Mode of Action of 1-Naphthylphthalamic Acid in Conspicuous Local Stem Swelling of Succulent Plant, Bryophyllum calycinum: Relevance to the Aspects of Its Histological Observation and Comprehensive Analyses of Plant Hormones.

The mode of action of 1-naphthylphthalamic acid (NPA) to induce conspicuous local stem swelling in the area of its application to the growing internode in intact Bryophyllum calycinum was studied based on the aspects of histological observation and comprehensive analyses of plant hormones. Histological analyses revealed that NPA induced an increase in cell size and numerous cell divisions in the cortex and pith, respectively, compared to untreated stem. In the area of NPA application, vascular tissues had significantly wider cambial zones consisting of 5-6 cell layers, whereas phloem and xylem seemed not to be affected. This indicates that stem swelling in the area of NPA application is caused by stimulation of cell division and cell enlargement mainly in the cambial zone, cortex, and pith. Comprehensive analyses of plant hormones revealed that NPA substantially increased endogenous levels of indole-3-acetic acid (IAA) in the swelling area. NPA also increased endogenous levels of cytokinins, jasmonic acid, and its precursor, 12-oxo-phytodienoic acid, but did not increase abscisic acid and gibberellin levels. It was shown, using radiolabeled 14C-IAA, that NPA applied to the middle of internode segments had little effect on polar auxin transport, while 2,3,5-triiodobenzoic acid substantially inhibited it. These results strongly suggest that NPA induces changes in endogenous levels of plant hormones, such as IAA, cytokinins, and jasmonic acid, and their hormonal crosstalk results in a conspicuous local stem swelling. The possible different mode of action of NPA from other polar auxin transport inhibitors in succulent plants is extensively discussed.

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.

Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus.

BACKGROUND: Interspecific hybridization is a useful tool in ornamental breeding to increase genetic variability and introduce new valuable traits into existing cultivars. The successful formation of interspecific hybrids is frequently limited by the presence of pre- and post-fertilization barriers. In the present study, we investigated the nature of hybridization barriers occurring in crosses between Kalanchoë species and evaluated possibilities of obtaining interspecific hybrids. RESULTS: The qualitative and quantitative analyses of pollen tube growth in situ were performed following intra- and interspecific pollinations. They revealed occurrence of pre-fertilization barriers associated with inhibition of pollen germination on the stigma and abnormal growth of pollen tubes. Unilateral incongruity related to differences in pistil length was also observed. The pollen quality was identified as a strong factor influencing the number of pollen tubes germinating in the stigma. In relation to post-fertilization barriers, endosperm degeneration was a probable barrier hampering production of interspecific hybrids. Moreover, our results demonstrate the relation of genetic distance estimated by AFLP marker analysis of hybridization partners with cross-compatibility of Kalanchoë species. At the same time, differences in ploidy did not influence the success of interspecific crosses. CONCLUSIONS: Our study presents the first comprehensive analysis of hybridization barriers occurring within Kalanchoë genus. Reproductive barriers were detected on both, pre- and post-fertilization levels. This new knowledge will contribute to further understanding of reproductive isolation of Kalanchoë species and facilitate breeding of new cultivars. For the first time, interspecific hybrids between K. nyikae as maternal plant and K. blossfeldiana as well as K. blossfeldiana and K. marnieriana were generated.

Origination of asexual plantlets in three species of Crassulaceae.

During asexual plant reproduction, cells from different organs can be reprogrammed to produce new individuals, a process that requires the coordination of cell cycle reactivation with the acquisition of other cellular morphological characteristics. However, the factors that influence the variety of asexual reproduction have not yet been determined. Here, we report on plantlet formation in Kalanchoe daigremontiana, Graptopetalum paraguayense, and Crassula portulacea (Crassulaceae) and analyse the effect of initiating cells on asexual reproduction in these three species. Additionally, the roles of WUSCHEL (WUS) and CUP-SHAPED COTYLEDON 1 (CUC1) in the asexual reproduction of these species were analysed through qRT-PCR. Our results indicated that pre-existing stem cell-like cells at the sites of asexual reproduction were responsible for the formation of plantlets. These cells were arrested in different phases of the cell cycle and showed different cell morphological characteristics and cell counts. The accumulation of auxin and cytokinin at the sites of asexual plantlet formation indicated their important functions, particularly for cell cycle reactivation. These differences may influence the pattern and complexity of asexual reproduction in these Crassulaceae species. Additionally, the dynamic expression levels of CUC1 and WUS may indicate that CUC1 functions in the formation of callus and shoot meristems; whereas, WUS was only associated with shoot induction.

Influence of blue light on the leaf morphoanatomy of in vitro Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae).

Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) (air plant, miracle leaf) is popularly used to treat gastrointestinal disorders and wounds. Recently, the species was tested to treat cutaneous leishmaniasis with successful results. This medicinal activity was associated with the phenolic fraction of the plant. Blue light induces biosynthesis of phenolic compounds and many changes in anatomical characteristics. We studied the effects of supplementary blue light on the leaf morphology of in vitro K. pinnata. Plants cultured under white light (W plants) only and white light plus blue light (WB plants) show petioles with plain-convex section, amphistomatic leaf blades with simple epidermis, homogeneous mesophyll with densely packed cells, and a single collateral vascular bundle in the midrib. W plants have longer branches, a larger number of nodes per branch, and smaller leaves, whereas WB plant leaves have a thicker upper epidermis and mesophyll. Leaf fresh weight and leaf dry weight were similar in both treatments. Phenolic idioblasts were observed in the plants supplemented with blue light, suggesting that blue light plays an important role in the biosynthesis of phenolic compounds in K. pinnata.

Ascorbic acid oxidase is dynamically regulated by light and oxygen. A tool for oxygen management in plants?

Ascorbic acid oxidase (AAO) is a plant blue-copper protein catalyzing dioxygen reduction to water using ascorbic acid as the electron donor. In spite of extensive molecular characterization the physiological role of AAO is still uncertain. Abundant mRNA, protein and activity of AAO were observed in illuminated leaves of Cucurbita pepo. AAO activity was found to be proportional to light intensity. The light effect was rapidly reversed in dark and activity remained low throughout the dark period. Activity was elicited in dark by increased oxygen concentration. AAO activity increased in the facultative CAM Kalanchoë blossfeldiana upon induction of the CAM cycle and decreased during germination of C. pepo and Zea mays under hypoxic conditions. These results strongly suggest that AAO activity could be part of a dynamic system for oxygen management in plants.

Vacuolar malate uptake is mediated by an anion-selective inward rectifier.

Electrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic-side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage-dependent gating of a 3-pS malate-selective ion channel, which showed a half-maximal open probability at -43 mV. Also, the time-averaged unitary currents following a step to a negative voltage corresponded to the time-dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion-selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent Km of 2.5 mm malate2- activity), a selectivity sequence of fumarate2- > malate2- > Cl- > maleate2- approximately citrate3-, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 microM niflumate reversibly blocked the activity of the 3-pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 microm-2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3-pS, inward-rectifying, anion-selective channel represents the principal pathway for malate influx into the vacuole.