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As one of the subtropical and tropical fruit trees, Citrus sinensis is sensitive to cold stress. However, most transcription factors (TFs) that regulate cold tolerance in citrus have not yet been reported. A phytochrome-interacting transcription factor (PIF) gene (CsPIF8) in citrus was significantly upregulated under cold stress. Overexpression of CsPIF8 increased cold tolerance in transgenic tomato plants and grapefruit callus, whereas virus-induced gene silencing-mediated suppression of PIF8 increased cold sensitivity in seedlings of Poncirus trifoliata. Superoxide dismutase (SOD) reduces the superoxide anion (O 2 - ) level to enhance cold tolerance in plants. Chromatin immunoprecipitation combined with high-throughput sequencing, yeast one hybrid, electrophoretic mobility shift and dual luciferase assays showed that CsPIF8 directly bound the E-box (CANNTG) of CsSOD promoter and activated the promoter of CsSOD. Furthermore, the expression level of CsSOD and CsSOD activity were significantly increased, whereas the level of O 2 - was significantly reduced in the transgenic lines. The Poncirus trifoliata seedlings with VIGS-mediated suppression of PIF8 exhibited the opposite effects. These results have shown that CsPIF8 improved cold tolerance in citrus through regulating the expression level of SOD and SOD activity. These findings may provide novel insights into the regulation of PIF8 in the response to cold stress in citrus., Competing Interests: Declaration of Competing Interest The authors have a patent pending related to this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This work reports the molecular cloning and heterologous expression of the genes coding for α and β subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg -1 for α and β subunits, respectively). On the other hand, co-expression rendered a α 3 β 3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism., (Copyright © 2018 Elsevier B.V. All rights reserved.)