Genomic footprints of repeated evolution of CAM photosynthesis in a Neotropical species radiation.

The adaptive radiation of Bromeliaceae (pineapple family) is one of the most diverse among Neotropical flowering plants. Diversification in this group was facilitated by shifts in several adaptive traits or "key innovations" including the transition from C3 to CAM photosynthesis associated with xeric (heat/drought) adaptation. We used phylogenomic approaches, complemented by differential gene expression (RNA-seq) and targeted metabolite profiling, to address the mechanisms of C3 /CAM evolution in the extremely species-rich bromeliad genus, Tillandsia, and related taxa. Evolutionary analyses of whole-genome sequencing and RNA-seq data suggest that evolution of CAM is associated with coincident changes to different pathways mediating xeric adaptation in this group. At the molecular level, C3 /CAM shifts were accompanied by gene expansion of XAP5 CIRCADIAN TIMEKEEPER homologs, a regulator involved in sugar- and light-dependent regulation of growth and development. Our analyses also support the re-programming of abscisic acid-related gene expression via differential expression of ABF2/ABF3 transcription factor homologs, and adaptive sequence evolution of an ENO2/LOS2 enolase homolog, effectively tying carbohydrate flux to abscisic acid-mediated abiotic stress response. By pinpointing different regulators of overlapping molecular responses, our results suggest plausible mechanistic explanations for the repeated evolution of correlated adaptive traits seen in a textbook example of an adaptive radiation.

Drought effects on resource partition and conservation among leaf ontogenetic stages in epiphytic tank bromeliads.

Studying the response to drought stress of keystone epiphytes such as tank bromeliads is essential to better understand their resistance capacity to future climate change. The objective was to test whether there is any variation in the carbon, water and nutrient status among different leaf ontogenetic stages in a bromeliad rosette subjected to a gradient of drought stress. We used a semi-controlled experiment consisting in a gradient of water shortage in Aechmea aquilega and Lutheria splendens. For each bromeliad and drought treatment, three leaves were collected based on their position in the rosette and several functional traits related to water and nutrient status, and carbon metabolism were measured. We found that water status traits (relative water content, leaf succulence, osmotic and midday water potentials) and carbon metabolism traits (carbon assimilation, maximum quantum yield of photosystem II, chlorophyll and starch contents) decreased with increasing drought stress, while leaf soluble sugars and carbon, nitrogen and phosphorus contents remained unchanged. The different leaf ontogenetic stages showed only marginal variations when subjected to a gradient of drought. Resources were not reallocated between different leaf ontogenetic stages but we found a reallocation of soluble sugars from leaf starch reserves to the root system. Both species were capable of metabolic and physiological adjustments in response to drought. Overall, this study advances our understanding of the resistance of bromeliads faced with increasing drought stress and paves the way for in-depth reflection on their strategies to cope with water shortage.

Involvement of aquaporins on nitrogen-acquisition strategies of juvenile and adult plants of an epiphytic tank-forming bromeliad.

MAIN CONCLUSION: Depending on the N source and plant ontogenetic state, the epiphytic tank-forming bromeliad Vriesea gigantea can modulate aquaporin expression to maximize the absorption of the most available nitrogen source. Epiphytic bromeliads frequently present a structure formed by the overlapping of leaf bases where water and nutrients can be accumulated and absorbed, called tank. However, this structure is not present during the juvenile ontogenetic phase, leading to differences in nutrient acquisition strategies. Recent studies have shown a high capacity of the bromeliad Vriesea gigantea, an epiphytic tank-forming bromeliad, to absorb urea by their leaves. Since plant aquaporins can facilitate the diffusion of urea through the membranes, we cloned three foliar aquaporin genes, VgPIP1;1, VgPIP1;2 and VgTIP2;1 from V. gigantea plants. Through functional studies, we observed that besides water, VgTIP2;1 was capable of transporting urea while VgPIP1;2 may facilitate ammonium/ammonia diffusion. Moreover, aiming at identifying urea and ammonium-induced changes in aquaporin expression in leaves of juvenile and adult-tank plants, we showed that VgPIP1;1 and VgPIP1;2 transcripts were up-regulated in response to either urea or ammonium only in juvenile plants, while VgTIP2;1 was up-regulated in response to urea only in adult-tank plants. Thereby, an ontogenetic shift from juvenile to adult-tank-forming-plant appears to occur with metabolic changes regarding nitrogen metabolism regulation. Investigating urea metabolism in wild species that naturally cope with organic N sources, such as V. gigantea, may provide the knowledge to modify nitrogen use efficiency of crop plants.

Beyond Porosity: 3D Leaf Intercellular Airspace Traits That Impact Mesophyll Conductance.

The leaf intercellular airspace (IAS) is generally considered to have high conductance to CO2 diffusion relative to the liquid phase. While previous studies accounted for leaf-level variation in porosity and mesophyll thickness, they omitted 3D IAS traits that potentially influence IAS conductance (gIAS). Here, we reevaluated the standard equation for gIAS by incorporating tortuosity, lateral path lengthening, and IAS connectivity. We measured and spatially mapped these geometric IAS traits for 19 Bromeliaceae species with Crassulacean acid metabolism (CAM) or C3 photosynthetic pathways using x-ray microcomputed tomography imaging and a novel computational approach. We found substantial variation in porosity (0.04-0.73 m3 m-3), tortuosity (1.09-3.33 m2 m-2), lateral path lengthening (1.12-3.19 m m-1), and IAS connectivity (0.81-0.97 m2 m-2) across all bromeliad leaves. The revised gIAS model predicted significantly lower gIAS in CAM (0.01-0.19 mol m-2 s-1 bar-1) than in C3 (0.41-2.38 mol m-2 s-1 bar-1) plants due to a coordinated decline in these IAS traits. Our reevaluated equation also generally predicted lower gIAS values than the former one. Moreover, we observed high spatial heterogeneity in these IAS geometric traits throughout the mesophyll, especially within CAM leaves. Our data show that IAS traits that better capture the 3D complexity of leaves strongly influence gIAS and that the impact of the IAS on mesophyll conductance should be carefully considered with respect to leaf anatomy. We provide a simple function to estimate tortuosity and lateral path lengthening in the absence of access to imaging tools such as x-ray microcomputed tomography or other novel 3D image-processing techniques.

Does oxidative stress determine the thermal limits of the regeneration niche of Vriesea friburgensis and Alcantarea imperialis (Bromeliaceae) seedlings?

The predicted environmental changes may be detrimental to initial seedling growth, particularly the expected increase in air temperature. We therefore investigated the thermal limits for growth and development of Vriesea friburgensis and Alcantarea imperialis seedlings in the context of oxidative stress. The optimal temperatures for the growth of V. friburgensis and A. imperialis were 25 and 25-30 °C, respectively. Extreme temperatures (15, 30, or 35 °C) induced oxidative stress in both species with significant accumulation of hydrogen peroxide (H2O2) and nitric oxide (NO). Under oxidative stress, the amount of chlorophyll decreased in both species, more prominently in V. friburgensis, while carotenoid levels dramatically increased in A. imperialis. Notably, the activities of superoxide dismutase, catalase (CAT), and ascorbate peroxidase increased in A. imperialis at extreme temperatures. Similar results were observed for V. friburgensis; however, the activity of CAT remained unaffected regardless of temperature. Seedlings of A. imperialis survived at a wider range of temperatures than V. friburgensis, which had greater than 40% mortality when growing at 30 °C. Overall, precise control of cellular H2O2 and NO levels takes place during the establishment of A. imperialis seedlings, allowing the species to cope with relatively high temperatures. The thermal limits of the fundamental niches of the species investigated, determined based on the ability of seedlings to cope with oxidative stress, were distinct from the realized niches of these species. The results suggest that recruitment success is dependent on the ability of seedlings to handle extreme temperature-triggered oxidative stress, which limits the regeneration niche.

Ammonium intensifies CAM photosynthesis and counteracts drought effects by increasing malate transport and antioxidant capacity in Guzmania monostachia.

Guzmania monostachia (Bromeliaceae) is a tropical epiphyte capable of up-regulating crassulacean acid metabolism (CAM) in its photosynthetic tissues in response to changing nutrient and water availability. Previous studies have shown that under drought there is a gradient of increasing CAM expression from the basal (youngest) to the apical (oldest) portion of the leaves, and additionally that nitrogen deficiency can further increase CAM intensity in the leaf apex of this bromeliad. The present study investigated the inter-relationships between nitrogen source (nitrate and/or ammonium) and water deficit in regulating CAM expression in G. monostachia leaves. The highest CAM activity was observed under ammonium nutrition in combination with water deficit. This was associated with enhanced activity of the key enzyme phosphoenolpyruvate carboxylase, elevated rates of ATP- and PPi-dependent proton transport at the vacuolar membrane in the presence of malate, and increased transcript levels of the vacuolar malate channel-encoding gene, ALMT. Water deficit was consistently associated with higher levels of total soluble sugars, which were maximal under ammonium nutrition, as were the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase). Thus, ammonium nutrition, whilst associated with the highest degree of CAM induction in G. monostachia, also mitigates the effects of water deficit by osmotic adjustment and can limit oxidative damage in the leaves of this bromeliad under conditions that may be typical of its epiphytic habitat.

Constitutive Expression of Aechmea fasciata SPL14 (AfSPL14) Accelerates Flowering and Changes the Plant Architecture in Arabidopsis.

Variations in flowering time and plant architecture have a crucial impact on crop biomass and yield, as well as the aesthetic value of ornamental plants. Aechmea fasciata, a member of the Bromeliaceae family, is a bromeliad variety that is commonly cultivated worldwide. Here, we report the characterization of AfSPL14, a squamosa promoter binding protein-like gene in A. fasciata. AfSPL14 was predominantly expressed in the young vegetative organs of adult plants. The expression of AfSPL14 could be upregulated within 1 h by exogenous ethephon treatment. The constitutive expression of AfSPL14 in Arabidopsis thaliana caused early flowering and variations in plant architecture, including smaller rosette leaves and thicker and increased numbers of main inflorescences. Our findings suggest that AfSPL14 may help facilitate the molecular breeding of A. fasciata, other ornamental and edible bromeliads (e.g., pineapple), and even cereal crops.

Potential N2O Emissions from the Tanks of Bromeliads Suggest an Additional Source of N2O in the Neotropics.

We studied the propensity of the tank bromeliad Werauhia gladioliflora to emit the greenhouse gas nitrous oxide (N2O) at current and at increased N deposition levels in the range of predicted future scenarios. Potential production rates and net accumulation of N2O from tank substrate corresponded to N availability. N2O was produced in excess at all N levels due to a low level of N2O reductase activity which agreed well with a low abundance of N2O reducers compared to nitrite reducers. Transcriptional activation, however, indicated that expression of denitrification genes may be enhanced with increasing N supply eventually leading to more efficient N2O turnover with potential for adaptation of denitrifier communities to higher N levels. Our findings indicate that tank bromeliads may constitute a novel source of N2O in Neotropical forest canopies but further studies are required to understand the size and significance of in situ N2O fluxes from tank bromeliads to the environment.

Trade-off between soluble protein production and nutritional storage in Bromeliaceae.

BACKGROUND AND AIMS: Bromeliads are able to occupy some of the most nutrient-poor environments especially because they possess absorptive leaf trichomes, leaves organized in rosettes, distinct photosynthetic pathways [C3, Crassulacean acid metabolism (CAM) or facultative C3-CAM], and may present an epiphytic habit. The more derived features related to these traits are described for the Tillandsioideae subfamily. In this context, the aims of this study were to evaluate how terrestrial predators contribute to the nutrition and performance of bromeliad species, subfamilies and ecophysiological types, whether these species differ in their ecophysiological traits and whether the physiological outcomes are consistent among subfamilies and types (e.g. presence/absence of tank, soil/tank/atmosphere source of nutrients, trichomes/roots access to nutrients). METHODS: Isotopic (15N-enriched predator faeces) and physiological methods (analyses of plant protein, amino acids, growth, leaf mass per area and total N incorporated) in greenhouse experiments were used to investigate the ecophysiological contrasts between Tillandsioideae and Bromelioideae, and among ecophysiological types when a predatory anuran contributes to their nutrition. KEY RESULTS: It was observed that Bromelioideae had higher concentrations of soluble protein and only one species grew more (Ananas bracteatus), while Tillandsioideae showed higher concentrations of total amino acids, asparagine and did not grow. The ecophysiological types that showed similar protein contents also had similar growth. Additionally, an ordination analysis showed that the subfamilies and ecophysiological types were discrepant considering the results of the total nitrogen incorporated from predators, soluble protein and asparagine concentrations, relative growth rate and leaf mass per area. CONCLUSIONS: Bromeliad subfamilies showed a trade-off between two strategies: Tillandsioideae stored nitrogen into amino acids possibly for transamination reactions during nutritional stress and did not grow, whereas Bromelioideae used nitrogen for soluble protein production for immediate utilization, possibly for fast growth. These results highlight that Bromeliaceae evolution may be directly associated with the ability to stock nutrients.

AfAP2-1, An Age-Dependent Gene of Aechmea fasciata, Responds to Exogenous Ethylene Treatment.

The Bromeliaceae family is one of the most morphologically diverse families with a pantropical distribution. To schedule an appropriate flowering time for bromeliads, ethylene is commonly used to initiate flower development in adult plants. However, the mechanism by which ethylene induces flowering in adult bromeliads remains unknown. Here, we identified an APETALA2 (AP2)-like gene, AfAP2-1, in Aechmea fasciata. AfAP2-1 contains two AP2 domains and is a nuclear-localized protein. It functions as a transcriptional activator, and the activation domain is located in the C-terminal region. The expression level of AfAP2-1 is higher in juvenile plants than in adult plants, and the AfAP2-1 transcript level was rapidly and transiently reduced in plants treated with exogenous ethylene. Overexpression of AfAP2-1 in Arabidopsis thaliana results in an extremely delayed flowering phenotype. These results suggested that AfAP2-1 responds to ethylene and is a putative age-dependent flowering regulator in A. fasciata.

Volatile compounds profile of Bromeliaceae flowers.

Volatile compounds play a vital role in the life cycle of plants, possessing antimicrobial and anti-herbivore activities, and with a significant importance in the food, cosmetic, chemical, and pharmaceutical industry. This study aimed to identify the volatile compounds emitted by flowers of thirteen species belonging to four genera of Bromeliaceae, using headspace solid-phase micro-extraction and detection by gas chromatography-mass spectrometry. A total of 71 volatile compounds belonging to nine chemical groups were identified. The compounds identified represented more than 97 % of the major components in Aechmea bicolor, Ae. bromeliifolia, Ae. distichantha, Ae. fasciata, and Vriesea friburgensis. In the Ananas varieties, over 99 % of the components were identified, and around 90 % in V. simplex. V. friburgensis presented the largest diversity of volatiles with 31 compounds, while Alcantarea nahoumii presented only 14. All three Ananas varieties presented the same 28 compounds in relatively similar abundance, which has been confirmed by principal component analysis. Current taxonomy and pollination syndrome studies available can adequately explain the variation in volatile compounds among species.

Temperature modulation of thermal tolerance of a CAM-tank bromeliad and the relationship with acid accumulation in different leaf regions.

Physiological changes that increase plant performance during exposure to high temperatures may play an inverse role during exposure to low temperatures. The objective of this study was to test variations in photosystem II response to heat and cold stress in the leaves of a bromeliad with crassulacean acid metabolism submitted to high or low temperatures. Leaves were maintained under constant temperatures of 10 and 35°C and used to examine possible relationships among physiological responses to high and low temperatures and organic acid accumulation. We also tested if distinct parts of bromeliad leaves show differences in photosynthetic thermotolerance. The samples from leaves maintained at 35°C showed greater heat tolerance values, while those from leaves maintained at 10°C showed lower cold tolerance values. Our results identified a strong negative relationship between the organic acid accumulation and thermal tolerance of bromeliad leaves that largely explained the differences in thermal tolerance among groups. One of these differences occurred among regions of a single leaf, with the base showing critical heat values of up to 8°C higher than the top region, suggesting a possible partitioning of leaf response among its regions. Differences in thermal tolerance were also observed between sampling times, with higher values observed in the morning.

Effects of Bromelia pinguin (Bromeliaceae) on soil ecosystem function and fungal diversity in the lowland forests of Costa Rica.

BACKGROUND: Bromelia pinguin (Bromeliaceae) is a terrestrial bromeliad commonly found under forest stands throughout the Neotropics that has been shown to have antifungal activity in vitro. We have hypothesized that this bromeliad would also have an effect on the fungal populations in nearby soil by decreasing fungaldiversity and negatively impacting C and N cycle-related activities. A previous study in the lowland forest of Costa Rica showed the soil beneath these bromeliads had decreased fungal ITS DNA and differences in C and N levels compared to adjacent primary forest soils. RESULTS: In this follow-up study, we found that the bromeliad soils had lower rates of C and N biomass development and lower phenol oxidase activity (suggesting less decreased fungal decomposition activity). The results of T-RFLP and cloning-based taxonomic analyses showed the community level diversity and abundance of fungal ITS DNA was less in bromeliad soils. Sequence analysis of fungal ITS DNA clones showed marked differences in fungal community structure between habitats of Basidiomycota (Tremellales, Agricales, Thelephorales), Ascomycota (Helotiales), and Zycomycota populations. CONCLUSIONS: The data show there to be differences in the soil nutrient dynamics and fungal community structure and activity associated with these bromeliads, as compared to the adjacent primary forest. This suggests the possibility that the anti-fungal activity of the bromeliad extends into the soil. The bromeliad-dense regions of these primary forest habitats provide a unique natural micro-habitat within the forests and the opportunity to better identify the role of fungal communities in the C and N cycles in tropical soils.

Drought memory in Acanthostachys strobilacea, a CAM epiphytic bromeliad.

Stress memory is the development of altered responses to stress due to previous exposure, which might result in increased tolerance. Biochemical and physiological parameters shown to be positively affected by stress memory include those of the antioxidant and nitrosative metabolism, photosynthetic pigments and osmolyte content. Epiphytic bromeliads likely present stress memory since they experience frequent droughts in the canopies. Thus, we aimed to evaluate if the epiphytic bromeliad Acanthostachys strobilacea (Schult. & Schult.f.) Klotzsch shows improved metabolic stress defence responses to a second drought and rewatering cycle compared to a single exposure. In a controlled environment chamber, 90-day-old plants were exposed to one or two drought-rewatering cycles of 14 days without irrigation and 5 days of rewatering each. Sampling occurred after the final drought and rewatering periods for one or two cycles treatments. The free amino acid, chlorophyll, and carotenoid levels and S-nitrosoglutathione reductase (GSNOR) activity were higher at the second drought than at the first exposure. The rise in nocturnal acidification (indicative of increased CAM activity) caused by the initial drought persisted through the second drought-rewatering cycle, implying a lasting memory effect on CAM activity. Furthermore, the second recovery did not induce glutathione accumulation, as in the first rewatering event, suggesting the pre-exposure to drought reduced this thiol's demand during a later recovery. Our results evidence metabolic changes related to drought stress memory in A. strobilacea, supporting this mechanism might be involved in the tolerance of epiphytic bromeliads to intermittent droughts.

Nutrient limitation restricts growth and reproductive output in a tropical montane cloud forest bromeliad: findings from a long-term forest fertilization experiment.

From studies in seasonal lowland tropical forests, bromeliad epiphytes appear to be limited mainly by water, and to a lesser extent by nutrient supply, especially phosphorous. Less is understood about the mineral nutrition of tropical montane cloud forest (TMCF) epiphytes, even though their highest diversity is in this habitat. Nutrient limitation is known to be a key factor restricting forest productivity in TMCF, and if epiphytes are nutritionally linked to their host trees, as has been suggested, we would expect that they are also nutrient limited. We studied the effect of a higher nutrient input on reproduction and growth of the tank bromeliad Werauhia sintenisii in experimental plots located in a TMCF in Puerto Rico, where all macro- and micronutrients had been added quarterly starting in 1989 and continuing throughout the duration of this study. We found that bromeliads growing in fertilized plots were receiving litterfall with higher concentrations of N, P, and Zn and had higher concentrations of P, Zn, Fe, Al, and Na in their vegetative body. The N:P ratios found (fertilized = 27.5 and non-fertilized = 33.8) suggest that W. sintenisii may also be phosphorous limited as are lowland epiphytes. Fertilized plants had slightly longer inflorescences, and more flowers per inflorescence, than non-fertilized plants, but their flowers produced nectar in similar concentrations and quantities. Fertilized plants produced more seeds per fruit and per plant. Frequency of flowering in two consecutive years was higher for fertilized plants than for controls, suggesting that fertilized plants overcome the cost of reproduction more readily than non-fertilized plants. These results provide evidence that TMCF epiphytic bromeliads are nutrient limited like their lowland counterparts.

Anatomical and physiological responses of Aechmea blanchetiana (Bromeliaceae) induced by silicon and sodium chloride stress during in vitro culture.

Salt stress is one of the most severe abiotic stresses affecting plant growth and development. The application of silicon (Si) is an alternative that can increase the tolerance of plants to various types of biotic and abiotic stresses. The objective was to evaluate salt stress's effect in vitro and Si's mitigation potential on Aechmea blanchetiana plants. For this purpose, plants already established in vitro were transferred to a culture medium with 0 or 14 µM of Si (CaSiO3). After growth for 30 days, a stationary liquid medium containing different concentrations of NaCl (0, 100, 200, or 300 µM) was added to the flasks. Anatomical and physiological analyses were performed after growth for 45 days. The plants cultivated with excess NaCl presented reduced root diameter and effective photochemical quantum yield of photosystem II (PSII) (ΦPSII) and increased non-photochemical dissipation of fluorescence (qN). Plants that grew with the presence of Si also had greater content of photosynthetic pigments and activity of the enzymes of the antioxidant system, as well as higher values of maximum quantum yield of PSII (FV/FM), photochemical dissipation coefficient of fluorescence (qP) and fresh weight bioaccumulation of roots and shoots. The anatomical, physiological and biochemical responses, and growth induced by Si mitigated the effect of salt stress on the A. blanchetiana plants cultivated in vitro, which can be partly explained by the tolerance of this species to grow in sandbank (Restinga) areas.

The genome of Aechmea fasciata provides insights into the evolution of tank epiphytic habits and ethylene-induced flowering.

Aechmea fasciata is one of the most popular bromeliads and bears a water-impounding tank with a vase-like rosette. The tank habit is a key innovation that has promoted diversity among bromeliads. To reveal the genomic basis of tank habit formation and ethylene-induced flowering, we sequenced the genome of A. fasciata and assembled 352 Mb of sequences into 24 chromosomes. Comparative genomic analysis showed that the chromosomes experienced at least two fissions and two fusions from the ancestral genome of A. fasciata and Ananas comosus. The gibberellin receptor gene GID1C-like was duplicated by a segmental duplication event. This duplication may affect GA signalling and promote rosette expansion, which may permit water-impounding tank formation. During ethylene-induced flowering, AfFTL2 expression is induced and targets the EIN3 binding site 'ATGTAC' by AfEIL1-like. The data provided here will serve as an important resource for studying the evolution and mechanisms underlying flowering time regulation in bromeliads.

Are vascular epiphytes nitrogen or phosphorus limited? A study of plant (15) N fractionation and foliar N : P stoichiometry with the tank bromeliad Vriesea sanguinolenta.

Although there is unambiguous evidence for vascular epiphytic plants to be limited by insufficient water and nutrient supply under natural conditions, it is an open debate whether they are primarily phosphorus (P) or nitrogen (N) limited. Plant (15) N fractionation and foliar N : P stoichiometry of a tank epiphyte (Vriesea sanguinolenta), and its response to combined N-P fertilization, were studied under semi-natural conditions over 334 d to clarify the type of nutrient limitation. Plants collected in the field and experimental plants with limited nutrient supply showed significant plant (15) N fractionation (mean 5‰) and plant N : P ratios of c. 13.5. Higher relative growth rates and declines in plant (15) N fractionation (0.5‰) and in foliar N : P ratios to 8.5 in the high N-P treatment indicated that these epiphytes were P limited in situ. The critical foliar N : P ratio was 10.4, as derived from the breakpoint in the relationship between plant (15) N fractionation and foliar N : P. We interpret the widespread (15) N depletion of vascular epiphytes relative to their host trees as deriving from (15) N fractionation of epiphytes as a result of P limitation. High foliar N : P ratios (> 12) corroborate widespread P limitation (or co-limitation by N and P) of epiphytic bromeliads and, possibly, other epiphyte species.

Crassulacean acid metabolism under severe light limitation: a matter of plasticity in the shadows?

Despite the increased energetic costs of CAM compared with C(3) photosynthesis, it is hypothesized that the inherent photosynthetic plasticity of CAM allows successful acclimation to light-limiting conditions. The present work sought to determine if CAM presented any constraints to short and longer term acclimation to light limitation and to establish if and how metabolic and photosynthetic plasticity in the deployment of the four phases of CAM might facilitate acclimation to conditions of deep shade. Measurements of leaf gas exchange, organic acids, starch and soluble sugar (glucose, fructose, and sucrose) contents were made in the leaves of the constitutive CAM bromeliad Aechmea 'Maya' over a three month period under severe light limitation. A. 'Maya' was not particularly tolerant of severe light limitation in the short term. A complete absence of net CO(2) uptake and fluctuations in key metabolites (i.e. malate, starch or soluble sugars) indicated a dampened metabolism whilst cell death in the most photosynthetically active leaves was attributed to an over-acidification of the cytoplasm. However, in the longer term, plasticity in the use of the different phases of gas exchange and different storage carbohydrate pools, i.e. a switch from starch to sucrose as the major carbohydrate source, ensured a positive carbon balance for this CAM species under extremely low levels of irradiance. As such, co-ordinated plasticity in the use of C(3) and C(4) carboxylases and different carbohydrate pools together with an increase in the abundance of light-harvesting complexes, appear to underpin the adaptive radiation of the energetically costly CAM pathway within light-limiting environments such as wet cloud forests and shaded understoreys of tropical forests.

Correlation between pollen aperture pattern and callose deposition in late tetrad stage in three species producing atypical pollen grains.

PREMISE OF THE STUDY: Pollen grains of flowering plants display a fascinating diversity of forms, in spite of their minute size. The observed diversity is determined by the developmental mechanisms implicated in the establishment of pollen morphological features. Pollen grains are generally surrounded by an extremely resistant wall interrupted in places by apertures that play a key role in reproduction, being the places at which pollen tube growth is initiated. Aperture shape, number, and position are determined during microsporogenesis (male meiosis), the earliest step in pollen ontogeny. We investigate in detail the unfolding of microsporogenesis in three species that present uncommon aperture pattern (i.e., disulculate in Calycanthus floridus [Calycanthaceae, magnoliids], tetraporate in Hohenbergia stellata [Bromeliaceae, monocots], and monoporate in Typha latifolia [Typhaceae, monocots]). METHODS: We performed a comparative analysis of microsporogenesis and aperture distribution within tetrads in these species with contrasting aperture arrangements. This was done using aniline blue coloration and UV light microscope observations. KEYS RESULTS: We show that aperture localization and features of callose deposition on intersporal walls produced during cytokinesis coincide in all three species examined. Such a correlation suggests that patterns of callose deposition are strongly involved in determining aperture localization. CONCLUSION: In flowering plants, patterns of male meiosis and especially callose deposition following meiosis may be implicated in the diversity of pollen aperture patterns.