Erwin Bünning and Wolfgang Engelmann: establishing the involvement of the circadian clock in photoperiodism.

In 1936, Erwin Bünning published his groundbreaking work that the endogenous clock is used to measure day length for initiating photoperiodic responses. His publication triggered years of controversial debate until it ultimately became the basic axiom of rhythm research and the theoretical pillar of chronobiology. Bünning's thesis is frequently quoted in the articles in this special issue on the subject of "A clock for all seasons". However, nowadays only few people know in detail about Bünning's experiments and almost nobody knows about the contribution of his former doctoral student, Wolfgang Engelmann, to his theory because most work on this topic is published in German. The aim of this review is to give an overview of the most important experiments at that time, including Wolfgang Engelmann's doctoral thesis, in which he demonstrated the importance of the circadian clock for photoperiodic flower induction in the Flaming Katy, Kalanchoë blossfeldiana, but not in the Red Morning Glory, Ipomoea coccinea.

Exposure to Light of the Abaxial versus Adaxial Side of Detached Kalanchoë blossfeldiana Leaves Affects Anthocyanin Content and Composition Differently.

The accumulation and composition of anthocyanins in leaves of Kalanchoë blossfeldiana, detached and kept for five days under natural light conditions, were investigated. The presence of fifteen derivatives of cyanidin, petunidin, and delphinidin was found. Changes in the content of each anthocyanin in the leaves before and after exposure to light on the abaxial (naturally upper) and adaxial (naturally lower) sides of the leaves were compared. When the adaxial side was exposed to light, the anthocyanin contents of the leaves did not change. In contrast, when the abaxial side of detached leaves was exposed to light, there was enhanced accumulation of delphinidin-rhamnoside-glucoside, cyanidin-rhamnoside-glucoside, cyanidin-glucoside-glucoside, and two unknown derivatives of petunidin and delphinidin. Application of methyl jasmonate (JA-Me) on the abaxial side exposed to light inhibited the accumulation of these anthocyanins. This effect could probably be due to the presence of these anthocyanins in the epidermal cells of K. blossfeldiana leaves and was visible in the microscopic view of its cross-section. These anthocyanins were directly exposed to JA-Me, leading to inhibition of their formation and/or accumulation. The lack of significant effects of JA-Me on anthocyanin mono- and tri-glycosides may indicate that they are mainly present in the mesophyll tissue of the leaf.

Accumulation of Anthocyanins in Detached Leaves of Kalanchoë blossfeldiana: Relevance to the Effect of Methyl Jasmonate on This Process.

Accumulation of anthocyanins in detached leaves and in excised stems of Kalanchoë blossfeldiana kept under natural light conditions in the presence or absence of methyl jasmonate (JA-Me) was investigated. When the abaxial surface of detached leaves was held lower than the adaxial surface (the normal or natural position) under natural light conditions, anthocyanins were not accumulated on the abaxial side of the leaves. In contrast, when the adaxial surface of detached leaves was held lower than the abaxial surface (inverted position), anthocyanins were highly accumulated on the abaxial side of the leaves. These phenomena were independent of the growth stage of K. blossfeldiana as well as photoperiod. Application of JA-Me in lanolin paste significantly inhibited anthocyanin accumulation induced on the abaxial side of detached leaves held in an inverted position in a dose-dependent manner. Anthocyanin accumulation in the excised stem in response to natural light was also significantly inhibited by JA-Me in lanolin paste. Possible mechanisms of anthocyanin accumulation on the abaxial side of detached K. blossfeldiana leaves held in an inverted position under natural light conditions and the inhibitory effect of JA-Me on this process are described. The accompanying changes in the content of primary metabolites and histological analyses were also described.

Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content.

CONTEXT: Kalanchoe species (Crassulaceae) are widely used in traditional medicine as remedies in infectious diseases and cancer treatment. OBJECTIVE: Cytotoxic and antimicrobial activities of Kalanchoe daigremontiana Raym.-Hamet & H. Perrier, K. pinnata (Lam.) Pers., and K. blossfeldiana Poelln. extracts were determined. The relationship between biological activities and the extracts bufadienolides content was also investigated. MATERIALS AND METHODS: Fresh leaves of Kalanchoe species were macerated with 95% ethanol or water. The quantitative analysis of bufadienolides in the extracts was carried out with mass spectrometry. Cytotoxicity tests were performed on human cancer cell lines - HeLa, SKOV-3, MCF-7, and A375 by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and Real-Time Cell Analysis system. The microbiological study was done using a few bacteria strains (ß-hemolytic Streptococcus, Corynebacterium diphtheriae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus hirae, Escherichia coli) and Candida albicans. RESULTS: The K. blossfeldiana ethanol extract and K. daigremontiana water extract exhibited the most potent cytotoxic activity (IC50 < 19 µg/mL for HeLa and SKOV-3 cells). The strongest antibacterial effects showed ethanol extract of K. blossfeldiana and K. pinnata (MIC values were 8.45, 8.45, 0.25 and <33.75 µg/mL for S. aureus, S. epidermidis, and E. hirae, respectively). The highest total amount of bufadienolides was in K. daigremontiana ethanol extract. In contrast, K. blossfeldiana ethanol extract did not show the presence of these compounds. CONCLUSIONS: Kalanchoe blossfeldiana ethanol extract is a potential candidate for cancer and bacterial infection treatment. Additionally, the biological effects of Kalanchoe extracts are not dependent on the presence and amount of bufadienolides in the plant extracts.

Kalanchoe sp. Extracts-Phytochemistry, Cytotoxic, and Antimicrobial Activities.

Kalanchoe species are succulents occurring in tropical regions. They have many biological and pharmacological properties. In this study, the cytotoxic and antimicrobial activities of water and dichloromethane Kalanchoe fractions obtained from ethanol extracts of three Kalanchoe species-K. daigremontiana, K. pinnata, and K. blossfeldiana were estimated. The cytotoxic effect was assessed on human cancer cell lines-ovarian SKOV-3, cervical HeLa, breast MCF-7, and melanoma A375-by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The antimicrobial activity was estimated on selected Gram-positive and Gram-negative bacteria strains and on Candida albicans. The phytochemical analysis of selected Kalanchoe extracts was conducted by LC-QTOF-MS. The obtained results showed that the water fraction of K. blossfeldiana was active both on the tested cancer cells (IC50 values were 28.28 ± 2.76 and 32.51 ± 0.69 µg/mL on HeLa and SKOV-3, respectively) and bacteria strains (MIC values were 16 and 32 µg/mL on S. epidermidis and S. aureus, respectively). The water fraction of K. pinnata also had a significant effect on S. epidermidis and S. aureus, with MIC values of 32 and 64 µg/mL, respectively. The water fraction of K. blossfeldiana triggered a decrease in mitochondrial membrane potential (MMP) and induced cell cycle arrest in the G2/M phase in the SKOV-3 and HeLa cells. This fraction did not significantly increase cellular oxidative stress level. The DPPH and ABTS assays revealed that the water fraction of K. blossfeldiana had a strong antioxidant effect (IC50 was 9.44 ± 0.06 and 3.17 ± 0.1 µg/mL, respectively). The phytochemical analysis of the extracts of K. blossfeldiana and K. pinnata revealed the presence of at least 218 main components. The most frequently occurring were flavonol glycosides (31 metabolites), phenylpropanoids (13 metabolites), gallic acid derivatives (13 compounds), benzoic acid derived compounds (14 metabolites), and acyclic alcohol glycosides (16 compounds). In addition, proanthocyanidins were detected mainly in K. blossfeldiana. The study indicates that the water fraction of K. blossfeldiana has significant biological potential and can be further investigated towards anticancer and antimicrobial application.

The Bacillus cereus Strain EC9 Primes the Plant Immune System for Superior Biocontrol of Fusarium oxysporum.

Antibiosis is a key feature widely exploited to develop biofungicides based on the ability of biological control agents (BCAs) to produce fungitoxic compounds. A less recognised attribute of plant-associated beneficial microorganisms is their ability to stimulate the plant immune system, which may provide long-term, systemic self-protection against different types of pathogens. By using conventional antifungal in vitro screening coupled with in planta assays, we found antifungal and non-antifungal Bacillus strains that protected the ornamental plant Kalanchoe against the soil-borne pathogen Fusarium oxysporum in experimental and commercial production settings. Further examination of one antifungal and one non-antifungal strain indicated that high protection efficacy in planta did not correlate with antifungal activity in vitro. Whole-genome sequencing showed that the non-antifungal strain EC9 lacked the biosynthetic gene clusters associated with typical antimicrobial compounds. Instead, this bacterium triggers the expression of marker genes for the jasmonic and salicylic acid defence pathways, but only after pathogen challenge, indicating that this strain may protect Kalanchoe plants by priming immunity. We suggest that the stimulation of the plant immune system is a promising mode of action of BCAs for the development of novel biological crop protection products.

Antimicrobial properties of Kalanchoe blossfeldiana: a focus on drug resistance with particular reference to quorum sensing-mediated bacterial biofilm formation.

OBJECTIVES: This study attempts to investigate the antimicrobial properties of Kalanchoe blossfeldiana with a particular reference to quorum sensing (QS)-mediated biofilm formation. METHODS: The methanol extract of K. blossfeldiana leaves (MEKB) was evaluated for antimicrobial properties including QS-controlled production of biofilm (including virulence factor, motility and lactone formation) in Pseudomonas aeruginosa. Methanol extract of K. blossfeldiana was also evaluated for anti-cytokine (tumour necrosis factor-alpha, interleukin-6 and interleukin-1 beta) properties in peripheral blood mononuclear cells (PBMC). KEY FINDINGS: Methanol extract of K. blossfeldiana exhibited antimicrobial effect on clinical isolates, as well as standard reference strains. Pseudomonas aeruginosa exposed to MEKB (subminimum inhibitory concentration (MIC)) displayed reduced biofilm formation, whereas supra-MIC produced destruction of preformed biofilms. Methanol extract of K. blossfeldiana reduced the secretion of virulence factors (protease and pyoverdin) along with generation of acyl homoserine lactone (AHL). Confocal laser scanning microscopy images indicate reduction of biofilm thickness. The extract also reduced cytokine formation in lipopolysaccharide-stimulated PBMC. CONCLUSIONS: Kalanchoe blossfeldiana was found to interfere with AHL production, which in turn may be responsible for downregulating QS-mediated production of biofilm and virulence. This first report on the antibiofilm and anticytokine properties of this plant may open up new vistas for future exploration of this plant for combating biofilm-related resistant infections.