Background TaCKX wheat gene family members (GFMs) encode the enzyme cytokinin oxidase/dehydrogenase (CKX), which irreversibly degrades cytokinins. The genes are important regulators of cytokinin content and take part in growth and development, with a major impact on yield-related traits. The goal of this research was to test whether these genes might be differentially expressed in the field compared to laboratory conditions and consequently differently affect plant development and yield. Results We compared expression and crosstalk of the TaCKX GFMs and TaNAC2-5A gene in modern varieties grown in a growth chamber (GC) and in the field and looked for differences in their impact on yield-related traits. The TaNAC2-5A gene was included in the research since it was expected to play an important role in co-regulation of these genes. The range of relative expression levels of TaCKX GFMs and TaNAC2-5A gene among tested cultivars was from 5 for TaCKX8 to more than 100 for TaCKX9 in the GC and from 6 for TaCKX8 to 275 for TaCKX10 in the field. The range was similar for four of them in the GC, but was much higher for seven others and TaNAC2-5A in the field. The TaCKX GFMs and TaNAC2-5A form co-expression groups, which differ depending on growth conditions. Consequently, the genes also differently regulate yield-related traits in the GC and in the field. TaNAC2-5A took part in negative regulation of tiller number and CKX activity in seedling roots only in controlled GC conditions. Grain number and grain yield were negatively regulated by TaCKX10 in the GC but positively by TaCKX8 and others in the field. Some of the genes, which were expressed in seedling roots, negatively influenced tiller number and positively regulated seedling root weight, CKX activity in the spikes, thousand grain weight (TGW) as well as formation of semi-empty spikes. Conclusions We have documented that: 1) natural variation in expression levels of tested genes in both environments is very high, indicating the possibility of selection of beneficial genotypes for breeding purposes, 2) to create a model of an ideotype for breeding, we need to take into consideration the natural environment.

BackgroundLymphotoxin β receptor (LTβR) plays important roles in the development of the immune system and immune response. At the cellular level, ligand-bound LTβR activates the pro-inflammatory NF-κB pathway but the detailed mechanisms regulating its signaling remain unknown. Understanding them is of high importance since LTβR and its ligands are promising therapeutic targets. Here, we studied the consequences of perturbed cellular cholesterol content on LTβR-induced NF-κB signaling.MethodsTo modulate cholesterol availability and/or level in lung carcinoma A549 and H2228, and endothelial HUVEC cells different treatment regimens with filipin, methyl-β-cyclodextrin and simvastatin were applied. LTβR localization was studied by confocal microscopy. The activity of LTβR-induced NF-κB pathway was assessed by measuring the levels of NF-κB pathway inhibitor IκBα and phosphorylation of RelA transcription factor by Western blotting. The NF-κB transcriptional response, production of chemokines and adhesion molecules were examined by qRT-PCR, ELISA, and Western blotting, respectively. Adherence of different types of primary immune cells to epithelial A549 cells and endothelial HUVECs was measured fluorometrically. Interactions of LTβR with its protein partners were investigated by immunoprecipitation.ResultsWe showed that filipin-mediated sequestration of cholesterol or its depletion from the plasma membrane with methyl-β-cyclodextrin impaired LTβR internalization and potentiated LTβR-dependent activation of the canonical branch of the NF-κB pathway. The latter was manifested by enhanced degradation of IκBα inhibitor, elevated RelA phosphorylation, substantial increase in the expression of NF-κB target genes encoding, among others, cytokines and adhesion molecules known to play important roles in immune response. It was followed by robust secretion of CXCL8 and upregulation of ICAM1, that favored the adhesion of immune cells (NK and T cells, neutrophils) to A549 cells and HUVECs. Mechanistically, we showed that cholesterol depletion stabilized interactions of ligand-stimulated LTβR with modified forms of TRAF2 and NEMO proteins.ConclusionsOur results showed that the reduction of the plasma membrane content of cholesterol or its sequestration strongly potentiated signaling outcome initiated by LTβR. Thus, drugs modulating cholesterol levels could potentially improve efficacy of LTβR-based therapies.