Gigantic chloroplasts, including bizonoplasts, are common in shade-adapted species of the ancient vascular plant family Selaginellaceae.

PREMISE: Unique among vascular plants, some species of Selaginella have single giant chloroplasts in their epidermal or upper mesophyll cells (monoplastidy, M), varying in structure between species. Structural variants include several forms of bizonoplast with unique dimorphic ultrastructure. Better understanding of these structural variants, their prevalence, environmental correlates and phylogenetic association, has the potential to shed new light on chloroplast biology unavailable from any other plant group. METHODS: The chloroplast ultrastructure of 76 Selaginella species was studied with various microscopic techniques. Environmental data for selected species and subgeneric relationships were compared against chloroplast traits. RESULTS: We delineated five chloroplast categories: ME (monoplastidy in a dorsal epidermal cell), MM (monoplastidy in a mesophyll cell), OL (oligoplastidy), Mu (multiplastidy, present in the most basal species), and RC (reduced or vestigial chloroplasts). Of 44 ME species, 11 have bizonoplasts, cup-shaped (concave upper zone) or bilobed (basal hinge, a new discovery), with upper zones of parallel thylakoid membranes varying subtly between species. Monoplastidy, found in 49 species, is strongly shade associated. Bizonoplasts are only known in deep-shade species (<2.1% full sunlight) of subgenus Stachygynandrum but in both the Old and New Worlds. CONCLUSIONS: Multiplastidic chloroplasts are most likely basal, implying that monoplastidy and bizonoplasts are derived traits, with monoplastidy evolving at least twice, potentially as an adaptation to low light. Although there is insufficient information to understand the adaptive significance of the numerous structural variants, they are unmatched in the vascular plants, suggesting unusual evolutionary flexibility in this ancient plant genus.

Comparative transcriptomics method to infer gene coexpression networks and its applications to maize and rice leaf transcriptomes.

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.

Histone deacetylase 2 (HDAC2) attenuates lipopolysaccharide (LPS)-induced inflammation by regulating PAI-1 expression.

BACKGROUND: Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection, and is primarily characterized by an uncontrolled systemic inflammatory response. In the present study, we developed an effective adjunct therapy mediated by a novel mechanism, to attenuate overt inflammation. LPS-treated macrophages were adopted as an in vitro model of endotoxin-induced inflammation during sepsis. Experiments were carried out using primary mouse peritoneal macrophages and the murine macrophage cell line RAW264.7, to elucidate the mechanisms by which HDAC2 modulates endotoxin-induced inflammation. RESULTS: Results revealed that PAI-1, TNF, and MIP-2 expression were inhibited by theophylline, an HDAC2 enhancer, in a RAW macrophage cell line, following LPS-induced inflammation. Thus, HDAC2 plays an important role in immune defense by regulating the expression of inflammatory genes via the c-Jun/PAI-1 pathway. During LPS-induced inflammation, overexpression of HDAC2 was found to inhibit PAI-1, TNF, and MIP-2 expression. Following LPS stimulation, HDAC2 knockdown increased nuclear translocation and DNA binding of c-Jun to the PAI-1 gene promoter, thereby activating PAI-1 gene transcription. Furthermore, inhibition of PAI-1 by TM5275 alone or in combination with theophylline notably suppressed TNF and MIP-2 expression. CONCLUSION: HDAC2 can attenuate lipopolysaccharide-induced inflammation by regulating c-Jun and PAI-1 expression in macrophages.

Elevated auxin biosynthesis and transport underlie high vein density in C4 leaves.

High vein density, a distinctive trait of C4 leaves, is central to both C3-to-C4 evolution and conversion of C3 to C4-like crops. We tested the hypothesis that high vein density in C4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. Up-regulation of genes in auxin biosynthesis pathways and higher auxin content were found in developing C4 leaves compared with developing C3 leaves. The same observation held for maize foliar (C4) and husk (C3) leaf primordia. Moreover, auxin content and vein density were increased in loss-of-function mutants of Arabidopsis MYC2, a suppressor of auxin biosynthesis. Treatment with an auxin biosynthesis inhibitor or an auxin transport inhibitor led to much fewer veins in new leaves. Finally, both Arabidopsis thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. Thus, development of high leaf vein density requires elevated auxin biosynthesis and transport.

Benzyl isothiocyanate promotes apoptosis of oral cancer cells via an acute redox stress-mediated DNA damage response.

Benzyl isothiocyanate (BITC) is a cruciferous vegetable-derived compound with anticancer properties in human cancer cells. However, its anticancer potential and underlying mechanisms remain absent in human oral cancer cells. Results indicate that BITC inhibits growth, promotes G2/M phase arrest and triggers apoptosis of OC2 cells with a minimal toxicity to normal cells. BITC-induced cell death was completely prevented by pretreatment with thiol-containing redox compounds including N-acetyl-l-cysteine (NAC), glutathione (GSH), dithiothreitol, and 2-mercaptoethanol, but not free radical scavengers mito-TEMPO, catalase, apocynin, l-NAME and mannitol. BITC rapidly produced reactive oxygen species and nitric oxide, triggered oxidative DNA damage. BITC effectively decreased the intracellular GSH and GSH/GSSG ratio and redox balance recovery by thiol-containing redox compounds, but not by free radical scavengers. Accordingly, redox stresses-DNA damage response (DDR) activated ATM, Chk2, p53, and p21 and subsequently resulted in G2/M phase arrest by inhibiting Cdc2 and cyclin B1. Notably, BITC-induced apoptosis was associated with reduced Mcl-1 and Bcl-2 expression, diminished mitochondrial membrane potential (ΔΨm), and increased PARP cleavage. These BITC-induced redox stress-mediated DDR and apoptosis could be blocked by NAC and GSH. Therefore, BITC can be a rational drug candidate for oral cancer and acted via a redox-dependent pathway.

A variation on chloroplast development: the bizonoplast and photosynthetic efficiency in the deep-shade plant Selaginella erythropus.

UNLABELLED: • PREMISE OF THE STUDY: Chloroplast development and structure are highly conserved in vascular plants, but the bizonoplast of Selaginella is a notable exception. In the shade plant S. erythropus, each dorsal epidermal cell contains one bizonoplast, while other cells have normal chloroplasts. Our quest was to (1) determine the origin of bizonoplasts, (2) explore developmental plasticity, and (3) correlate developmental changes with photosynthetic activity to provide insights unavailable in other green plants with more constrained development.• METHODS: Bizonoplast development was studied in juvenile prostrate and older erect shoots of S. erythropus. Plastid plasticity was studied in plants cultivated under different light conditions. Chlorophyll fluorescence was measured and correlated with photosynthetic activity.• KEY RESULTS: The bizonoplast originates from a proplastid, forming a distinctive upper zone rapidly after exposure to low light. In the prostrate shoots, the proplastid develops through early stages only. When the shoot becomes erect, the proplastid soon develops into a mature bizonoplast. Erect shoots have significantly higher photosynthetic efficiency than prostrate shoots. No bizonoplasts were found in the plants growing in high light, where 2-4 spheroidal chloroplasts formed, or with light from below.• CONCLUSIONS: The upper zone develops above a normal-looking chloroplast structure to produce a bizonoplast. Bizonoplast developmental plasticity suggests that regular lamellar structure and monoplastidy are adaptations to deep shade environments. Such novel variation in S. erythropus is in stark contrast to known plastid development in other vascular plants, possibly reflecting retention of developmental flexibility in the basal clade, Lycophyta, to which it belongs.

Phenethyl isothiocyanate induces DNA damage-associated G2/M arrest and subsequent apoptosis in oral cancer cells with varying p53 mutations.

Phenethyl isothiocyanate (PEITC) is a naturally occurring cruciferous vegetable-derived compound that inhibits cell growth and induces apoptosis in oral cancer cells. However, the exact mechanism of PEITC action has not been fully elucidated. This study investigated the molecular mechanism and anticancer potential of PEITC in oral squamous cell carcinoma (OSCC) cells with various p53 statuses. PEITC inhibited the growth of OC2, SCC4, and SCC25 cells (functional p53 mutants) in a dose-dependent manner with low toxicity to normal cells. Treatment with PEITC induced reactive oxygen species production, nitric oxide generation, and GSH depletion and triggered DNA damage response as evidenced by flow cytometry, 8-OHdG formation, and comet assay. Furthermore, the subsequent activation of ATM, Chk2, and p53 as well as the increased expression of downstream proteins p21 and Bax resulted in a G2/M phase arrest by inhibiting Cdc25C, Cdc2, and cyclin B1. The PEITC-induced apoptotic cell death, following a diminished mitochondrial transmembrane potential, reduced the expression of Bcl-2 and Mcl-1, released mitochondrial cytochrome c, and activated caspase 3 and PARP cleavage. The p53 inhibitor pifithrin-α and the antioxidants N-acetylcysteine and glutathione (GSH) protected the cells from PEITC-mediated apoptosis. However, mito-TEMPO, catalase, apocynin, and L-NAME did not prevent PEITC-induced cell death, suggesting that PEITC induced G2/M phase arrest and apoptosis in oral cancer cells via a GSH redox stress and oxidative DNA damage-induced ATM-Chk2-p53-related pathway. These results provide new insights into the critical roles of both GSH redox stress and p53 in the regulation of PEITC-induced G2/M cell cycle arrest and apoptosis in OSCCs.

Biochanin a promotes osteogenic but inhibits adipogenic differentiation: evidence with primary adipose-derived stem cells.

Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1-1 µM) were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPAR γ ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 µM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

Caffeine inhibits adipogenic differentiation of primary adipose-derived stem cells and bone marrow stromal cells.

Caffeine consumption has been related to loss of body weight and modulates lipid metabolism. However, impacts of caffeine on adipogenic differentiation have not been well determined yet. The present study evaluated the effects of caffeine on adipogenesis using primary rat adipose-derived stem cells (ADSCs) and a mouse bone marrow stromal cell line (M2-10B4) in vitro. ADSCs and M2-10B4 were continuously exposed to caffeine (0.1-1mM) during adipogenic differentiation for 7 and 12 days, respectively. Oil red O and Nile red staining showed that caffeine reduced lipid droplet and adipocyte levels in both cell types. In addition, Nile red staining and FACScan flow cytometry showed that caffeine dose-dependently decreased adipocyte differentiation from 20% to 50% of the control ADSCs and M2-10B4 cells. Caffeine decreased the expression of adipogenesis-related genes including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, adipocyte lipid binding protein, lipoprotein lipase, leptin, and TNFα in a dose-dependent manner. Rather, low concentration of caffeine (0.1mM) significantly increased IL-6 expression, but unexpectedly inhibited that at a concentration more than 0.3mM. Taken together, caffeine was able to effectively inhibit adipogenic differentiation of ADSCs and M2-10B4 cells partly through its inhibition of adipogenesis-related factors.