MicroRNA408 negatively regulates salt tolerance by affecting secondary cell wall development in maize.

Although microRNA408 (miR408) is a highly conserved miRNA, the miR408 response to salt stress differs among plant species. Here, we show that miR408 transcripts are strongly repressed by salt stress and methyl viologen treatment in maize (Zea mays). Application of N, N1-dimethylthiourea partly relieved the NaCl-induced down-regulation of miR408. Transgenic maize overexpressing MIR408b is hypersensitive to salt stress. Overexpression of MIR408b enhanced the rate of net Na+ efflux, caused Na+ to locate in the inter-cellular space, reduced lignin accumulation, and reduced the number of cells in vascular bundles under salt stress. We further demonstrated that miR408 targets ZmLACCASE9 (ZmLAC9). Knockout of MIR408a or MIR408b or overexpression of ZmLAC9 increased the accumulation of lignin, thickened the walls of pavement cells, and improved salt tolerance of maize. Transcriptome profiles of the wild-type and MIR408b-overexpressing transgenic maize with or without salt stress indicated that miR408 negatively regulates the expression of cell wall biogenesis genes under salt conditions. These results indicate that miR408 negatively regulates salt tolerance by regulating secondary cell wall development in maize.

The auxin signaling pathway contributes to phosphorus-mediated zinc homeostasis in maize.

Although the interaction between P and Zn has long been recognized in plants, the physiological and molecular mechanisms underlying P and Zn interactions are poorly understood. We show here that P supply decreases the Zn concentration in maize shoots and roots. Compared to +P + Zn (addition of both P and Zn), +P-Zn reduced and -P-Zn increased the total length of 1° lateral roots (LRs). Under +P + Zn, both P and Zn concentrations were lower in the sl1 mutant roots than in wild-type (WT) maize roots, and P accumulation did not reduce the Zn concentration in ll1 mutant roots. Transcriptome profiling showed that the auxin signaling pathway contributed to P-mediated Zn homeostasis in maize. Auxin production and distribution were altered by changes in P and Zn supply. Cytosolic Zn co-localized with auxin accumulation under +P + Zn. Exogenous application of 1-NAA and L-Kyn altered the P-mediated root system architecture (RSA) under Zn deficiency. -P-Zn repressed the expression of miR167. Overexpression of ZmMIR167b increased the lengths of 1° LRs and the concentrations of P and Zn in maize. These results indicate that auxin-dependent RSA is important for P-mediated Zn homeostasis in maize.HighlightAuxin-dependent RSA is important for P-mediated Zn homeostasis in maize.

MIR164b represses iron uptake by regulating the NAC domain transcription factor5-Nuclear Factor Y, Subunit A8 module in Arabidopsis.

Recent findings have revealed the important roles of microRNAs (miRNAs) in the secondary responses to oxidative damage caused by iron (Fe) excess. However, the functional importance of miRNAs in plant responses to Fe deficiency remains to be explored. Here, we show that the expression level of miR164 in Arabidopsis (Arabidopsis thaliana) roots was repressed by Fe deficiency. Primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2) were higher in the mir164b mutant than in the wild-type (WT) under Fe-deficient conditions. Analysis of the Fe concentrations and ferric reductase activities in the roots of miR164 knockdown transgenic plants showed that members of the miR164 family had different functions in Fe-deficiency responses. Promoter::GUS analysis showed that NAM/ATAF/CUC (NAC) domain transcription factor5 (NAC5) is regulated at both transcriptional and posttranscriptional levels under Fe-deficient conditions. Transgenic Arabidopsis plants overexpressing NAC5 were more tolerant of Fe deficiency than the WT. NAC5 has transactivation activity and directly transactivates the expression of Nuclear Factor Y, Subunit A8 (NFYA8), as demonstrated by chromatin immunoprecipitation followed by quantitative polymerase chain reaction, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay. Like overexpression of NAC5, overexpression of NFYA8 increases primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRT1 and FRO2 under Fe-deficient conditions. Thus, MIR164b is important for Fe-deficiency responses by its regulation of the NAC5-NFYA8 module.

Maize Dek407 Encodes the Nitrate Transporter 1.5 and Is Required for Kernel Development.

The kernel serves as the storage organ and harvestable component of maize, and it plays a crucial role in determining crop yield and quality. Understanding the molecular and genetic mechanisms of kernel development is of considerable importance for maize production. In this study, we obtained a mutant, which we designated defective kernel 407 (dek407), through ethyl methanesulfonate mutagenesis. The dek407 mutant exhibited reduced kernel size and kernel weight, as well as delayed grain filling compared with those of the wild type. Positional cloning and an allelism test revealed that Dek407 encodes a nitrate transporter 1/peptide transporter family (NPF) protein and is the allele of miniature 2 (mn2) that was responsible for a poorly filled defective kernel phenotype. A transcriptome analysis of the developing kernels showed that the mutation of Dek407 altered the expression of phytohormone-related genes, especially those genes associated with indole-3-acetic acid synthesis and signaling. Phytohormone measurements and analysis indicated that the endogenous indole-3-acetic acid content was significantly reduced by 66% in the dek407 kernels, which may be the primary cause of the defective phenotype. We further demonstrated that natural variation in Dek407 is associated with kernel weight and kernel size. Therefore, Dek407 is a potential target gene for improvement of maize yield.

ZmEHD1 Is Required for Kernel Development and Vegetative Growth through Regulating Auxin Homeostasis.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize.

Complex Genetic System Involved in Fusarium Ear Rot Resistance in Maize as Revealed by GWAS, Bulked Sample Analysis, and Genomic Prediction.

Fusarium ear rot (FER) caused by Fusarium verticillioides is one of the most prevalent maize diseases in China and worldwide. Resistance to FER is a complex trait controlled by multiple genes highly affected by environment. In this paper, genome-wide association study (GWAS), bulked sample analysis (BSA), and genomic prediction were performed for understanding FER resistance using 509 diverse inbred lines, which were genotyped by 37,801 high-quality single-nucleotide polymorphisms (SNPs). Ear rot evaluation was performed using artificial inoculation in four environments in China: Xinxiang, Henan, and Shunyi, Beijing, during 2017 and 2018. Significant phenotypic and genetic variation for FER severity was observed, and FER resistance was significantly correlated among the four environments with a generalized heritability of 0.78. GWAS identified 23 SNPs that were associated with FER resistance, 2 of which (1_226233417 on chromosome 1 and 10_14501044 on chromosome 10) were associated at threshold of 2.65 × 10-7 [-log(0.01/37,801)]. Using BSA, resistance quantitative trait loci were identified on chromosomes 3, 4, 7, 9, and 10 at the 90% confidence level and on chromosomes 3 and 10 at the 95% confidence level. A key region, bin 10.03, was detected by both GWAS and BSA. Genomic prediction for FER resistance showed that the prediction accuracy by trait-related markers was higher than that by randomly selected markers under different levels of marker density. Marker-assisted selection using genomic prediction could be an efficient strategy for genetic improvement for complex traits like FER resistance.

The PILNCR1-miR399 Regulatory Module Is Important for Low Phosphate Tolerance in Maize.

The regulation of adaptive responses to phosphorus (P) deficiency by the microRNA399 (miR399)/PHOSPHATE2 (PHO2) pathway has been well studied in Arabidopsis (Arabidopsis thaliana) but not in maize (Zea mays). Here, we show that miR399 transcripts are strongly induced in maize by phosphate (Pi) deficiency. Transgenic maize plants that overexpressed MIR399b accumulated excessive amounts of P in their shoots and displayed typical Pi-toxicity phenotypes. We reannotated ZmPHO2 with an additional 1,165 bp of the 5' untranslated region. miR399-guided posttranscriptional repression of ZmPHO2 was mainly observed in the P-efficient lines. We identified Pi-deficiency-induced long-noncoding RNA1 (PILNCR1) from our strand-specific RNA libraries. Transient expression assays in Nicotiana benthamiana and maize leaf protoplasts demonstrated that PILNCR1 inhibits ZmmiR399-guided cleavage of ZmPHO2 The abundance of PILNCR1 was significantly higher in P-inefficient lines than in P-efficient lines, which is consistent with the abundance of ZmmiR399 transcripts. These results indicate that the interaction between PILNCR1 and miR399 is important for tolerance to low Pi in maize.

microRNA/microRNA* complementarity is important for the regulation pattern of NFYA5 by miR169 under dehydration shock in Arabidopsis.

MicroRNAs regulate gene expression at the mRNA and translational levels. Although our previous research showed that expression of miR169 and one of its targets, NFYA5, is down- and up-regulated by drought stress, respectively, the current study shows that expression of both miR169 and NFYA5 are induced by dehydration shock. Unlike overexpression of MIR169a/b, overexpression of MIR169i/l did not decrease NFYA5 transcripts but increased NFYA5 protein levels. NFYA5 protein abundance also increased in mir169a knock-out mutants and 35S::MIR169l mir169a double mutants. When bulge #11 and bulge #16 in the miR169/miR169* duplex were mutated, both NFYA5 transcripts and the corresponding protein levels were lower in 35S::MIR169l*-mut transgenic plants than in wild-type (WT) plants, and the 35S::MIR169l*-mut transgenic plants were as sensitive to drought stress as the 35S::MIR169a plants. The mRNA and protein levels of NFYA5 did not differ substantially between WT and 35S::MIR169a*-mut transgenic plants when the two bulges were introduced in the miR169a/miR169a* duplex. Both bulge #11 and bulge #16 in the miR169/miR169* duplex were essential for different regulation patterns of NFYA5 by miR169a and miR169l. These results increase the understanding of regulatory specialization in one MIR family, and also increase our understanding of the importance of microRNA/microRNA* secondary structure.

Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing.

Ribosome biogenesis is a fundamental process in eukaryotic cells. Although Urb2 protein has been implicated in ribosome biogenesis in yeast, the Urb2 domain is loosely conserved between plants and yeast, and the function of Urb2 protein in plants remains unknown. Here, we isolated a maize mutant, designated as urb2, with defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that urb2 encodes an Urb2 domain-containing protein. Compared with the wild-type (WT), the urb2 mutant showed decreased ratios of 60S/40S and 80S/40S and increased ratios of polyribosomes. The pre-rRNA intermediates of 35/33S rRNA, P-A3 and 18S-A3 were significantly accumulated in the urb2 mutant. Transcriptome profiling of the urb2 mutant indicated that ZmUrb2 affects the expression of a number of ribosome-related genes. We further demonstrated that natural variations in ZmUrb2 are significantly associated with maize kernel length. The overall results indicate that, by affecting pre-rRNA processing, the Urb2 protein is required for ribosome biogenesis in maize.

Genome-wide association study dissects yield components associated with low-phosphorus stress tolerance in maize.

KEY MESSAGE: Phosphorus deficiency in soil is a worldwide constraint threatening maize production. Through a genome-wide association study, we identified molecular markers and associated candidate genes and molecular pathways for low-phosphorus stress tolerance. Phosphorus deficiency in soils will severely affect maize (Zea mays L.) growth and development, thus decreasing the final yield. Deciphering the genetic basis of yield-related traits can benefit our understanding of maize tolerance to low-phosphorus stress. However, considering that yield-related traits should be evaluated under field condition with large populations rather than under hydroponic condition at a single-plant level, searching for appropriate field experimental sites and target traits for low-phosphorus stress tolerance is still very challenging. In this study, a genome-wide association analysis using two natural populations was performed to detect candidate genes in response to low-phosphorus stress at two experimental sites representative of different climate and soil types. In total, 259 candidate genes were identified and these candidate genes are mainly involved in four major pathways: transcriptional regulation, reactive oxygen scavenging, hormone regulation, and remodeling of cell wall. Among these candidate genes, 98 showed differential expression by transcriptome data. Based on a haplotype analysis of grain number under phosphorus deficiency condition, the positive haplotypes with favorable alleles across five loci increased grain number by 42% than those without favorable alleles. For further verifying the feasibility of genomic selection for improving maize low-phosphorus tolerance, we also validated the predictive ability of five genomic selection methods and suggested that moderate-density SNPs were sufficient to make accurate predictions for low-phosphorus tolerance traits. All these results will facilitate elucidating genetic basis of maize tolerance to low-phosphorus stress and improving marker-assisted selection efficiency in breeding process.

Nitrogen Limitation Adaptation (NLA) is involved in source-to-sink remobilization of nitrate by mediating the degradation of NRT1.7 in Arabidopsis.

Recent studies on nitrate transporters (NRTs) have greatly increased our knowledge of the mechanisms regulating nitrogen (N) homeostasis in plants. However, an understanding of how these NRTs are regulated is still lacking. The nitrogen limitation adaptation (nla) mutant is hypersensitive to N limitation. In the nla mutant, 15 N-nitrate spotted on old leaves preferentially accumulated in the youngest leaves. Analysis of leaf cross-sections indicated that NLA expression was expressed in the sieve element and companion cell system. The results of bimolecular fluorescence complementation (BiFC), split-ubiquitin yeast two-hybrid and co-immunoprecipitation (CoIP) assays demonstrated that NLA interacts with NRT1.7 in the plasma membrane. The following findings suggest that NLA directs the ubiquitination of NRT1.7: the down-regulation of NRT1.7 protein abundance in 35S::NLA/35S::Myc-NRT1.7 double transgenic plants compared with 35S::Myc-NRT1.7 transgenic plants; the up-regulation of NRT1.7 protein abundance in the nla mutant compared with wild-type plants; and the direct degradation of truncated NRT1.7 recombinant protein by NLA. Furthermore, analysis of NLA and NRT1.7 protein abundance in mirna827 knock-out plants showed that N deficiency-guided translational repression of NLA depends on miRNA827. Our findings reveal that plants regulate source-to-sink remobilization of nitrate by the ubiquitin-mediated post-translational regulatory pathway.

NERF encodes a RING E3 ligase important for drought resistance and enhances the expression of its antisense gene NFYA5 in Arabidopsis.

NFYA5 is an important drought-stress inducible transcription factor gene that is targeted by miR169 in Arabidopsis. We show here that the cis-natural antisense transcript gene of NFYA5, NFYA5 Enhancing RING FINGER (NERF), can produce siRNAs from their overlapping region (OR) and affect NFYA5 transcripts by functioning together with miR169. The NERF protein functions as an E3 ligase for ubiquitination. Overexpression of NERF or OR cDNA leads to siRNANERF accumulation, miR169 repression, and NFYA5 transcript enhancement; knock-down of NERF transcripts by an artificial miRNA enhances miR169 abundance and reduces NFYA5 transcripts. Overexpression of NFYA5 does not affect the NERF mRNA level. Deep sequencing of the small RNA library from 35S::OR plants identifies 960 sequences representing 323 unique siRNAs that originate from OR; the sequences of some siRNANERF are similar/complementary to those of miR169. Overexpression of the 195- to 280-bp OR cDNA-containing siRNAs similar/complementary to miR169 also leads to the accumulation of NFYA5 transcripts. Analysis of NERF knock-down plants and NERF overexpression lines showed that, like NFYA5, NERF is important for controlling stomatal aperture and drought resistance. This regulatory model might apply to other natural antisense transcripts with positively correlated expression patterns.

RepSox improves viability and regulates gene expression in rhesus monkey-pig interspecies cloned embryos.

OBJECTIVE: To investigate the effect of the small molecule, RepSox, on the expression of developmentally important genes and the pre-implantation development of rhesus monkey-pig interspecies somatic cell nuclear transfer (iSCNT) embryos. RESULTS: Rhesus monkey cells expressing the monomeric red fluorescent protein 1 which have a normal (42) chromosome complement, were used as donor cells to generate iSCNT embryos. RepSox increased the expression levels of the pluripotency-related genes, Oct4 and Nanog (p < 0.05), but not of Sox2 compared with untreated embryos at the 2-4-cell stage. Expression of the anti-apoptotic gene, Bcl2, and the pro-apoptotic gene Bax was also affected at the 2-4-cell stage. RepSox treatment also increased the immunostaining intensity of Oct4 at the blastocyst stage (p < 0.05). Although the blastocyst developmental rate was higher in the group treated with 25 µM RepSox for 24 h than in the untreated control group (2.4 vs. 1.2%, p > 0.05), this was not significant. CONCLUSION: RepSox can improve the developmental potential of rhesus monkey-pig iSCNT embryos by regulating the expression of pluripotency-related genes.

The developmental competence of oocytes parthenogenetically activated by an electric pulse and anisomycin treatment.

OBJECTIVE: The aim of this study was to investigate the developmental competence of oocytes parthenogenetically activated by an electric pulse (EP) and treated with anisomycin and to determine whether this method is applicable to somatic cell nuclear transfer (SCNT). RESULTS: Embryos derived from porcine oocytes parthenogenetically activated by an EP and treatment with 0.01 µg/mL anisomycin had a significantly improved in vitro developmental capacity. Furthermore, 66.6% of blastocysts derived from these embryos had a diploid karyotype. The blastocyst formation rate of cloned embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 0.01 µg/mL anisomycin for 4 h. The level of maturation-promoting factor was significantly decreased in oocytes activated by an EP and treated with anisomycin. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR. CONCLUSION: Our results demonstrate that porcine oocyte activation via an EP in combination with anisomycin treatment can lead to a high blastocyst formation rate in parthenogenetic activation and SCNT experiments.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment.

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

Strand-specific RNA-Seq transcriptome analysis of genotypes with and without low-phosphorus tolerance provides novel insights into phosphorus-use efficiency in maize.

BACKGROUND: Phosphorus (P) stress is a global problem in maize production. Although macro/microarray technologies have greatly increased our general knowledge of maize responses to P stress, a greater understanding of the diversity of responses in maize genotypes is still needed. RESULTS: In this study, we first evaluated the tolerance to low P of 560 accessions under field conditions, and selected the low P-tolerant line CCM454 and the low P-sensitive line 31778 for further research. We then generated 24 strand-specific RNA libraries from shoots and roots of CCM454 and 31778 that had been subjected to P stress for 2 and 8 days. The P deficiency-responsive genes common to CCM454 and 31778 were involved in various metabolic processes, including acid phosphatase (APase) activity. Determination of root-secretory APase activities showed that the induction of APase by P stress occurred much earlier in CCM454 than that in 31778. Gene Ontology analysis of differentially expressed genes (DEGs) and CAT/POD activities between CCM454 and 31778 under P-sufficient and -deficient conditions demonstrated that CCM454 has a greater ability to eliminate reactive oxygen species (ROS) than 31778. In addition, 16 miRNAs in roots and 12 miRNAs in shoots, including miRNA399s, were identified as DEGs between CCM454 and 31778. CONCLUSIONS: The results indicate that the tolerance to low P of CCM454 is mainly due to the rapid responsiveness to P stress and efficient elimination of ROS. Our findings increase the understanding of the molecular events involved in the diversity of responses to P stress among maize accessions.

Identification and functional characterization of the AGO1 ortholog in maize.

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1-27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild-type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.

[Sexual satisfaction and its associated factors among individuals with hearing disability].

OBJECTIVE: To investigate sexual satisfaction (SS) and the factors associated with decreased SS among individuals with hearing disability. METHODS: We conducted an investigation on SS among 439 individuals (268 males and 171 females, aged ≥18 yr) with hearing disability using a general information questionnaire, Center for Epidemiologic Studies Depression Scale, Social Support Rating Scale, and a self-report on SS. We identified the factors of decreased SS by multivariate ordinal logistic regression analysis. RESULTS: Totally 76 (17.3%) of the hearing-disability individuals investigated were dissatisfied with their sexual life. SS reduction was significantly correlated with the status of being single (OR=1.72), grade-1 or -2 disability (OR=1.78), physical diseases (OR=2.46), depression (OR=6.61), or inadequate subjective social support (OR=3.28). CONCLUSIONS: SS of hearing-disability persons is relatively low, which can be improved by treating physical diseases, promoting mental health, and providing psycho-social support.

Cadmium Induced Apoptosis in MG63 Cells by Increasing ROS, Activation of p38 MAPK and Inhibition of ERK 1/2 Pathways.

BACKGROUND/AIMS: Cadmium (Cd) induces apoptosis in different kinds of cells, including osteoblasts, both in vivo and in vitro. However, little is known about the mechanisms by which Cd induces apoptosis. METHODS: In the present study, we used the human osteosarcoma cell line MG63, which has characteristics similar to human osteoblasts, as an in vitro model to determine the cellular mechanisms by which Cd induces apoptosis. RESULTS: We found that short-term exposure to CdCl2 induced apoptosis in MG63 cells. Furthermore, the incubation of cells with CdCl2 significantly increased the level of phosphorylated p38MAPK and significantly decreased the phosphorylation of ERK1/2 in a concentration-dependent manner. Additionally, the inhibition of the phosphorylation of p38 MAPK by SB202190 protected MG63 cells from Cd-induced apoptosis. The incubation of MG63 cells with the ERK1/2 inhibitor PD98059 significantly increased apoptosis in MG63 cells. CdCl2 also significantly increased the intracellular levels of ROS. N-acetylcysteine (NAC) significantly reduced ROS levels and reversed the effects of CdCl2 on MAPK signaling. CONCLUSION: Our results suggested that Cd induced apoptosis in MG63 cells by increasing ROS, activation of p38 MAPK and inhibition of ERK1/2 pathways.

bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism.

• Although proteins in the basic helix-loop-helix (bHLH) family are universal transcription factors in eukaryotes, the biological roles of most bHLH family members are not well understood in plants. • The Arabidopsis thaliana bHLH122 transcripts were strongly induced by drought, NaCl and osmotic stresses, but not by ABA treatment. Promoter::GUS analysis showed that bHLH122 was highly expressed in vascular tissues and guard cells. Compared with wild-type (WT) plants, transgenic plants overexpressing bHLH122 displayed greater resistance to drought, NaCl and osmotic stresses. In contrast, the bhlh122 loss-of-function mutant was more sensitive to NaCl and osmotic stresses than were WT plants. • Microarray analysis indicated that bHLH122 was important for the expression of a number of abiotic stress-responsive genes. In electrophoretic mobility shift assay and chromatin immunoprecipitation assays, bHLH122 could bind directly to the G-box/E-box cis-elements in the CYP707A3 promoter, and repress its expression. Further, up-regulation of bHLH122 substantially increased cellular ABA levels. • These results suggest that bHLH122 functions as a positive regulator of drought, NaCl and osmotic signaling.