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.

Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops).

Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.

Physiological alterations and genotoxic damage under combined aluminum and cadmium treatments in Bryophyllum daigremontianum clones.

BACKGROUND: Cadmium (Cd) is one of the most important stress factors in plants, with its high mobility in soils, ease of uptake by plants and toxicity at low concentrations. Aluminum (Al) is another phytotoxic metal, the accumulation of which is a crucial agricultural complication for plants, especially in acidic soils. METHODS AND RESULTS: In this study, Bryophyllum daigremontianum clone plantlets were obtained from bulbiferous spurs of a mother plant and separated into four different groups and watered with Hoagland solution and mixtures containing 0, 50, 100, and 200 µM of AlCl3 and CdCl2 each for 75 days. Control groups were maintained under the same conditions without Al and Cd treatment. To simulate acidic soil conditions typical of environments where Al toxicity is prevalent, the soil pH was adjusted to 4.5 by spraying the sulphuric acid (0.2%) with 2-day intervals after each irrigation day. After harvesting, growth parameters such as shoot length and thickness, root, shoot and leaf fresh and dry weights were measured, along with physiological parameters like mineral nutrient status, total protein, and photosynthetic pigment concentrations (chlorophyll a, b, a/b, total chlorophyll, and carotenoid) in both control and experimental groups of B. daigremontianum clones. In response to Al and Cd applications, the plant height, shoot thickness and carotenoid levels were declined, whereas the increments were found in leaf/shoot/root fresh weight, root dry weight, and total protein content. Moreover, differences in genomic alterations were investigated using 21 ISSR and 19 RAPD markers, which both have been used extensively as genetic markers to specify phylogenetic relationships among different cultivars as well as stress-dependent genetic alterations. RAPD primers were used due to their arbitrary sequences and the unknown genome sequence of the plant material used. In contrast, ISSR primers were preferred for a genome-wide genotoxic effect scan via non-arbitrary and more common genetic markers. Distinct types of band polymorphisms detected via RAPD and ISSR markers include band loss, and new band formation under a combination of Al and Cd stress. 17 ISSR and 14 RAPD primers generated clear electrophoretic bands. CONCLUSION: The study revealed that combined application of Al and Cd affect B. daigremontianum clones in terms of growth, physiology and genotoxicity related to the increasing concentrations.

In Vitro Antiviral Activity of Kalanchoe daigremontiana Extract against Human Herpesvirus Type 1.

Plant polyphenols possess diverse bioactivities, including antiviral activity against a broad spectrum of viruses. Here, we investigated the virucidal properties of an Kalanchoe daigremontiana extract using an in vitro model of human herpesvirus type 1 (HHV-1) infection. Chromatographic analysis indicated that the extract of Kalanchoe daigremontiana is rich in various compounds, among which are polyphenols with virucidal activity confirmed in the literature. We found that Kalanchoe daigremontiana extract shows an ability to prevent HHV-1 infection by direct inhibition of the virus attachment, penetration, and blocking of infection when used in pretreatment or post-entry treatment. Our results indicate that Kalanchoe daigremontiana extract may be a good candidate drug against HHV-1, both as a substance to prevent infection and to treat an already ongoing infection. Our findings illustrate that Kalanchoe daigremontiana could be a potential new candidate for clinical consideration in the treatment of HHV-1 infection alone or in combination with other therapeutics.

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.

Anti-ulcerogenic Potential of Kalanchoë gastonis-bonnieri Extracts in Male ICR Mice Model of Ethanol-induced Gastric Ulcers.

Peptic ulcers (PU) are a breach in the mucosa of the digestive tract and are related to several factors including an altered immune system and an unbalanced diet. Current treatment carries to long-term complications; therefore, the use of medicinal plants is an alternative for several inflammatory diseases including ulcerative lesions. Kalanchoë gastonis-bonnieri, is a succulent plant and, has been used in traditional medicine against gastric ulcers, inflammation, and cancer among others. The main goal of this work was to analyze the anti-ulcerogenic potential of extracts from leaves of K. gastonis-bonnieri in an assay of ethanol-induced gastric ulcers. An ethanolic extract was obtained by maceration from fresh leaves of K. gastonis-bonnieri, and fractions were obtained through bipartition and chemical fractioning. The chemical characterization of the extract was made through HPLC, GC-MS, and NMR. Total extract and fractions showed an anti-ulcerogenic effect in specimens of male ICR mice with a gastric ulcer index (UI) of 3.27-5.47. The recorded effect is attributed to the presence of terpenoid compounds such as ß-Amyrin acetate, which showed antioxidant properties and lessened formations of ulcers induced by ethanol administration in mice stomach.

Exploring the therapeutic mechanism of potential phytocompounds from Kalanchoe pinnata in the treatment of diabetes mellitus by integrating network pharmacology, molecular docking and simulation approach.

Since ancient times, bioactive phytocompounds from different parts of medicinal plants have been used to heal various disease ailments and they are now regarded as a valuable source of disease prevention globally. Kalanchoe pinnata is a member of the Crassulaceae family; it has a long history of usage in traditional ayurvedic treatment. Analysis of bioactive compounds for their potential anti-type-2 diabetes mellitus (T2DM) mechanism along with in-vitro and in-silico approaches was studied in the present research. The alpha-amylase and alpha-glucosidase inhibitory activity of methanolic extract of Kalanchoe pinnata (α-amylase: IC50 29.50 ± 0.04 µg/ml; α-glucosidase IC50 32.04 ± 0.35 µg/ml) exhibit a high degree of similarity to the standard drug acarbose (IC50 35.82 ± 0.14 µg/ml). Different biological databases were used to list phytocompounds from the plant, and ADME analysis using swissADME was carried out to screen compounds that obeyed the Lipinski rule of 5 and were employed further. STRING and KEGG pathway analysis was performed for gene enrichment analysis followed by network pharmacology to identify key target proteins involved in DM. AMY2A, NOX4, RPS6KA3, ADRA2A, CHRM5, and IL2 were identified as core targets for luteolin, kaempferol, alpha amyrin, stigmasterol compounds by modulating neuroactive ligand interaction, P13-AKT, MAPK, and PPAR signaling pathways. Molecular docking was performed to study the binding affinity among bioactive compounds of K. pinnata against aldose reductase, alpha-amylase, alpha-glucosidase, and dipeptidyl peptidase IV. Alpha-amylase-friedelin [FRI] and alpha-amylase-acarbose [STD] complexes were subjected to molecular simulation for a 200 ns duration that depicted the stability of the compounds and proteins. In the current study, employing dual approach in-silico and in-vitro enzyme assays has yielded a comprehensive and strong understanding of its potential therapeutic properties, making a significant step towards the development of novel anti-diabetic treatment.

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

BACKGROUND AND AIMS: Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as a substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating daytime stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure. METHODS: Biochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants. KEY RESULTS: Starch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. CONCLUSIONS: Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

First report of Kalanchoe Latent Virus naturally infecting common bean (Phaseolus vulgaris L.) in South Korea.

The common bean (Phaseolus vulgaris; family: Fabaceae) is an economically and nutritionally important food crop worldwide (Ganesan et al. 2017). In 2021, several plants collected from different provinces in South Korea had symptoms of viral infections (e.g., mild yellow-greenish speckling, stunting, crinkling, and deformed leaves). To identify the causal pathogens, total RNA was isolated from pooled leaf tissues from all samples (n = 29) for paired-end high-throughput sequencing (HTS). The cDNA library was constructed after eliminating ribosomal RNA using the TruSeq RNA Sample Prep Kit and then sequenced using the Illumina NovaSeq 6000 platform (Macrogen, Korea). The 297,868,156 paired-end clean reads (150 nt) were de novo assembled using Trinity with default parameters. BLASTx was used for the contig analysis, which revealed the pooled samples were infected with several plant viruses (e.g., turnip mosaic virus, zucchini yellow mosaic virus, cucumber mosaic virus, lily mottle virus). Notably, the assembled contigs included a single viral contig (8,472 nt) comprising the nearly complete KLV genome (HTS mean coverage: 39.46%). Kalanchoe latent virus (KLV; genus: Carlavirus; family: Betaflexiviridae) has been detected in Kalanchoë blossfeldiana (Hearon 1982), Chenopodium quinoa (Dinesen et al. 2009), and Graptopetalum paraguayense (Sorrentino et al. 2017). The sequence was most similar (96.28% nucleotide identity; 99% query coverage) to KLV isolate DSMZ PV-0290 (GenBank: OP525283) from Denmark. The contig sequence was validated via reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from the 29 individually stored samples and nine primer sets specific for the KLV contig. All nine contig-specific overlapping fragments were amplified from only a P. vulgaris plant with mild yellowing mosaic symptoms collected on July 6, 2021, in Jeongseon County, South Korea. Additionally, 5' and 3' rapid amplification of cDNA ends (RACE)-specific primers were designed for the KLV contig sequence to determine the terminal ends of the genome of the South Korean KLV isolate using the 5'/3' RACE System (Invitrogen, Carlsbad, CA, USA). All of the amplified and overlapping fragments were cloned into the RBC T&A Cloning Vector (RBC Bioscience, Taipei, Taiwan) and sequenced using the Sanger method. The obtained full-length genomic sequence of the KLV isolate (KLV-SK22) was 8,517 nt long and was deposited in GenBank OQ718816. According to the BLASTn analysis, KLV-SK22 was highly similar (96.30% sequence identity; 100% query coverage) to the DSMZ PV-0290 isolate. Phylogenetic trees constructed on the basis of coat protein and RNA-dependent RNA polymerase amino acid sequences revealed that KLV-SK22 is closely related to the DSMZ PV-0290 and PV-0290B isolates from Denmark, respectively. At the genome and gene levels, the individual sequence identities between the carlaviruses and other KLV isolates were 96.29% to 100% (Adams et al. 2004). Additionally, an RT-PCR analysis using detection primers specific for KLV-SK22 did not detect KLV in 15 samples (P. vulgaris = 3, Glycine max = 8, Pueraria montana = 2, Trifolium repens = 1, and Vigna angularis = 1) randomly collected from different regions in South Korea. Based on these results, KLV infection may not be widespread at this time in South Korea. To the best of our knowledge, this is the first report of KLV in P. vulgaris in South Korea or elsewhere. Our findings will aid future research on the epidemiology and long-term management of KLV-related diseases.

A Review of the Popular Uses, Anatomical, Chemical, and Biological Aspects of Kalanchoe (Crassulaceae): A Genus of Plants Known as "Miracle Leaf".

Species of the genus Kalanchoe have a long history of therapeutic use in ethnomedicine linked to their remarkable healing properties. Several species have chemical and anatomical similarities, often leading to confusion when they are used in folk medicine. This review aims to provide an overview and discussion of the reported traditional uses, botanical aspects, chemical constituents, and pharmacological potential of the Kalanchoe species. Published scientific materials were collected from the PubMed and SciFinder databases without restriction regarding the year of publication through April 2023. Ethnopharmacological knowledge suggests that these species have been used to treat infections, inflammation, injuries, and other disorders. Typically, all parts of the plant are used for medicinal purposes either as crude extract or juice. Botanical evaluation can clarify species differentiation and can enable correct identification and validation of the scientific data. Flavonoids are the most common classes of secondary metabolites identified from Kalanchoe species and can be correlated with some biological studies (antioxidant, anti-inflammatory, and antimicrobial potential). This review summarizes several topics related to the Kalanchoe genus, supporting future studies regarding other unexplored research areas. The need to conduct further studies to confirm the popular uses and biological activities of bioactive compounds is also highlighted.

Cytotoxic and genotoxic effects of Kalanchoe pinnata (Lam.) Pers. fresh leaf juice in the cultured human blood lymphocytes.

This is the first investigation on the in vitro cytotoxicological and genotoxicological effects of Kalanchoe pinnata (Lam.) Pers. fresh leaf juice, for establishing a safe and effective quantity for use. Peripheral blood of 6 healthy, non-addicted males between 20 and 25 years of age was used for toxicity assessment by 3-(4, 5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), mitotic index (MI), sister chromatid exchanges (SCEs) and Cytokinesis Blocked Micronucleus (CBMN) assays. The IC50 of the leaf juice calculated by MTT assay was 155.1 µl. The 50 and70 µl of the juice concentrations were decided after standardization by MI and showed non-significant and significant decrease respectively when compared to control. The SCEs/Cell and SCEs/Chromosome were increased non-significantly (50 µl) and significantly (70 µl),while cell cycle proliferative index, Average generation time and Population doubling time values were non-significant for both doses when compared to the controls. In CBMN assay, the Cytokinesis block proliferation index, cytotoxicity, Micronuclei, Nuclear bud, Nucleoplasmic bridge frequencies and total DNA damage biomarkers showed non-significant changes for both 50 and 70 µl. The changes observed were significant only at 70 µl for MI and SCEs, which were significantly lower than that by positive control indicating a non genotoxic effect. Hence, the fresh leaf juice can be used pharmaceutically as well as traditionally, but for long durations and higher doses should be used with caution, as it can have mutagenic effect at particularly high levels.

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.

Potential of Kalanchoe pinnata as a Cancer Treatment Adjuvant and an Epigenetic Regulator.

Cancer is a global public health problem that is related to different environmental and lifestyle factors. Although the combination of screening, prevention, and treatment of cancer has resulted in increased patient survival, conventional treatments sometimes have therapeutic limitations such as resistance to drugs or severe side effects. Oriental culture includes herbal medicine as a complementary therapy in combination with chemotherapy or radiotherapy. This study aimed to identify the bioactive ingredients in Kalanchoe pinnata, a succulent herb with ethnomedical applications for several diseases, including cancer, and reveal its anticancer mechanisms through a molecular approach. The herb contains gallic acid, caffeic acid, coumaric acid, quercetin, quercitrin, isorhamnetin, kaempferol, bersaldegenin, bryophyllin a, bryophyllin c, bryophynol, bryophyllol and bryophollone, stigmasterol, campesterol, and other elements. Its phytochemicals participate in the regulation of proliferation, apoptosis, cell migration, angiogenesis, metastasis, oxidative stress, and autophagy. They have the potential to act as epigenetic drugs by reverting the acquired epigenetic changes associated with tumor resistance to therapy-such as the promoter methylation of suppressor genes, inhibition of DNMT1 and DNMT3b activity, and HDAC regulation-through methylation, thereby regulating the expression of genes involved in the PI3K/Akt/mTOR, Nrf2/Keap1, MEK/ERK, and Wnt/ß-catenin pathways. All of the data support the use of K. pinnata as an adjuvant in cancer treatment.

Three bufadienolides induce cell death in the human lung cancer cell line CL1-5 mainly through autophagy.

The effects of 3 bufadienolides, namely kalantuboside B, kalantuboside A, and bryotoxin C, isolated from Kalanchoe tubiflora (Harvey) were evaluated and characterized in CL1-5 highly metastatic human lung cancer cells. In contrast to their apoptosis-promoting activity in other cancer cells, these bufadienolides only slight or did not induce apoptosis in CL1-5 cancer cells. Instead, they activated an autophagy pathway, as indicated by increased autophagosome formation. Autophagy induced by these bufadienolides was demonstrated to be linked to the down-regulation of p-mTOR and the up-regulation of LC3-II, ATG5, ATG7, and Beclin-1. Our findings revealed an autophagy as the alternative mechanism of drug action by bufadienolides in CL1-5 lung cancer cells and provided evidence that bufadienolides are a potential therapeutic strategy for highly metastatic human lung cancer.

Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells.

CONTEXT: Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. OBJECTIVE: The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate - a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. MATERIALS AND METHODS: The cytotoxic activity of the compound (at 0.1-20.0 µg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. RESULTS: The compound had strong effect on the cells (IC50 = 0.55 µg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. CONCLUSIONS: Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

Crassulacean acid metabolism guard cell anion channel activity follows transcript abundance and is suppressed by apoplastic malate.

Plants utilising crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO. CO2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light. In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild-type and RNAi mutants with impaired Mal metabolism. We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle.

Differential Distribution of Flavonoids and Phenolic Acids in Leaves of Kalanchoe delagoensis Ecklon & Zeyher (Crassulaceae).

Kalanchoe delagoensis is adapted to intense solar irradiation, drought, and heat, partially due to the presence of phenols, important photo-protective compounds and antioxidants. This study aimed to evaluate the distribution of flavonoids and phenolic acid derivatives throughout the erect-tubular leaves of K. delagoensis. Specimens grown under sunny conditions were used for histochemical and high-performance liquid chromatography coupled with diode array detection (liquid HPLC-DAD) analysis. The NP (2-aminoethyl diphenylborinate) test suggested the presence of phenolic acids throughout the leaf blade below the epidermis and in chloroplasts, mainly in the leaf base. Flavonoids were detected specifically in chloroplasts, on the adaxial side of the middle third and at the leaf apex, near the meristematic cells. There was a tendency of flavonoid accumulation from the middle third to the apex, especially surrounding the gem, while phenolic acids were observed mainly in the base. This can be explained by the more exposed leaf apex and to the presence of apical buds (high production and regulation sites of ROS). The HPLC-DAD analysis showed different classes of flavonoids and phenolic acid derivatives in the leaf extracts, agreeing with the NP test results. This is the first time that the substitution of phenolic acids by flavonoids from the leaf base to the apex has been 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.

Ecophysiology of constitutive and facultative CAM photosynthesis.

In plants exhibiting crassulacean acid metabolism (CAM), CAM photosynthesis almost always occurs together with C3 photosynthesis, and occasionally with C4 photosynthesis. Depending on species, ontogeny, and environment, CAM input to total carbon gain can vary from values of <1% to 100%. The wide range of CAM phenotypes between and within species is a fascinating example of functional diversity and plasticity, but poses a significant challenge when attempting to define CAM. CO2 gas exchange experiments designed for this review illustrate key patterns of CAM expression and highlight distinguishing features of constitutive and facultative CAM. Furthermore, they help to address frequently recurring questions on CAM terminology. The functional and evolutionary significance of contrasting CAM phenotypes and of intermediate states between extremes is discussed. Results from a study on nocturnal malate accumulation in 50 species of Aizoaceae exposed to drought and salinity stress suggest that facultative CAM is more widespread amongst vascular plants than previously thought.

Cyclodextrin-Elicited Bryophyllum Suspension Cultured Cells: Enhancement of the Production of Bioactive Compounds.

The rates of production of secondary metabolites obtained by employing conventional plant breeding may be low for practical purposes. Thus, innovative approaches for increasing their rates of production are being developed. Here, we propose the use of elicited plant suspension cultured cells (PSCC) with cyclodextrins (CDs) as an alternative method for the production of bioactive compounds from Bryophyllum species. For this purpose, we analyzed the effects of methyl-ß-cyclodextrin and 2-hydroxypropyl-ß-cyclodextrin on cell culture growth and on the intra- and extracellular production of phenols and flavonoids. Results clearly show that CDs enhance the biosynthesis of polyphenols by PSCC favoring their accumulation outside the cells. CDs shift the homeostatic equilibrium by complexing extracellular phenolics, causing stress in cells that respond by increasing the production of intracellular phenolics. We also analyzed the radical scavenging activity of the culture medium extracts against 2,2-diphenyl-1-pycrilhydrazyl (DPPH) radical, which increased with respect to the control samples (no added CDs). Our results suggest that both the increase in the production of polyphenols and their radical scavenging activity are a consequence of their inclusion in the CD cavities. Overall, based on our findings, CDs can be employed as hosts for increasing the production of polyphenols from Bryophyllum species.