatory cells, junction proteins, various factors including adhesion factors, interleukins and scf/MGF.

ZmNRAMP4 Enhances the Tolerance to Aluminum Stress in Arabidopsis thaliana.

Aluminum (Al) toxicity causes severe reduction in crop yields in acidic soil. The natural resistance-associated macrophage proteins (NRAMPs) play an important role in the transport of mineral elements in plants. Recently, OsNrat1 and SbNrat1 were reported specifically to transport trivalent Al ions. In this study, we functionally characterized ZmNRAMP4, a gene previously identified from RNA-Seq data from Al-treated maize roots, in response to Al exposure in maize. ZmNRAMP4 was predominantly expressed in root tips and was specifically induced by Al stress. Yeast cells expressing ZmNRAMP4 were hypersensitive to Al, which was associated with Al accumulation in yeast. Furthermore, overexpression of ZmNRAMP4 in Arabidopsis conferred transgenic plants with a significant increase in Al tolerance. However, expression of ZmNRAMP4, either in yeast or in Arabidopsis, had no effect on the response to cadmium stress. Taken together, these results underlined an internal tolerance mechanism involving ZmNRAMP4 to enhance Al tolerance via cytoplasmic sequestration of Al in maize.

Overexpression of the Aldehyde Dehydrogenase Gene ZmALDH Confers Aluminum Tolerance in Arabidopsis thaliana.

Aluminum (Al) toxicity is the main factor limiting plant growth and the yield of cereal crops in acidic soils. Al-induced oxidative stress could lead to the excessive accumulation of reactive oxygen species (ROS) and aldehydes in plants. Aldehyde dehydrogenase (ALDH) genes, which play an important role in detoxification of aldehydes when exposed to abiotic stress, have been identified in most species. However, little is known about the function of this gene family in the response to Al stress. Here, we identified an ALDH gene in maize, ZmALDH, involved in protection against Al-induced oxidative stress. Al stress up-regulated ZmALDH expression in both the roots and leaves. The expression of ZmALDH only responded to Al toxicity but not to other stresses including low pH and other metals. The heterologous overexpression of ZmALDH in Arabidopsis increased Al tolerance by promoting the ascorbate-glutathione cycle, increasing the transcript levels of antioxidant enzyme genes as well as the activities of their products, reducing MDA, and increasing free proline synthesis. The overexpression of ZmALDH also reduced Al accumulation in roots. Taken together, these findings suggest that ZmALDH participates in Al-induced oxidative stress and Al accumulation in roots, conferring Al tolerance in transgenic Arabidopsis.

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.

ZmMATE6 from maize encodes a citrate transporter that enhances aluminum tolerance in transgenic Arabidopsis thaliana.

The yields of cereal crops grown on acidic soils are often reduced by aluminum (Al) toxicity because the prevalence of toxic Al3+ cations increases as pH falls below 5.0. The Al-dependent release of citrate from resistant lines of maize is controlled by ZmMATE1 which encodes a multidrug and toxic compound extrusion (MATE) transporter protein. ZmMATE6 is another member of this family in maize whose expression is also increased by Al treatment. We investigated the function of this gene in more detail to determine whether it also contributes to Al resistance. Quantitative RT-PCR measurements found that ZmMATE6 was expressed in the roots and leaves of Al-resistant and sensitive inbred lines. Treatment with Al induced ZmMATE6 expression in all tissues but several other divalent or trivalent cations tested had no effect on expression. This expression pattern and the induction by Al treatment was confirmed in ZmMATE6 promoter-ß-glucuronidase fusion lines. Heterogeneous expression of ZmMATE6 displayed a greater Al-activated release of citrate from the roots and was significantly resistant to Al toxicity than controls. This was associated with reduced accumulation of Al in the root tissues. Our results demonstrated that ZmMATE6 expression is induced by Al and functions as a citrate transporter.

Transcriptomic and genome-wide association study reveal long noncoding RNAs responding to nitrogen deficiency in maize.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in essential biological processes. However, our understanding of lncRNAs as competing endogenous RNAs (ceRNAs) and their responses to nitrogen stress is still limited. RESULTS: Here, we surveyed the lncRNAs and miRNAs in maize inbred line P178 leaves and roots at the seedling stage under high-nitrogen (HN) and low-nitrogen (LN) conditions using lncRNA-Seq and small RNA-Seq. A total of 894 differentially expressed lncRNAs and 38 different miRNAs were identified. Co-expression analysis found that two lncRNAs and four lncRNA-targets could competitively combine with ZmmiR159 and ZmmiR164, respectively. To dissect the genetic regulatory by which lncRNAs might enable adaptation to limited nitrogen availability, an association mapping panel containing a high-density single-nucleotide polymorphism (SNP) array (56,110 SNPs) combined with variable LN tolerant-related phenotypes obtained from hydroponics was used for a genome-wide association study (GWAS). By combining GWAS and RNA-Seq, 170 differently expressed lncRNAs within the range of significant markers were screened. Moreover, 40 consistently LN-responsive genes including those involved in glutamine biosynthesis and nitrogen acquisition in root were identified. Transient expression assays in Nicotiana benthamiana demonstrated that LNC_002923 could inhabit ZmmiR159-guided cleavage of Zm00001d015521. CONCLUSIONS: These lncRNAs containing trait-associated significant SNPs could consider to be related to root development and nutrient utilization. Taken together, the results of our study can provide new insights into the potential regulatory roles of lncRNAs in response to LN stress, and give valuable information for further screening of candidates as well as the improvement of maize resistance to LN stress.

A Maize ZmAT6 Gene Confers Aluminum Tolerance via Reactive Oxygen Species Scavenging.

Aluminum (Al) toxicity is the primary limiting factor that affects crop yields in acid soil. However, the genes that contribute to the Al tolerance process in maize are still poorly understood. Previous studies have predicted that ZmAT6 is a novel protein which could be upregulated under Al stress condition. Here, we found that ZmAT6 is expressed in many tissues and organs and can be dramatically induced by Al in both the roots and shoots but particularly in the shoots. The overexpression of ZmAT6 in maize and Arabidopsis plants increased their root growth and reduced the accumulation of Al, suggesting the contribution of ZmAT6 to Al tolerance. Moreover, the ZmAT6 transgenic maize plants had lower contents of malondialdehyde and reactive oxygen species (ROS), but much higher proline content and even lower Evans blue absorption in the roots compared with the wild type. Furthermore, the activity of several enzymes of the antioxidant system, such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), increased in ZmAT6 transgenic maize plants, particularly SOD. Consistently, the expression of ZmSOD in transgenic maize was predominant upregulated by Al stress. Taken together, these findings revealed that ZmAT6 could at least partially confer enhanced tolerance to Al toxicity by scavenging ROS in maize.

The taxane-based chemotherapy triplet is superior to the doublet in one to nine node-positive but not node-negative triple-negative breast cancer: results from a retrospective analysis.

Background: Taxane-based regimens that are frequently used in adjuvant chemotherapy in early triple-negative breast cancer (TNBC) include a three-drug regimen (TAC and AC-T) and a two-drug regimen (TA and TC). Whether pathological lymph node stage guides taxane-based de-escalating chemotherapies in TNBC in adjuvant setting is still unclear. Methods: We retrospectively examined 381 patients with early TNBC over a median follow-up period of 75.9 months and compared the disease-free survival (DFS) and overall survival (OS) of patients who received adjuvant taxane-based three-drug chemotherapy and two-drug chemotherapy according to pathological lymph node stage (negative, pN0; 1-3 positive, pN1; 4-9 positive, pN2). Results: In 222 pN0 patients, the taxane-based three-drug regimen was not superior to the two-drug regimen. In 159 pN1-2 patients, the taxane-based three-drug regimen significantly improved DFS (5-year DFS rate, 78.2% vs. 46.9%; log-rank p=0.0002) and OS (5-year OS rate, 90.7% vs. 64.3%; log-rank p=0.0001) compared with the two-drug regimen. In a multivariable Cox regression analysis of pN1-2 patients, after adjustment for clinical characteristics, the taxane-based three-drug regimen significantly decreased the risk of recurrence (adjusted HR, 0.37; 95% CI, 0.22 to 0.64; p=0.0004) and death (adjusted HR, 0.22; 95% CI, 0.10 to 0.48; p=0.0001) compared with the two-drug regimen. Conclusions: The taxane-based chemotherapy triplet is superior to the chemotherapy doublet in patients with one to nine positive lymph nodes but not node-negative TNBC in adjuvant setting. These data suggest that pathological lymph node stage leads to de-escalating chemotherapy strategies in operable TNBC patients.

qRf8-1, a Novel QTL for the Fertility Restoration of Maize CMS-C Identified by QTL-seq.

C-type cytoplasmic male sterility (CMS-C), one of the three major CMS types in maize, has a promising application prospect in hybrid seed production. However, the complex genetic mechanism underlying the fertility restoration of CMS-C remains poorly understood. The maize inbred line A619 is one of the rare strong restorer lines carrying the restorer gene Rf4, but different fertility segregation ratios are found in several F2 populations derived from crosses between isocytoplasmic allonucleus CMS-C lines and A619. In the present study, the segregation ratios of fertile to sterile plants in the (CHuangzaosi × A619) F2 and BC1F1 populations (36.77:1 and 2.36:1, respectively) did not follow a typical monogenic model of inheritance, which suggested that some F2 and BC1F1 plants displayed restored fertility even without Rf4 To determine the hidden locus affecting fertility restoration, next-generation sequencing-based QTL-seq was performed with two specific extreme bulks consisting of 30 fertile and 30 sterile rf4rf4 individuals from the F2 population. A major QTL related to fertility restoration, designated qRf8-1, was detected on the long arm of chromosome 8 in A619. Subsequently, qRf8-1 was further validated and narrowed down to a 17.93-Mb genomic interval by insertion and deletion (InDel) and simple sequence repeat (SSR) marker-based traditional QTL mapping, explaining 12.59% (LOD = 25.06) of the phenotypic variation. Thus, using genetic analyses and molecular markers, we revealed another fertility restoration system acting in parallel with Rf4 in A619 that could rescue the male sterility of CHuangzaosi. This study not only expands the original fertility restoration system but also provides valuable insights into the complex genetic mechanisms underlying the fertility restoration of CMS-C.

Development of a handheld dual-channel optical fiber fluorescence sensor based on a smartphone.

In order to meet the needs of on-site, accurate, fast, and remote detection, we design a smartphone-based handheld dual-channel optical fiber fluorescence sensor (DOFFS), which is composed of a semiconductor laser for exciting fluorescence signals, a smartphone with a dual-bandpass filter for collecting fluorescence signals, a fiber coupler for transmitting light, and batteries for laser power supply. All the components are integrated into a 3D printed shell, on the side of which there are two fiber flanges used for fiber probe connection. The fluorescence signals of green and red quantum dots modified on the fiber probes can be captured by the smartphone camera and calculated by a self-developed Android application. The comparisons of single-channel and dual-channel fluorescence signals with pH show that the performance of the sensor is good. The proposed sensor not only can simultaneously detect dual-channel signals for fast detection needs, but it also is handheld with a small size of 79×57×154mm3 and inner power supply, and the fiber probes can be easily replaced, supporting remote and on-site applications. It is a potential tool for many occasions in many fields.

Metabolite profiling and genome-wide association studies reveal response mechanisms of phosphorus deficiency in maize seedling.

Inorganic phosphorus (Pi) is an essential element in numerous metabolic reactions and signaling pathways, but the molecular details of these pathways remain largely unknown. In this study, metabolite profiles of maize (Zea mays L.) leaves and roots were compared between six low-Pi-sensitive lines and six low-Pi-tolerant lines under Pi-sufficient and Pi-deficient conditions to identify pathways and genes associated with the low-Pi stress response. Results showed that under Pi deprivation the concentrations of nucleic acids, organic acids and sugars were increased, but that the concentrations of phosphorylated metabolites, certain amino acids, lipid metabolites and nitrogenous compounds were decreased. The levels of secondary metabolites involved in plant immune reactions, including benzoxazinoids and flavonoids, were significantly different in plants grown under Pi-deficient conditions. Among them, the 11 most stable metabolites showed significant differences under low- and normal-Pi conditions based on the coefficient of variation (CV). Isoleucine and alanine were the most stable metabolites for the identification of Pi-sensitive and Pi-resistant maize inbred lines. With the significant correlation between morphological traits and metabolites, five low-Pi-responding consensus genes associated with morphological traits and simultaneously involved in metabolic pathways were mined by combining metabolites profiles and genome-wide association study (GWAS). The consensus genes induced by Pi deficiency in maize seedlings were also validated by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Moreover, these genes were further validated in a recombinant inbred line (RIL) population, in which the glucose-6-phosphate-1-epimerase encoding gene mediated yield and correlated traits to phosphorus availability. Together, our results provide a framework for understanding the metabolic processes underlying Pi-deficient responses and give multiple insights into improving the efficiency of Pi use in maize.

Comparative transcriptome analysis reveals that tricarboxylic acid cycle-related genes are associated with maize CMS-C fertility restoration.

BACKGROUND: C-type cytoplasmic male sterility (CMS-C) is one of the three major types of cytoplasmic male sterility (CMS) in maize. Rf4 is a dominant restorer gene for CMS-C and has great value in hybrid maize breeding, but little information concerning its functional mechanism is known. RESULTS: To reveal the functional mechanism of Rf4, we developed a pair of maize near-isogenic lines (NILs) for the Rf4 locus, which included a NIL_rf4 male-sterile line and a NIL_Rf4 male fertility-restored line. Genetic analysis and molecular marker detection indicated that the male fertility of NIL_Rf4 was controlled by Rf4. Whole-genome sequencing demonstrated genomic differences between the two NILs was clustered in the Rf4 mapping region. Unmapped reads of NILs were further assembled to uncover Rf4 candidates. RNA-Seq was then performed for the developing anthers of the NILs to identify critical genes and pathways associated with fertility restoration. A total of 7125 differentially expressed genes (DEGs) were identified. These DEGs were significantly enriched in 242 Gene Ontology (GO) categories, wherein 100 DEGs were involved in pollen tube development, pollen tube growth, pollen development, and gametophyte development. Homology analysis revealed 198 male fertility-related DEGs, and pathway enrichment analysis revealed that 58 DEGs were enriched in cell energy metabolism processes involved in glycolysis, the pentose phosphate pathway, and pyruvate metabolism. By querying the Plant Reactome Pathway database, we found that 14 of the DEGs were involved in the mitochondrial tricarboxylic acid (TCA) cycle and that most of them belonged to the isocitrate dehydrogenase (IDH) and oxoglutarate dehydrogenase (OGDH) enzyme complexes. Transcriptome sequencing and real-time quantitative PCR (qPCR) showed that all the above TCA cycle-related genes were up-regulated in NIL_Rf4. The results of our subsequent enzyme-linked immunosorbent assay (ELISA) experiments pointed out that the contents of both the IDH and OGDH enzymes accumulated more in the spikelets of NIL_Rf4 than in those of NIL_rf4. CONCLUSION: The present research provides valuable genomic resources for deep insight into the molecular mechanism underlying CMS-C male fertility restoration. Importantly, our results indicated that genes involved in energy metabolism, especially some mitochondrial TCA cycle-related genes, were associated with maize CMS-C male fertility restoration.

Identification and functional characterization of the ZmCOPT copper transporter family in maize.

Copper (Cu) is an essential micronutrient for plant growth and development; Cu homeostasis in plant is maintained by the important functions of Ctr/COPT-type Cu transporters. Although the COPT genes have been identified in Arabidopsis thaliana and rice, little is known about Cu transporters in maize. In this study, three-members of putative maize Cu transporters (ZmCOPT 1, 2 and 3) are identified. ZmCOPT genes have expression in all of the tested tissues, including roots, stems, leaves and flowers (male and female), and their expression levels vary responding to stress due to Cu-deficiency and excess. Functional complementation and overexpression together with Cu uptake measurements in ZmCOPTs-transformed ctr1⊿ctr2⊿mutant strain or the wild type strain of Saccharomyces cerevisiae show that the three ZmCOPT members possess the ability to be Cu transporters. Among these, ZmCOPT1 and ZmCOPT2 have high-affinity while ZmCOPT3 has low-affinity. In addition, ZmCOPT2 tend to specifically transport Cu (I) but no other bivalent metal ions.

Molecular and functional characterization of the magnesium transporter gene ZmMGT12 in maize.

Magnesium (Mg) is an essential mineral element for normal plant growth and development, and the CorA/MRS2/MGT-type Mg transporters play a significant role in maintaining Mg homeostasis in plants. In total, 12 maize CorA-like Mg2+ transporters have been identified, but none of them had been functionally characterized. Accordingly, we cloned and functionally characterized ZmMGT12 from the maize CorA-like gene family. ZmMGT12 exhibited the structure typical of Mg2+ transporters, i.e., two conserved TM domains and a GMN tripeptide motif. ZmMGT12, Arabidopsis AtMGT6, and rice OsMRS2-6 shared high protein sequence identity and thus clustered in the same phylogenetic branch, suggesting that they could be homologs. A functional complementation assay in the Salmonella typhimurium MM281 mutant indicated that ZmMGT12 possessed Mg2+ transport ability. ZmMGT12 was expressed in roots, stems, and leaves, with the highest expression in leaves. Moreover, ZmMGT12 expression was induced by light and exhibited a circadian expression pattern. In addition, the expression level of ZmMGT12 in leaf tissue was related to chlorophyll synthesis. Overexpression of ZmMGT12 in Arabidopsis caused no phenotypic change in transgenic plants, including in fresh shoot weight, chlorophyll content, shoot Mg2+ content, and chloroplast Mg2+ content. Together, these results suggest that ZmMGT12 is a Mg2+ transporter and may play a role in Mg transport into chloroplasts.

Cloning, molecular evolution and functional characterization of ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142).

The transcription factor ZmbHLH16, the maize ortholog of OsTIP2 (OsbHLH142), was isolated in the present study. Tissue expression analysis showed that ZmbHLH16 is preferentially expressed in male reproductive organs. Subcellular location analysis of ZmbHLH16 via rice protoplast indicated that it is located in the nucleus. Through nucleotide variation analysis, 36 polymorphic sites in ZmbHLH16, including 23 single nucleotide polymorphisms and 13 InDels, were detected among 78 maize inbred lines. Neutrality tests and linkage disequilibrium analysis showed that ZmbHLH16 experienced no significant evolutionary pressure. Yeast one-hybrid experiment showed that the first 80 residues in the N-terminus of ZmbHLH16 had transactivation activity, whereas the full length did not. Genome-wide coexpression analysis showed that 395 genes were coexpressed with ZmbHLH16. Among these genes, the transcription factor ZmbHLH51 had similar expression pattern and identical subcellular localization to those of ZmbHLH16. Subsequently, the interaction between ZmbHLH51 and ZmbHLH16 was verified by yeast two-hybrid experiment. Through yeast two-hybrid analysis of series truncated ZmbHLH16 fragments, we found not only the typical bHLH domain [175-221 amino acids (a.a.)], but also that the 81-160 a.a. and 241-365 a.a. of ZmbHLH16 could interact with ZmbHLH51. All these results lay the foundation for further understanding the functions of ZmbHLH16.

The maize CorA/MRS2/MGT-type Mg transporter, ZmMGT10, responses to magnesium deficiency and confers low magnesium tolerance in transgenic Arabidopsis.

KEY MESSAGE: ZmMGT10 was specifically expressed in maize roots and induced by a deficiency of magnesium. Overexpression of ZmMGT10 restored growth deficiency of the Salmonella typhimurium MM281 strain and enhanced the tolerance in Arabidopsis to stress induced by low magnesium levels by increasing uptake of Mg2+ via roots. CorA/MRS2/MGT-type Mg2+ transporters play a significant role in maintaining magnesium (Mg) homeostasis in plants. Although the maize CorA/MRS2/MGT family comprises of 12 members, currently no member has been functionally characterized. Here, we report the isolation and functional characterization of ZmMGT10 from the maize MRS2/MGT gene family. ZmMGT10 has a typical structure feature which includes two conserved TMs near the C-terminal end and an altered AMN tripeptide motif. The high sequence similarity and close phylogenetic relationship indicates that ZmMGT10 is probably the counterpart of Arabidopsis AtMGT6. The complementation of the Salmonella typhimurium mutated MM281 strain indicates that ZmMGT10 possesses the ability to transport Mg2+. ZmMGT10 was specifically expressed in the plant roots and it can be stimulated by a deficiency of Mg. Transgenic Arabidopsis plants which overexpressed ZmMGT10 grew more vigorously than wild-type plants under low Mg conditions, exhibited by longer root length, higher plant fresh weight and chlorophyll content, suggesting ZmMGT10 was essential for plant growth and development under low Mg conditions. Further investigations found that high accumulation of Mg2+ occurred in transgenic plants attributed to improved Mg2+ uptake and thereby enhanced tolerance to Mg deficiency. Results from this investigation illustrate that ZmMGT10 is a Mg transporter of maize which can enhance the tolerance to Mg deficient conditions by improving Mg2+ uptake in the transgenic plants of Arabidopsis.