The WRKY6 Transcription Factor Modulates PHOSPHATE1 Expression in Response to Low Pi Stress in Arabidopsis

Arabidopsis thaliana WRKY family comprises 74 members and some of them are involved in plant responses to biotic and abiotic stresses. This study demonstrated that WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. WRKY6 overexpression lines, similar to the pho1 mutant, were more sensitive to low Pi stress and had lower Pi contents in shoots compared with wild-type seedlings and the wrky6-1 mutant. Immunoprecipitation assays demonstrated that WRKY6 can bind to two W-boxes of the PHO1 promoter. RNA gel blot and β-glucuronidase activity assays showed that PHO1 expression was repressed in WRKY6-overexpressing lines and enhanced in the wrky6-1 mutant. Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression. Protein gel blot analysis showed that the decrease in WRKY6 protein induced by low Pi treatment was inhibited by a 26S proteosome inhibitor, MG132, suggesting that low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated proteolysis. In addition, WRKY42 also showed binding to W-boxes of the PHO1 promoter and repressed PHO1 expression. Our results demonstrate that WRKY6 and WRKY42 are involved in Arabidopsis responses to low Pi stress by regulation of PHO1 expression.     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

Pericytes augment glioblastoma cell resistance to temozolomide through CCL5-CCR5 paracrine signaling

Glioblastoma (GBM) is a prevalent and highly lethal form of glioma, with rapid tumor progression and frequent recurrence. Excessive outgrowth of pericytes in GBM governs the ecology of the perivascular niche, but their function in mediating chemoresistance has not been fully explored. Herein, we uncovered that pericytes potentiate DNA damage repair (DDR) in GBM cells residing in the perivascular niche, which induces temozolomide (TMZ) chemoresistance. We found that increased pericyte proportion correlates with accelerated tumor recurrence and worse prognosis. Genetic depletion of pericytes in GBM xenografts enhances TMZ-induced cytotoxicity and prolongs survival of tumor-bearing mice. Mechanistically, C-C motif chemokine ligand 5 (CCL5) secreted by pericytes activates C-C motif chemokine receptor 5 (CCR5) on GBM cells to enable DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-mediated DDR upon TMZ treatment. Disrupting CCL5-CCR5 paracrine signaling through the brain-penetrable CCR5 antagonist maraviroc (MVC) potently inhibits pericyte-promoted DDR and effectively improves the chemotherapeutic efficacy of TMZ. GBM patient-derived xenografts with high CCL5 expression benefit from combined treatment with TMZ and MVC. Our study reveals the role of pericytes as an extrinsic stimulator potentiating DDR signaling in GBM cells and suggests that targeting CCL5-CCR5 signaling could be an effective therapeutic strategy to improve chemotherapeutic efficacy against GBM.Subject terms: Cancer microenvironment, CNS cancer, Cancer therapy     

Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His₆ fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49°C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58°C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg⁻¹, respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.