External application of brassinolide enhances cold resistance of tea plants (Camellia sinensis L.) by integrating calcium signals.

MAIN CONCLUSION: Exogenous brassinolide can activate the expression of key genes in the calcium signalling pathway to enhance cold resistance of tea plants. Brassinolide is an endogenous sterol phytohormone containing multiple hydroxyl groups that has the important function of improving plant cold resistance and alleviating freeze damage. To explore the molecular mechanism of how brassinolide improves the cold resistance of tea plants, "Qiancha 1" was used as the material, and the method of spraying brassinolide on the leaves was adopted to explore its effects on the tea plants under 4 °C low-temperature treatment. The results showed that brassinolide can significantly increase the protective enzyme activity of tea plants under cold stress and reduce cold damage. At the transcriptome level, brassinolide significantly enhanced the expression of key genes involved in calcium signal transduction, Calmodulin (CaM), Calcium-dependent protein kinase (CDPK), calcineurin B-like protein (CBL) and calmodulin-binding transcriptional activators (CAMTA), which then activated the downstream key genes transcriptional regulator CBF1 (CBF1) and transcription factor ICE1 (ICE1) during cold induction. Quantitative real-time PCR (qRT‒PCR) results showed that the expression of these genes was significantly induced after treatment with brassinolide, especially CaM and CBF1. When calcium signalling was inhibited, the upregulated expression of CBF1 and ICE1 disappeared, and when CAMTA was knocked down, the expression of other genes under cold stress was also significantly reduced. The above results indicate that brassinolide combined with the calcium signalling pathway can improve the cold resistance of tea plants. This study provides a new theoretical basis for the study of the cold resistance mechanism of brassinolide.

Exogenous calcium regulates the growth and development of Pinus massoniana detecting by physiological, proteomic, and calcium-related genes expression analysis.

Pinus massoniana is an important industrial crop tree species commonly used for timber and wood pulp for papermaking, rosin, and turpentine. This study investigated the effects of exogenous calcium (Ca) on P. massoniana seedling growth, development, and various biological processes and revealed the underlying molecular mechanisms. The results showed that Ca deficiency led to severe inhibition of seedling growth and development, whereas adequate exogenous Ca markedly improved growth and development. Many physiological processes were regulated by exogenous Ca. The underlying mechanisms involved diverse Ca-influenced biological processes and metabolic pathways. Calcium deficiency inhibited or impaired these pathways and processes, whereas sufficient exogenous Ca improved and benefited these cellular events by regulating several related enzymes and proteins. High levels of exogenous Ca facilitated photosynthesis and material metabolism. Adequate exogenous Ca supply relieved oxidative stress that occurred at low Ca levels. Enhanced cell wall formation, consolidation, and cell division also played a role in exogenous Ca-improved P. massoniana seedling growth and development. Calcium ion homeostasis and Ca signal transduction-related gene expression were also activated at high exogenous Ca levels. Our study facilitates the elucidation of the potential regulatory role of Ca in P. massoniana physiology and biology and is of guiding significance in Pinaceae plant forestry.

Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins.

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

Mechanical Stress Induces Ca2+-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis.

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.

[Regulatory mechanism underlying mycelium aggregation during filamentous fungi submerged fermentation].

Filamentous fungi are one of the platforms for producing fermented products. The specific characteristic of their submerged fermentation is the aggregation of mycelia that is affected by environmental conditions, leading to significantly different rheology for fermentation broth. Such a rheological change not only affects the transfer of mass, heat and momentum, but also the biosynthesis of target products and the efficiency of their production. In this article, strategies for morphological regulation of filamentous fungi are reviewed, and the impact of calcium signal transduction and chitin biosynthesis on apical growth of hyphae and branching of mycelia for their aggregation are further commented.

Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry.

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca2+ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca2+-binding protein, with Ca2+-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

TNF-α promotes colon cancer cell migration and invasion by upregulating TROP-2.

High levels of tumor-associated calcium signal transduction protein (TROP)-2 have been demonstrated to be strongly associated with tumor necrosis factor (TNF)-α levels in colon cancer. In the present study, the effect of TNF-α on the regulation of TROP-2 expression and its effect in colon cancer cell migration and invasion were investigated in vitro. TROP-2 protein levels were evaluated in HCT-116 human colon cancer cells cultured with various concentrations of TNF-α using western blot analysis. Changes in the migratory and invasive potential of the cells were evaluated using a wound healing and transwell assay, respectively. Then, TROP-2 expression was downregulated in cells by use of siRNA, and TROP-2 knockdown was confirmed at the mRNA and protein level by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The migration and invasion potential of cells transfected with TROP-2 siRNA was also evaluated. Levels of several mitogen-activated protein kinase proteins, namely p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), were detected in cells treated with TNF-α using western blot analysis. The results demonstrated that TROP-2 protein levels increased in cells treated with lower concentrations of TNF-α, but decreased in cells treated with higher concentrations of TNF-α, compared with untreated control. Maximum TROP-2 levels were observed in cells treated with 20 µg/l TNF-α. Migration and invasion were enhanced in cells treated with 20 µg/l TNF-α. When TROP-2 was silenced in colon cancer cells by siRNA, migration and invasion were significantly decreased compared with control cells. TNF-α stimulation activated the ERK1/2 pathway, but did not significantly affect p38 and JNK phosphorylation levels. Treatment with a specific ERK1/2 inhibitor suppressed the TNF-α-induced upregulation of TROP-2 and the TNF-α-induced colon cancer cell migration and invasion. In conclusion, the present results demonstrated that low concentrations of TNF-α significantly enhanced colon cancer cell migration and invasion by upregulating TROP-2 via the ERK1/2 signaling pathway.