Small RNA sequencing revealed various microRNAs involved in ethylene-triggered flowering process in Aechmea fasciata.

Ethylene-triggered flowering is a common phenomenon in plants of the family Bromeliaceae, but its molecular mechanism remains unclear. As a classical group of small RNAs, microRNAs play an essential role in the regulation of flowering. In this study, we found that various miRNAs participate in the ethylene-triggered flowering process in Aechmea fasciata via small RNA sequencing using juvenile and adult plants treated with ethylene for 24 hours. Finally, 63 known miRNAs, 52 novel miRNAs and 1721 target genes were identified or predicted. Expression changes of specific miRNAs were validated by qRT-PCR and northern blotting. Some predicted targets, including SPL, GAMYB and ARF, were verified in RLM-RACE experiments. Gene Ontology (GO) and KEGG analysis showed that numerous developmental and RNA-related processes were enriched. Integrated analysis of the transcriptomic data with small RNA sequencing revealed that numerous miRNAs and targets involved in ethylene-triggered flowering in A. fasciata. Our study is helpful for illuminating the molecular basis of the ethylene-triggered flowering phenomenon in Bromeliaceae.

Morpho-physiological changes in Billbergia zebrina due to the use of silicates in vitro.

The use of silicon in Billbergia zebrina cultivation in vitro is an alternative for optimizing micropropagation of this important ornamental plant species. The objective of the present study was to evaluate the growth and anatomical and physiological alterations in Billbergia zebrina (Herbert) Lindley plants as a function of different sources and concentrations of silicon during in vitro cultivation and acclimatization. The experimental design was completely randomized, with a double factorial arrangement and an additional control treatment (2 x 3 + 1). The first factor was relative to calcium silicate and sodium silicate added to the Murashige & Skoog culture medium; the second factor was related to its concentrations, 0.5, 1.0, and 2.0 mg L-1. After 100 days, their growth, anatomical characteristics, level of silicon and chlorophyll content were evaluated. Growth characteristics were assessed after 60 days of acclimatization period. Plants absorbed more sodium silicate than calcium silicate. This source also stressed the plants impairing their growth, but the highest silicon absorption at 1 mg L-1 attenuated the stressful conditions. The supplementation of the culture medium with calcium silicate led to improved growth, anatomical, and physiological characteristics, which benefited the development of more resistant seedlings with better performance during acclimatization.

Viabilidad y germinación de semillas de Hechtiaperotensis (Bromeliaceae) / Viability and germination of Hechtia perotensis (Bromeliaceae) seed

ResumenPoblaciones endémicas de Hechtiaperotensis se han descrito en Puebla y Veracruz, México. La recolección de semillas de buena calidad permite su uso en conservación, investigación y restauración ecológica. Algunos compuestos para evaluar la calidad de las semillas silvestres y endémicas, como nitrato de potasio (KNO3) y ácido giberílico (AG3), se utilizan para incrementar la germinación de las semillas y disminuir la latencia. La prueba de cloruro de trifeniltetrazolio (tetrazolio) se correlaciona con la viabilidad de las semillas porque se basa en la actividad de las deshidrogenasas de tejidos vivos que catalizan la respiración mitocondrial. El objetivo de este estudio fue conocer el tamaño y el peso de las cápsulas y las semillas y la germinación y viabilidad de las semillas de H. perotensis recolectadas en Veracruz en 2012 y 2015. Las hipótesis fueron (1) que la germinación y la viabilidad de las semillas son independientes del año de recolecta, (2) que hay una concentración adecuada de tetrazolio para identificar la viabilidad de las semillas y (3) que el pretratamiento con KNO3 o AG3 incrementa la germinación de las semillas. La germinación se evaluó con un diseño completamente al azar con tres tratamientos (testigo y promotores de la germinación KNO3 0.2 % y AG3 500 mg/L), cuatro tratamientos para la prueba de viabilidad (testigo, 0.2, 0.5 y 1.0 % de tetrazolio) y seis repeticiones de cada tratamiento. La germinación se evaluó en 100 semillas y la viabilidad en 25. Los resultados entre y dentro de años se analizaron con ANDEVA y prueba de comparación múltiple de medias de Tukey. La proporción de semillas no germinadas se cuantificó junto con el número de plántulas normales y anormales, semillas con embrión viable o sin él, y con viabilidad baja o sin viabilidad. En promedio la muestra recolectada en 2012 tuvo 36 % de semillas con embrión viable, 7 con viabilidad baja, 24 % no viables y 33 % sin embrión. Este resultado fue significativamente diferente al de 2015, que presentó 87 % de embriones viables, 10 % con viabilidad baja, 0 % no viables y 3 % sin embrión. La germinación también fue significativamente diferente entre los años (22 y 92 %). Los tratamientos pregerminativos no modificaron la germinación. La germinación y la viabilidad de las semillas de H. perotensis varían significativamente entre los años de recolecta.

Abstract:Endemic populations of Hechtiaperotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H.perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection. Rev. Biol. Trop. 65 (1): 153-165. Epub 2017 March 01.

[Viability and germination of Hechtia perotensis (Bromeliaceae) seed]. / Viabilidad y germinación de semillas de Hechtia perotensis (Bromeliaceae).

Endemic populations of Hechtia perotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H. perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection.

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.

Spatial division of phosphoenolpyruvate carboxylase and nitrate reductase activity and its regulation by cytokinins in CAM-induced leaves of Guzmania monostachia (Bromeliaceae).

Crassulacean acid metabolism (CAM) is a physiological adaptation of plants that live in stress environment conditions. A good model of CAM modulation is the epiphytic bromeliad, Guzmania monostachia, which switches between two photosynthetic pathways (C3-CAM) in response to different environmental conditions, such as light stress and water availability. Along the leaf length a gradient of acidity can be observed when G. monostachia plants are kept under water deficiency. Previous studies showed that the apical portions of the leaves present higher expression of CAM, while the basal regions exhibit lower expression of this photosynthetic pathway. The present study has demonstrated that it is possible to induce the CAM pathway in detached leaves of G. monostachia kept under water deficit for 7 d. Also, it was evaluated whether CAM expression can be modulated in detached leaves of Guzmania and whether some spatial separation between NO3(-) reduction and CO2 fixation occurs in basal and apical portions of the leaf. In addition, we analyzed the involvement of endogenous cytokinins (free and ribosylated forms) as possible signal modulating both NO3(-) reduction and CO2 fixation along the leaf blade of this bromeliad. Besides demonstrating a clear spatial and functional separation of carbon and nitrogen metabolism along G. monostachia leaves, the results obtained also indicated a probable negative correlation between endogenous free cytokinins - zeatin (Z) and isopentenyladenine (iP) - concentration and PEPC activity in the apical portions of G. monostachia leaves kept under water deficit. On the other hand, a possible positive correlation between endogenous Z and iP levels and NR activity in basal portions of drought-exposed and control leaves was verified. Together with the observations presented above, results obtained with exogenous cytokinins treatments, strongly suggest that free cytokinins might act as a stimulatory signal involved in NR activity regulation and as a negative regulator of PEPC activity in CAM-induced leaves of G. monostachia during a diel cycle.

Abscisic acid and nitric oxide signaling in two different portions of detached leaves of Guzmania monostachia with CAM up-regulated by drought.

Guzmania monostachia is an epiphyte tank bromeliad capable of up-regulating crassulacean acid metabolism (CAM) in response to several environmental stimuli, including drought and light stress. In other plant species, abscisic acid (ABA) and nitric oxide (NO) seem to be involved in CAM induction. Because the leaves of tank bromeliads perform different functions along their length, this study attempted to investigate whether ABA and NO are involved in regulation of CAM expression in this species by quantifying these compounds in apical and basal portions of the leaf, and whether there would be differences in this event for each leaf portion. Detached leaves exposed to a 30% polyethylene glycol solution showed a significant upregulation of CAM on the seventh day of treatment only in the apical portion, as indicated by nocturnal acid accumulation and phosphoenolpyruvate carboxylase (PEPC) activity. On the three days prior to CAM induction, ABA, NO and H2O2 were quantified. The amounts of ABA were higher in PEG-exposed leaves, along their entire length. NO, however, was higher only in the apical portion, precisely where CAM was up-regulated. H2O2 was higher only in the basal portion of PEG-exposed leaves. Our results suggest that ABA might be a systemic signal to drought, occurring in the entire leaf. NO and H2O2, however, may be signals restricted only to the apical or basal portions, respectively.

Physiological responses of root-less epiphytic plants to acid rain.

Selected physiological responses of Tillandsia albida (Bromeliaceae) and two lichens (Hypogymnia physodes and Xanthoria parietina) exposed to simulated acid rain (AR) over 3 months were studied. Pigments were depressed in all species being affected the most in Tillandsia. Amounts of hydrogen peroxide and superoxide were elevated and soluble proteins decreased only in AR-exposed Hypogymnia. Free amino acids were slightly affected among species and only glutamate sharply decreased in AR-exposed Xanthoria. Slight increase in soluble phenols but decrease in flavonoids in almost all species suggests that the latter are not essential for tolerance to AR. Almost all phenolic acids in Tillandsia leaves decreased in response to AR and activities of selected enzymes (phenylalanine ammonia-lyase, polyphenol oxidase, ascorbate- and guaiacol-peroxidase) were enhanced by AR. In lichens, considerable increase in metabolites (physodalic acid, atranorin and parietin) in response to AR was found but amount of ergosterol was unchanged. Macronutrients (K, Ca, Mg) decreased more pronouncedly in comparison with micronutrients in all species. Xanthoria showed higher tolerance in comparison with Hypogymnia, suggesting that could be useful for long-term biomonitoring.

Highly efficient uptake of phosphorus in epiphytic bromeliads.

BACKGROUND AND AIMS: Vascular epiphytes which can be abundant in tree crowns of tropical forests have to cope with low and highly intermittent water and nutrient supply from rainwater, throughfall and stem flow. Phosphorus rather than nitrogen has been suggested as the most limiting nutrient element, but, unlike nitrogen, this element has received little attention in physiological studies. This motivated the present report, in which phosphate uptake kinetics by leaves and roots, the subsequent distribution within plants and the metabolic fate of phosphate were studied as a step towards an improved understanding of physiological adaptations to the conditions of tree canopies. METHODS: Radioactively labelled [(32)P]phosphate was used to study uptake kinetics and plant distribution of phosphorus absorbed from bromeliad tanks. The metabolism of low molecular phosphorus metabolites was analysed by thin-layer chromatography followed by autoradiography. KEY RESULTS: Uptake of phosphate from tanks is an ATP-dependent process. The kinetics of phosphorus uptake suggest that epiphytes possess effective phosphate transporters. The K(m) value of 1.05 microm determined for leaves of the bromeliad Aechmea fasciata is comparable with values obtained for the high affinity phosphate transporters in roots of terrestrial plants. In this species, young leaves are the main sink for phosphate absorbed from tank water. Within these leaves, phosphate is then allocated from the basal uptake zone into distal sections of the leaves. More than 80 % of the phosphate incorporated into leaves is not used in metabolism but stored as phytin. CONCLUSIONS: Tank epiphytes are adapted to low and intermittent nutrient supply by different mechanisms. They possess an effective mechanism to take up phosphate, minimizing dilution and loss of phosphorus captured in the tank. Available phosphorus is taken up from the tank solution almost quantitatively, and the surplus not needed for current metabolism is accumulated in reserves, i.e. plants show luxury consumption. Young, developing leaves are preferentially supplied with this nutrient element. Taken together, these features allow epiphytes the efficient use of scarce and variable nutrient supplies.