Ectopic expression of MmCYP1A1, a mouse cytochrome P450 gene, positively regulates stress tolerance in apple calli and Arabidopsis.

KEY MESSAGE: Ectopic expression of MmCYP1A1 gene from Mus musculus in apple calli and Arabidopsis increased the levels of melatonin and 6-hydroxymelatonin, and improved their stress resistance. Melatonin occurs widely in organisms, playing a key regulatory role. CYP1A1 is a cytochrome P450 monooxygenase, involved in the melatonin metabolism, and is responsible for the synthesis of 6-hydroxymelatonin from melatonin. Melatonin and 6-hydroxymelatonin have strong antioxidant activities in animals. Here, we cloned MmCYP1A1 from Mus musculus and found that ectopic expression of MmCYP1A1 improved the levels of melatonin and 6-hydroxymelatonin in transgenic apple calli and Arabidopsis. Subsequently, we observed that MmCYP1A1 increased the tolerance of transgenic apple calli and Arabidopsis to osmotic stress simulated by polyethylene glycol 6000 (PEG 6000), as well as resistance of transgenic Arabidopsis to drought stress. Further, the number of lateral roots of MmCYP1A1 transgenic Arabidopsis were enhanced significantly after PEG 6000 treatment. The expression of MmCYP1A1 remarkably reduced malondialdehyde (MDA) content, electrolyte leakage, accumulation of H2O2 and O2- during stress treatment. Moreover, MmCYP1A1 enhanced stress tolerance in apple calli and Arabidopsis by increasing the expression levels of resistance genes. MmCYP1A1 also promoted stomatal closure in transgenic Arabidopsis to reduce leaf water loss during drought. Our results indicate that MmCYP1A1 plays a key role in plant stress tolerance, which may provide a reference for future plant stress tolerance studies.

[Chemical constituents in different parts of Ixeris sonchifolia based on UPLC-LTQ-Orbitrap-MS~n].

The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Interaction of BTB-TAZ protein MdBT2 and DELLA protein MdRGL3a regulates nitrate-mediated plant growth.

Nitrate acts as a vital signal molecule in the modulation of plant growth and development. The phytohormones gibberellin (GA) is also involved in this process. However, the exact molecular mechanism of how nitrate and GA signaling pathway work together in regulating plant growth remains poorly understood. In this study, we found that a nitrate-responsive BTB/TAZ protein MdBT2 participates in regulating nitrate-induced plant growth in apple (Malus × domestica). Yeast two-hybridization, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with a DELLA protein MdRGL3a, which is required for the ubiquitination and degradation of MdRGL3a proteins via a 26S proteasome-dependent pathway. Furthermore, heterologous expression of MdBT2 partially rescued growth inhibition caused by overexpression of MdRGL3a in Arabidopsis. Taken together, our findings indicate that MdBT2 promotes nitrate-induced plant growth partially through reducing the abundance of the DELLA protein MdRGL3a.

Apple MdSAT1 encodes a bHLHm1 transcription factor involved in salinity and drought responses.

MAIN CONCLUSION: This study identified a new bHLHm1 transcription factor MdSAT1 which functioned in mediating tolerance to salt and drought resistance. Changes in the expression of stress-related genes play crucial roles in response to environmental stress. Basic helix-loop-helix (bHLH) proteins are the largest superfamily of transcription factors and a large number of bHLH proteins function in plant responses to abiotic stresses. We identified a new bHLHm1 transcription factor from apple and named it MdSAT1. ß-Glucuronidase (GUS) staining showed that MdSAT1 expressed in various tissues with highly expressed in leaves. Promoter analysis revealed that MdSAT1 contained multiple response elements and its transcription was induced by several environmental cues, particularly salt and drought stresses. Overexpression of MdSAT1 in apple calli and Arabidopsis resulted in a phenotype of increased tolerance to salt and drought. Altering abscisic acid (ABA) treatment increased the sensitivity of MdSAT1-OE Arabidopsis to ABA, and heavy metal stress, osmotic stress, and ethylene did not participate in MdSAT1 mediated plant development. These findings reveal the abiotic stress functions of MdSAT1 and pave the way for further functional investigation.

Ubiquitination-Related MdBT Scaffold Proteins Target a bHLH Transcription Factor for Iron Homeostasis.

Iron (Fe) homeostasis is crucial for plant growth and development. A network of basic helix-loop-helix (bHLH) transcription factors positively regulates Fe uptake during iron deficiency. However, their up-regulation or overexpression leads to Fe overload and reactive oxygen species generation, thereby damaging the plants. Here, we found that two BTB/TAZ proteins, MdBT1 and MdBT2, interact with the MbHLH104 protein in apple. In addition, the function of MdBT2 was characterized as a regulator of MdbHLH104 degradation via ubiquitination and the 26S proteasome pathway, thereby controlling the activity of plasma membrane H+-ATPases and the acquisition of iron. Furthermore, MdBT2 interacted with MdCUL3 proteins, which were required for the MdBT2-mediated ubiquitination modification of MdbHLH104 and its degradation. In sum, our findings demonstrate that MdBT proteins interact with MdCUL3 to bridge the formation of the MdBTsMdCUL3 complex, which negatively modulates the degradation of the MdbHLH104 protein in response to changes in Fe status to maintain iron homeostasis in plants.

Overexpression of MdbHLH104 gene enhances the tolerance to iron deficiency in apple.

Fe deficiency is a widespread nutritional disorder in plants. The basic helix-loop-helix (bHLH) transcription factors (TFs), especially Ib subgroup bHLH TFs which are involved in iron uptake, have been identified. In this study, an IVc subgroup bHLH TF MdbHLH104 was identified and characterized as a key component in the response to Fe deficiency in apple. The overexpression of the MdbHLH104 gene noticeably increased the H(+) -ATPase activity under iron limitation conditions and the tolerance to Fe deficiency in transgenic apple plants and calli. Further investigation showed that MdbHLH104 proteins bonded directly to the promoter of the MdAHA8 gene, thereby positively regulating its expression, the plasma membrane (PM) H(+) -ATPase activity and Fe uptake. Similarly, MdbHLH104 directly modulated the expression of three Fe-responsive bHLH genes, MdbHLH38, MdbHLH39 and MdPYE. In addition, MdbHLH104 interacted with 5 other IVc subgroup bHLH proteins to coregulate the expression of the MdAHA8 gene, the activity of PM H(+) -ATPase and the content of Fe in apple calli. Therefore, MdbHLH104 acts together with other apple bHLH TFs to regulate Fe uptake by modulating the expression of the MdAHA8 gene and the activity of PM H(+) -ATPase in apple.

The apple 14-3-3 protein MdGRF11 interacts with the BTB protein MdBT2 to regulate nitrate deficiency-induced anthocyanin accumulation.

Nitrogen is an important factor that affects plant anthocyanin accumulation. In apple, the nitrate-responsive BTB/TAZ protein MdBT2 negatively regulates anthocyanin biosynthesis. In this study, we found that MdBT2 undergoes posttranslational modifications in response to nitrate deficiency. Yeast two-hybrid, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with MdGRF11, a 14-3-3 protein; 14-3-3 proteins compose a family of highly conserved phosphopeptide-binding proteins involved in multiple physiological and biological processes. The interaction of MdGRF11 negatively regulated the stability of the MdBT2 protein via a 26S proteasome-dependent pathway, which increased the abundance of MdMYB1 proteins to activate the expression of anthocyanin biosynthesis-related genes. Taken together, the results demonstrate the critical role of 14-3-3 proteins in the regulation of nitrate deficiency-induced anthocyanin accumulation. Our results provide a novel avenue to elucidate the mechanism underlying the induction of anthocyanin biosynthesis in response to nitrate deficiency.

Functional identification of apple MdMYB2 gene in phosphate-starvation response.

Inorganic phosphate (Pi) starvation severely affects the normal growth and development of plants. Here, a Pi-responsive gene, named MdMYB2 (MDP0000823458), was cloned and functionally identified in apple. Overexpression of MdMYB2 regulated the expression of Pi starvation-induced (PSI) genes and then promoted phosphate assimilation and utilization. The ectopic expression of MdMYB2 in Arabidopsis influenced plant growth and flowering, which was partially rescued by application of exogenous gibberellin (GA). These results indicated that MdMYB2 may be an essential regulator in phosphate utilization and GA-regulated plant growth and development.

MdGRF11, an apple 14-3-3 protein, acts as a positive regulator of drought and salt tolerance.

The 14-3-3 proteins are a family of highly conserved phosphoserine-binding proteins that participate in the regulation of diverse physiological and developmental processes. In this research, twenty 14-3-3 genes in apples, which contained a highly conserved 14-3-3 domain, were identified and divided into two subgroups. Among them, MdGRF11 was further cloned and investigated. qRT-PCR analyses and GUS staining show that MdGRF11 is expressed in various organs and tissues with the highest expression levels found in the fruit. MdGRF11 was upregulated by polyethylene glycol 6000 (PEG 6000), NaCl, abscisic acid (ABA) and low temperature (4 °C) treatments. MdGRF11-overexpressing transgenic Arabidopsis and apple calli exhibited reduced sensitivity to salt and PEG 6000 treatments. Moreover, the ectopic expression of MdGRF11 improved the tolerance of transgenic tobacco to salt and drought stresses, which grew longer roots, underwent more growth, and presented higher chlorophyll levels than the wild-type control under salt and drought stress conditions. Furthermore, MdGRF11 expression remarkably reduced electrolyte leakage, malondialdehyde content levels, H2O2 and O2- accumulation under salt and drought stress conditions, which relied on the regulation of ROS-scavenging signaling to reduce oxidative damage of cells after salt and drought stress treatment. MdGRF11 also enhanced tolerance to stress by upregulating expression levels of ROS-scavenging and stress-related genes, especially improving responses to drought stress by modifying the water loss rates and stomatal aperture. Moreover, MdGRF11 could interact with MdAREB/ABF transcription factors through yeast two hybrid analyses. In conclusion, our results indicate that MdGRF11 acts as a positive regulator of salt and drought stress responses through regulating ROS scavenging and other signaling systems.

Screening, verification, and analysis of biomarkers for drug-induced cardiac toxicity in vitro based on RTCA coupled with PCR Array technology.

Cardiotoxicity is one of the most serious side effects of new drugs. Early detection of the drug induced cardiotoxicity based on the biomarkers provides an important preventative strategy for detecting potential cardiotoxicity of candidate drugs. In this study, we aim to identify the predictive genomics biomarkers for drug-induced cardiac toxicity based on the RTCA coupled with PCR Array technology in primary cells. Three prototypical cardiotoxic compounds (doxorubicin, isoproterenol, ouabain) with different mechanisms were firstly real-time monitored to diagnose the cytotoxicity by using the RTCA, while the functional alterations of cardiomyocytes were also monitored by analyzing the beating frequency of cardiomyocytes. Then cardiac specific toxicity gene expression changes were studied by using the technology of PCR Array, which can detect the changes of 84 cardiac functions related genes. Rps6kb1 was identified to be the common cardiac biomarkers by using multivariate statistical and integration analyses. The biomarker was further verified by selecting other drugs with or without cardiotoxicity, and the results showed that the gene exhibited specific changes in cardiac toxicity. Moreover, IPA was applied to combine relevant pathways of Rps6kb1, and identify the main types of cardiac toxicity. These results would further enrich the evaluating strategy of drug-induced cardiotoxicity in vitro, and Rps6kb1 could be used as the specific biomarker of cardiotoxcity during safety assessment of the novel drug candidates.