Helenice Mercier

Specific leaf areas of the tank bromeliad Guzmania monostachia perform distinct functions in response to water shortage.

Leaves comprise most of the vegetative body of tank bromeliads and are usually subjected to strong longitudinal gradients. For instance, while the leaf base is in contact with the water accumulated in the tank, the more light-exposed middle and upper leaf sections have no direct access to this water reservoir. Therefore, the present study attempted to investigate whether different leaf portions of Guzmania monostachia, a tank-forming C(3)-CAM bromeliad, play distinct physiological roles in response to water shortage, which is a major abiotic constraint in the epiphytic habitat. Internal and external morphological features, relative water content, pigment composition and the degree of CAM expression were evaluated in basal, middle and apical leaf portions in order to allow the establishment of correlations between the structure and the functional importance of each leaf region. Results indicated that besides marked structural differences, a high level of functional specialization is also present along the leaves of this bromeliad. When the tank water was depleted, the abundant hydrenchyma of basal leaf portions was the main reservoir for maintaining a stable water status in the photosynthetic tissues of the apical region. In contrast, the CAM pathway was intensified specifically in the upper leaf section, which is in agreement with the presence of features more suitable for the occurrence of photosynthesis at this portion. Gas exchange data indicated that internal recycling of respiratory CO(2) accounted for virtually all nighttime acid accumulation, characterizing a typical CAM-idling pathway in the drought-exposed plants. Altogether, these data reveal a remarkable physiological complexity along the leaves of G. monostachia, which might be a key adaptation to the intermittent water supply of the epiphytic niche.

Nitric Oxide Mediates the Hormonal Control of Crassulacean Acid Metabolism Expression in Young Pineapple Plants

Genotypic, developmental, and environmental factors converge to determine the degree of Crassulacean acid metabolism (CAM) expression. To characterize the signaling events controlling CAM expression in young pineapple (Ananas comosus) plants, this photosynthetic pathway was modulated through manipulations in water availability. Rapid, intense, and completely reversible up-regulation in CAM expression was triggered by water deficit, as indicated by the rise in nocturnal malate accumulation and in the expression and activity of important CAM enzymes. During both up- and down-regulation of CAM, the degree of CAM expression was positively and negatively correlated with the endogenous levels of abscisic acid (ABA) and cytokinins, respectively. When exogenously applied, ABA stimulated and cytokinins repressed the expression of CAM. However, inhibition of water deficit-induced ABA accumulation did not block the up-regulation of CAM, suggesting that a parallel, non-ABA-dependent signaling route was also operating. Moreover, strong evidence revealed that nitric oxide (NO) may fulfill an important role during CAM signaling. Up-regulation of CAM was clearly observed in NO-treated plants, and a conspicuous temporal and spatial correlation was also evident between NO production and CAM expression. Removal of NO from the tissues either by adding NO scavenger or by inhibiting NO production significantly impaired ABA-induced up-regulation of CAM, indicating that NO likely acts as a key downstream component in the ABA-dependent signaling pathway. Finally, tungstate or glutamine inhibition of the NO-generating enzyme nitrate reductase completely blocked NO production during ABA-induced up-regulation of CAM, characterizing this enzyme as responsible for NO synthesis during CAM signaling in pineapple plants.

Endogenous auxin and cytokinin contents associated with shoot formation in leaves of pineapple cultured in vitro

The in vitro culture of pineapple leaves on a shoot induction medium (SIM) results in the formation of protuberances and further development in shoots, and plantlets. The contents of endogenous indoleacetic acid (IAA) and five cytokinins (Cks), N6(2-isopentenyl)adenine (iP), N6(2-isopentenyl)adenosine (iPR), zeatin (Z), zeatin riboside (ZR) and N6-benzyladenine (BA), present in the basal portion of those leaves, were correlated to the organogenic response that occurs over 15 days of culture. The endogenous auxin/cytokinins ratio was lowest on the 3rd day, mainly due to a strong increase in the iP level. It seems that endogenous iP concentration triggered the induction signal for an organogenic response in pineapple leaf bases. The rise in iP content required the presence of BA and a-naphthaleneacetic acid (NAA) in the medium, suggesting that endogenous iP production is regulated in response to these growth regulator uptakes.

TEMPERATURE DETERMINES THE OCCURRENCE OF CAM OR C3 PHOTOSYNTHESIS IN PINEAPPLE PLANTLETS GROWN IN VITRO

SummaryAnanas comosus (L.) Merr. var. Smooth Cayenne plants when grown in vitro under different temperature regimes developed as CAM or as C3 plants. The plants used in this study were developed from the lateral buds of the nodal etiolated stem explants cultured on Murashige and Skoog medium for 3 mo. The cultures were maintained under a 16-h photoperiod for different thermoperiods. With 28°C light/15°C dark thermoperiod, as compared with constant 28°C light and dark, pineapple plants had a succulence index two times greater, and also a greater nocturnal titratable acidity and phosphoenolpyruvate carboxylase (PEPCase) activity, indicating CAM-type photosynthesis. The highest abscisic acid (ABA) level occurred during the light period, 8 h prior to maximum PEPCase activity, while the indole-3-acetic acid (IAA) peak was found during the dark period, coinciding with the time of highest PEPCase activity. These plants were also smaller with thicker leaves and fewer roots, but had greater dry weight. Their leaves showed histological characteristics of CAM plants, such as the presence of greater quantities of chlorenchyma and hypoderm. In addition, their vascular system was more conspicuous. In contrast, under constant temperature (28°C light/dark) plants showed little succulence in the leaves. There was no significant acid oscillation and diurnal variation in PEPCase activity in these plants, suggesting the occurrence of C3 photosynthesis. Also, no diurnal variation in ABA and IAA contents was observed. The results of this study clearly indicate a role for temperature in determining the type of carbon fixation pathway in in vitro grown pineapple. Evidence that ABA and IAA participate in CAM signaling is provided.

The Control of Storage Xyloglucan Mobilization in Cotyledons of Hymenaea courbaril

Hymenaea courbaril is a leguminous tree species from the neotropical rain forests. Its cotyledons are largely enriched with a storage cell wall polysaccharide (xyloglucan). Studies of cell wall storage polymers have been focused mostly on the mechanisms of their disassembly, whereas the control of their mobilization and the relationship between their metabolism and seedling development is not well understood. Here, we show that xyloglucan mobilization is strictly controlled by the development of first leaves of the seedling, with the start of its degradation occurring after the beginning of eophyll (first leaves) expansion. During the period of storage mobilization, an increase in the levels of xyloglucan hydrolases, starch, and free sugars were observed in the cotyledons. Xyloglucan mobilization was inhibited by shoot excision, darkness, and by treatment with the auxin-transport inhibitor N-1-naphthylphthalamic acid. Analyses of endogenous indole-3-acetic acid in the cotyledons revealed that its increase in concentration is followed by the rise in xyloglucan hydrolase activities, indicating that auxin is directly related to xyloglucan mobilization. Cotyledons detached during xyloglucan mobilization and treated with 2,4-dichlorophenoxyacetic acid showed a similar mobilization rate as in attached cotyledons. This hormonal control is probably essential for the ecophysiological performance of this species in their natural environment since it is the main factor responsible for promoting synchronism between shoot growth and reserve degradation. This is likely to increase the efficiency of carbon reserves utilization by the growing seedling in the understorey light conditions of the rain forest.

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic (15N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (δ15N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (±0.07 SE) and 49.6% (±0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian–bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.

Effects of Auxin, Cytokinin and Ethylene Treatments on the Endogenous Ethylene and Auxin-to-cytokinins Ratio Related to direct Root Tip Conversion of Catasetum fimbriatum Lindl. (Orchidaceae) into Buds

Summary The present investigation attempts to correlate the effect of the exogenous auxins, cytokinins and ethylene with the endogenous ethylene and auxin-to-cytokinin ratio, and with the direct root-to-bud conversion in Catasetum fimbriatum . Incubation of root tips with IAA and NAA strongly inhibited both root elongation and bud formation. On the other hand, treatment with IBA resulted in a conspicuous stimulation of longitudinal root growth, while bud formation was retarded. High levels of exogenous zeatin, iP and BA enhanced bud formation, and inhibited root growth. Ethylene production was measured in IAA, IBA and zeatin-treated roots. All growth regulators elicited ethylene evolution, which decreased considerably along the period of incubation. Addition of AVG to the media reduced ethylene evolution. Endogenous auxin, cytokinins, and ABA contents were determined by means of HPLC and ELISA methods in root tips of C. fimbriatum pre-incubated with ethylene (CEPA) and cytokinin (iP). Both exogenous substances stimulated bud formation and conduced to an endogenous IAA/Cks ratio favourable to Cks. From these results it is proposed that the stimulatory effect of exogenous Cks and ethylene on root-to-bud conversion of C. fimbriatum seems to be mediated by the establishment of an endogenous auxin-to-cytokinins balance favourable to shoot formation.

Ammonium and urea as nitrogen sources for bromeliads

Summary Epiphytic bromeliads have no contact with the pedosphere, so they need to draw their nutrients from the atmosphere as well as from the host tree and animal debris. Terrestrial bromeliads, like Ananas comosus, generally depend on the soil as their main nutrient source. The aim of this study was to investigate and compare some aspects of the nitrogen metabolism of two bromeliads with different growth habits: Ananas comosus, a terrestrial bromeliad, and Vriesea gigantea, an epiphytic tank bromeliad. Nitrogen-starved plants were grown in vitro for 3, 7, 15, 30, and 60 days, either with 5 mmol L −1 ammonium [(NH 4 ) 2 SO 4 ] or urea as the sole nitrogen source. When NH 4 + was supplied to the plants, it stimulated a faster increase of chlorophyll content in A. comosus than in V. gigantea . In the presence of urea, after 15 days of the plants in culture, there was a significant increase in tissue free-NH 4 + and total amino acids for V. gigantea only. V. gigantea presented a higher level of total free amino acids than A. comosus when nitrogen was supplied to the plants. Asparagine was the main amino acid accumulated in both bromeliads when plants were transferred to the medium with nitrogen. When the ratio of the main individual free amino acids between the bromeliads grown in NH 4 + and urea was compared, values such as 7.2 for asparagine, 5.3 for glutamate, and 1.8 for aspartate in A. comosus, and values such as 2.3 for asparagine, 1.1 for glutamate and 0.7 for aspartate in V. gigantea were observed, demonstrating that the last is more efficient in assimilating urea. The results prompted us to support the idea that V. gigantea, an epiphytic tank bromeliad, is better adapted to absorb and assimilate organic nitrogen, such as urea, while A. comosus, a terrestrial plant, is better adapted to inorganic nitrogen forms, such as ammonium. The natural exposure of tank bromeliads to urea is discussed in the paper.

Relationships between endogenous hormonal levels and axillary bud development of Ananas comosus nodal segments

The effects of some endogenous hormones on the control of axillary bud development of pineapple Ananas comosus (L.) Merr. nodal segments cultivated in vitro were verified. Nodal segments with the apex (control) and decapitated nodal segments were used as explants and were cultured on hormone-free medium. Histological modifications occurring during the developmental process were also observed. Endogenous levels of indole-3-acetic acid (IAA) and four cytokinins (Cks) (isopentenyladenine (iP), isopentenyladenine 9-riboside (iPR), zeatin (Z) and zeatin riboside (ZR)) were quantified by means of high performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). During the first 4 h of culture, a marked reduction in the level of IAA (309%) of the decapitated nodal segment was detected and this coincided with the beginning of cell division in the leaf axil. Cks levels decreased during the first 12 h of culture in both explants. However, this reduction was 150% higher in the control, and occurred mainly due to a decrease in the level of ZR. The reduction in the level of IAA probably favored Cks biosynthesis and/or inhibited its degradation in the nodal tissue of the decapitated segment. After 16 h of culture, there was an increase in the level of IAA probably as a consequence of the establishment of the new auxin-producing shoot apex. After 1 d, a progressive increase in ZR occurred, suggesting that Z-type Cks have an important role in the leaf development of the new plant. Comparatively, auxin/total Cks ratio was always lower in the decapitated nodal segment throughout the process of the axillary bud development.

Effects of Nitrogen Source on Growth Rates and Levels of Endogenous Cytokinins and Chlorophyll in Protocorms of Epidendrum fulgens

Summary Effects of nitrogen source on development, endogenous cytokinin levels and chlorophyll content in protocorms of Epidendrum fulgens , as well as cytokinin content of matura seeds, were studied. A significant increase in growth rates of protocorms was observed with NH 4 NO 3 and urea. Chlorophyll content increased with NH 4 NO 3 and (NH 4 ) 2 SO 4 . In seeds, levels of 2iP exceeded levels of zeatin. In protocorms, NO 3 - as the only nitrogen source increased 2iP activity, while NH 4 + alone increased zeatin activity. This cytokinin was the only one detected in protocorms incubated with urea. The highest endogenous cytokinin levels were found in protocorms supplied with NH 4 NO 3 and (NH 4 ) 2 SO 4 . Correlations among cytokinin activity, development, and chlorophyll content in protocorms were described.