Acessibilidade / Reportar erro

Nitrogen assimilation in the bromeliad Ananas comosus var. ananassoides (Baker) Coppens & F.Leal grown in vitro with different sources of inorganic nitrogen

Assimilação de nitrogênio na bromélia Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultivada in vitro com diferentes fontes de nitrogênio inorgânico

ABSTRACT

Ananas comosus var. ananassoides (Baker) Coppens & F.Leal is a native ornamental bromeliad of the endangered biome Cerrado. Therefore, approaches aimed at the preservation of this species, such as in vitro cultivation and micropropagation are needed. Nitrogen (N) is absorbed by plants, mainly as NO3- and/or NH4+, and assimilated into amino acids. The aim of this work was to evaluate the N assimilation in this bromeliad. Plants were grown in vitro for seven months in modified MS medium with 15, 30, 60, and 90 mM of N as NO3-, NH4+ or NH4NO3, and then transferred to ex vitro conditions for acclimatization. Plants grown with NH4+ had high mortality. During acclimatization plants cultivated with 30, 60, and 90 mM of N as NH4NO3 showed higher biomass. With regard to N assimilation, GS and NR showed the highest activity in plants cultivated with NH4NO3, whereas plants cultivated with NH4+ had the highest GDH activity. Consequently, in vitro and ex vitro cultivation of this species with 60 mM N as NH4NO3 is recommended.

Keywords:
acclimatization; Bromeliaceae; in vitro culture; micropropagation; pineapple

RESUMO

Ananas comosus var. ananassoides (Baker) Coppens & F.Leal é uma bromélia ornamental nativa do Cerrado, um bioma ameaçado de extinção, sendo necessários estudos para sua preservação, como o uso de micropropagação. O nutriente nitrogênio (N) é absorvido pelas plantas, principalmente, como NO3- e/ou NH4+ e é assimilado em aminoácidos. O objetivo deste trabalho foi avaliar a assimilação de N nesta bromélia. As plantas foram cultivadas in vitro por sete meses em meio MS modificado com 15, 30, 60 e 90 mM de N como NO3-, NH4+ ou NH4NO3, depois as plantas foram aclimatizadas ex vitro. Plantas cultivadas com NH4+ tiveram alta mortalidade. Durante a aclimatização, plantas cultivadas com 30, 60 e 90 mM de N como NH4NO3 apresentaram maior biomassa. Em relação à assimilação do N, GS e NR apresentaram as maiores atividades em plantas cultivadas com NH4NO3, e as adubadas apenas com NH4+ tiveram alta atividade da GDH. Consequentemente, recomenda-se cultivar esta espécie in vitro e ex vitro com 60 mM de N como NH4NO3.

Palavras-chave:
abacaxi; aclimatização; Bromeliaceae; cultivo in vitro; micropropagação

Introduction

Ananas comosus var. ananassoides (Baker) Coppens & F.Leal, also known as field pineapple (Silva 2006, Crestani et al. 2010), is a terricolous bromeliad, native to Cerrado (Proença & Sajo 2007Proença, S.L. & Sajo, M.G. 2007. Anatomia foliar de bromélias ocorrentes em áreas de cerrado do Estado de São Paulo, Brasil. Acta Botanica Brasilica 21(3): 657-673.). This species, like other bromeliads from this biome, has ornamental and landscape potential (Zucchi et al. 2019) but is still little explored economically (Silva 2006, Souza et al. 2007, Crestani et al. 2010). It is characterized by the presence of leaves covered with trichomes, well-developed absorbent roots as it is terricolous, a globose inflorescence bearing bracts with complete and small flowers, and an 8-10 cm and creamy to pink-yellow infructescence located in the apical part of the stem which can measure up to 80 cm (Paula & Silva 2004, Souza et al. 2007). This ornamental bromeliad has been traditionally cultivated for export as a cut flower (Souza et al. 2012). Besides its economic importance as an ornamental plant, this species is native to a biome that belongs to the 34 global biodiversity hotspots, which are areas of high biodiversity and endemism (Myer et al. 2000Myers, N., Mittermeier, R.A., Mittermeier C.G., Fonseca, G.A.B.da & Kent, J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858., Orme et al. 2005Orme, C.D.R., Davies, R.G., Burgess, M., Eigenbrod, F., Pickup, N., Olson, V.A., Webster, A.J., Ding, T-S., Rasmussen, P.C., Ridgely, R.S., Statterfield, A.J., Bennett, P.M., Blackburn, T.M., Gaston, K.J. & Owens, I.P.F. 2005. Global hotspots of species richness are not congruent with endemism or threat. Nature 436(7053): 1016-1019.). Therefore, conservation measures of their species are important and necessary. One way of contributing to the biome preservation, could be through the development of micropropagation studies of plants.

A key factor for in vitro cultivation is the selection of a suitable culture medium for the growth and development of the species of interest (Bunn et al. 2011Bunn, E., Turner, S.R. & Dixon, K. 2011. Biotechnology for saving rare and threatened florain a biodiversity hotspot. In Vitro Cellular & Developmental Biology Plant 47(1): 188-200.). Most culture mediums are basic formulations such as the MS medium formulated by Murashige and Skoog (1962)Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497., which is the most commonly used for in vitro culture (Aranda-Peres et al. 2009Aranda-Peres, A.N., Peres, L.E.P., Higashi, E.N. & Martinelli, A.P. 2009. Adjustment of mineral elements in the culture medium for the micropropagation of three Vriesea bromeliads from the brazilian atlantic forest: the importance of calcium. Hortscience 44: 106-112., Werner et al. 2010Werner, E.T., Milanez, C.R.D., Mengarda, L.H.G., Vendrame, W.A. & Cuzzuol, G.R.F. 2010. Meios de cultura, reguladores de crescimento e fontes de nitrogênio na regulação da calogênese do pau-brasil (Caesalpinia echinata Lam.). Acta Botanica Brasílica 24(4): 1046-1051., Lima et al. 2020Lima, A.P.P.S., Brito, A.L. & Santana, J.R.F. 2020. Micropropagation of Chapada Diamantina ornamental bromeliad. Ciência Rural 50(2): e20180922.). Among the nutrients, that compose the culture medium, the nitrogen (N) is required in a major amount by most plants (Marschner & Marschner 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Buchanan et al. 2015Buchanan, B.B., Gruissem, W. & Jones, R.L. 2015. Biochemistry and Molecular Biology of Plants. 2nd ed. John Wiley & Sons, West Sussex/UK., Esteban et al. 2016Esteban, R., Ariz, I., Cruz, C. & Moran, J.F. 2016. Review: Mechanisms of ammonium toxicity and the quest for tolerance. Plant Science 248: 92-101.).

Ammonium (NH4+) and nitrate (NO3-) ions are the most available N sources in the soil solution to be absorbed by plant roots (Epstein & Bloom 2006Epstein, E. & Bloom, A.J. 2006. Nutrição mineral de plantas: princípios e perpectivas. 2 ed. Trad. Editora Planta, Londrina., Marschner & Marschner 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Esteban et al. 2016Esteban, R., Ariz, I., Cruz, C. & Moran, J.F. 2016. Review: Mechanisms of ammonium toxicity and the quest for tolerance. Plant Science 248: 92-101., Hachiya & Sakakibara, 2017Hachiya, T. & Sakakibara, H. 2017. Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. Journal of Experimental Botany 68(10): 2501-2512.). The roots absorb these two ions by high- (HATS) and low- (LATS) affinity transporters that are specific for each N form (Miller et al. 2007Miller, A.J., Fan, X., Orsel, M., Smith, S.J. & Wells, D.M. 2007. Nitrate transport and signaling Journal of Experimental Botany 58(9): 2297-2306., Kraiser et al. 2011Kraiser, T., Gras, D.E., Gutierrez, A.G., Gonzalez, B. & Gutierrez, R.A. 2011. A holistic view of nitrogen acquisition in plants. Journal of Experimental Botany 62: 1455-1466., Xu et al. 2012Xu, G., Fan, X. & Miller, A.J. 2012. Plant nitrogen assimilation and use efficiency. Annual Review of Plant Biology 63: 153-182.). Once absorbed, the N can be either readily assimilated in the roots or stored in the vacuoles, and also transported via the xylem to be assimilated in the shoot (Rennenberg et al. 2010Rennenberg, H., Wildhagen, H. & Ehlting, B. 2010. Nitrogen nutrition of pop- lar trees. Plant Biology 12:275-291., Braun et al. 2013Braun, H., Coelho, S.F., Silva, M.C.C., Fontes, P.C.R., Cecon, P.R. & Busato, C. 2013. Absorção, metabolismo e diagnóstico do estado de nitrogênio em plantas de batata. Revista de Ciências Agrárias 56(3): 185-195.). To enter in the assimilation pathway, the N in the form of NO3- is reduced to nitrite (NO2-) in the cell cytosol by the nitrate reductase (NR), and then transported inside the plastid, where it is reduced to NH4+ by the nitrite reductase (NiR) (Marschner & Marschner 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Taiz & Zeiger 2013Taiz, L & Zeiger, E. 2013. Fisiologia Vegetal. 5 ed. Artmed, Porto Alegre.). The NH4+ originated is assimilated into glutamine (GLN) by the glutamine synthetase (GS), and then transformed into glutamate (GLU) by the glutamine oxoglutarate aminotransferase (GOGAT) (Marschner & Marschner 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Taiz & Zeiger 2013Taiz, L & Zeiger, E. 2013. Fisiologia Vegetal. 5 ed. Artmed, Porto Alegre., Buchanan et al. 2015Buchanan, B.B., Gruissem, W. & Jones, R.L. 2015. Biochemistry and Molecular Biology of Plants. 2nd ed. John Wiley & Sons, West Sussex/UK., Liu & Wirén 2019Liu, Y. & Wirén, N.von. 2017. Ammonium as a signal for physiological and morphological responses in plants. Journal of Experimental Botany 68: 2581-2592.). In addition, the glutamate dehydrogenase (GDH), which normally catalyzes the reversible deamination of glutamate to 2-oxoglutarate, it might be collaborating in the assimilation of N into GLU under certain conditions (Skopelitis et al. 2006Skopelitis, D.S., Paranychianakis, N.V., Paschalidis, K.A., Plia- konis, E.D., Delis, I.D., Yakoumakis, D.I., Kouvarakis, A., Papa- dakis, A.K., Stephanou, E.G. e Roubelakis-Angelakisa, K.A. 2006. Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 18: 2767-2781., Andrews et al. 2013Andrews, M., Raven, J.A. & Lea, P.J. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Annals of Applied Biology 163(2): 1-26., Luo et al. 2015Luo, J., Zhou, J., Li, H., Shi, W., Polle, A., Lu, M., Sun, X. & Luo, Z.B. 2015. Global poplar root and leaf transcriptomes reveal links between growth and stress responses under nitrogen starvation and excess. Tree Physiology 35: 1283-1302.). Subsequently, GLU and GLN will be used for the biosynthesis of other organic N forms essential for cellular metabolism, promoting plant growth and development (Marschner & Marschner 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Andrews et al. 2013)Andrews, M., Raven, J.A. & Lea, P.J. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Annals of Applied Biology 163(2): 1-26.. Although the process of N assimilation in plants is well-known, much information is still lacking to better understand this metabolic activity (Krapp 2015Krapp, A. 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Current Opinion in Plant Biology 25: 115-122.).

After in vitro production, plants need to be acclimatized before their transfer to the field (Grattapaglia & Machado 1998Grattapaglia, D. & Machado, M.A. 1998. Micropropagação. In: A.C. Torres, L.S. Caldas & J.A. Buso (eds.). Cultura de tecidos e transformação genética de plantas. 1 ed. Embrapa-CNPH, Brasília, pp.183-260., Berilli et al. 2011Berilli, S.S., Carvalho, A.J.C., Freitas, S.J., Faria, D.C. & Marinho., C.S. 2011. Avaliação do desenvolvimento de diferentes tamanhos de mudas micropropagadas de abacaxizeiro, após aclimatação. Revista Brasileira de Fruticultura 33(1): 208-214.). Transfer to ex vitro conditions implies morphological and physiological changes that confer plants resistance to external environmental factors, such as to increased transpiration and luminous flux, temperature fluctuations, interaction with microorganisms, among others (Calvete et al. 2002Calvete, E.O., Azevedo, M., Bordignon, M.H. & Suzin, M. 2002. Análises anatômicas e da biomassa em plantas de morangueiro cultivadas in vitro e ex vitro. Horticultura Brasileira 20(4): 649-653., Hartmann et al. 2002Hartmann, H.T., Kester, D.E., Davis Júnior, F.T. & Geneve, R.L. 2002. Plant propagation: principles and practices. Englewood Clipps, New York., Albert 2004Albert, L.H.B. 2004. Aspectos morfoanatômicos de mudas de abacaxizeiro ‘Smooth Cayenne’ micropropagadas. Tese Doutorado, Universidade Federal de Lavras, Lavras.). Sudden changes in these conditions might be limiting for the propagation of some species (Grattapaglia & Machado 1998Grattapaglia, D. & Machado, M.A. 1998. Micropropagação. In: A.C. Torres, L.S. Caldas & J.A. Buso (eds.). Cultura de tecidos e transformação genética de plantas. 1 ed. Embrapa-CNPH, Brasília, pp.183-260., Bregonci et al. 2008Bregonci, S.I.; Reis, E.S., Almeida, G.D., Brum, V.J. & Zucoloto, M. 2008. Avaliação do crescimento foliar e radicular de mudas micropropagadas do abacaxizeiro cv. gold em aclimatação. Idesia 26(3): 87-96., Berilli et al. 2011Berilli, S.S., Carvalho, A.J.C., Freitas, S.J., Faria, D.C. & Marinho., C.S. 2011. Avaliação do desenvolvimento de diferentes tamanhos de mudas micropropagadas de abacaxizeiro, após aclimatação. Revista Brasileira de Fruticultura 33(1): 208-214.), and therefore, highlight the importance of acclimatization studies.

Some studies showed that the joint use of NH4+ and NO3- promotes plant growth (Hachiya & Sakakibara, 2017Hachiya, T. & Sakakibara, H. 2017. Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. Journal of Experimental Botany 68(10): 2501-2512.), including some bromeliad species, such as Ananas comosus (L.) Merr., Vriesea fosteriana L.B.Sm., Tillandsia pohliana Mez, and Nidularium minutum Mez (Endres & Mercier 2001Endres, L. & Mercier, H. 2001. Ammonium and urea as nitrogen sources for bromeliads. Journal of Plant Physiology 158: 205-212., Nievola et al. 2001Nievola, C.C., Mercier, H. & Majerowisz, N. 2001. Levels of nitrogen assimilation in bromeliads with different growth habits. Journal of Plant Nutrition 24(9): 1387-1387., Tamaki & Mercier 2007Tamaki, V., Mercier, H. & Nievola, C.C. 2007. Cultivo in vitro de clones de Ananas comosus (L.) Merril cultivar Smooth Cayene em diferentes concentrações de macronutrientes. Hoehnea 34(1): 67-73., Silva et al. 2010Silva, P., Couto, J. & Santos, A. 2010. Absorção dos íons amônio e nitrato e seus efeitos no desenvolvimento do girassol em solução nutritiva. Revista de Biologia e Ciências da Terra 10(2): 97-104., Andrade & Tamaki 2016Andrade, S.V. & Tamaki, V. 2016. In vitro growth of Nidularium Minutum Mez ( Bromeliaceae ) in different concentrations of nitrogen, phosphorus, potassium and calcium. Journal of Plant Nutrition 39(11): 1634-1643.). Some studies that cultivated A. comosus var. ananassoides in vitro in MS medium with modified concentrations of NO₃- and NH4+ proved that NH4+ is not appropriate for the growth of this species (Dias et al. 2008Dias, G.deM.G., Carvalho, A.C.P.P.de, Pinheiro, M.V.M. & Morais, J.P.S. 2008. Micropropagação de abacaxi ornamental (Ananas comosus var. ananassoides) por estiolamento e regeneração de plântulas. Plant Cell Culture & Micropropagation 4(1): 1-7., Dias et al. 2010Dias, M.M., Pasqual M., Araújo, A.G., Santos, V.A.dos, Custódio T.N. & Costa, F.H.S.da. 2010. Enraizamento ex vitro e aclimatização de plantas micropropagadas de abacaxizeiro ornamental. Tecnologia & Ciência Agropecuária 4(2): 29-33., Dias et al. 2011aDias, M.M., Pasqual, M., Araújo, A.G.de & Santos, V.A. 2011a. Reguladores de crescimento na propagação in vitro de abacaxizeiro ornamental. Revista Brasileira de Ciências Agrárias 6(3): 383-390., bDias, M.M., Pasqual, M., Araújo, A.G.de, Santos, V.A.dos, Oliveira, A.C.de & Rodrigues, V.A. 2011b. Concentrações de reguladores vegetais no estiolamento in vitro de ananás do campo. Semina: Ciências Agrárias 32(2): 513-520., Carvalho et al. 2012Carvalho, A.C.P.P.de, Pinheiro, M.V.M., Dias, G.deM.G. & Morais, J.P.S. 2012. Estiolamento in vitro de plantas: alternativa para a produção de mudas micropropagadas de abacaxizeiro ornamental. Embrapa Agroindústria Tropical, Circular Técnica 42: 1-12., Silva & Tamaki 2012Silva, P.P.A. & Tamaki, V. 2012. Micropropagação da bromélia Ananas ananassoides (Baker) L.B. Sm. In: XVI Encontro de Iniciação Científica do Instituto de Botânica. Instituto de Botânica da Secretaria do Meio Ambiente, São Paulo, pp 22., Dias et al. 2013Dias, M.M., Nietsche, S. & Pereira, M.C.T. 2013. Concentrações de NPK no cultivo in vitro de abacaxizeiro ornamental. Tecnologia & Ciência Agropecuária 7(4): 1-6., Silva et al. 2017Silva, P.P.A., Kurita, F.M.K. & Tamaki, V. 2017. In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45(3): 313-320.). However, there are no studies published hitherto that evaluate the influence of different sources of inorganic nitrogen on the growth, nitrogen assimilation, and ex vitro acclimatization of this bromeliad. Therefore, this study aimed at evaluating the growth and nitrogen assimilation in the bromeliad A. comosus var. ananassoides cultivated in vitro and acclimatized ex vitro with different sources and concentrations of inorganic N.

Material and methods

Plant material - Seeds of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal, whose fruits were harvested at the Biological Reserve of Mogi Guaçu, São Paulo State, Brazil, were used in the experiments.

Obtaining nodal segments - Approximately 1,000 nodal segments were obtained from in vitro germination of approximately 400 seeds. These seeds were previously disinfested and subsequently deposited in Petri dishes for germination according to Araujo et al. (2012)Araújo, N.A.B.de, Kurita, F.M.K. & Tamaki, V. 2012. Nutritional reserves in seeds of bromeliad species. Communications in Plant Sciences 2(3-4): 57-59.. Posteriorly, the seedlings were transferred to 250 mL glass flasks containing 30 mL of modified Murashige & Skoog (1962)Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497. (MS) culture medium with only 50% of macronutrient concentration (MS/2), 30% of sucrose and 5% of agar. Seedlings were maintained in a culture room with a 12 h photoperiod, 30 µmol m−2.s−1 of photosynthetically active radiation, and a temperature of 26 ± 2 °C for two months. Leaves of plants grown in aseptic conditions were cut leaving the shoot with only 1 cm long, and transferred to 500 ml glass flasks containing 40 ml of MS/2 culture medium, in which they remained in the absence of light for five months to obtain nodal segments.

Plants obtained from nodal segments and grown in vitro with different nitrogen sources and concentrations - The nodal segments obtained were isolated and maintained in 350 mL glass flasks containing 30 mL MS/2 culture medium for two months in a culture room under the same conditions described previously. After this period, the grown plants were transferred to 350 mL glass flasks (five plants per flask) containing 30 mL of modified MS medium with different N sources (NH4+, NO3- and NH4NO3) and concentrations (15, 30, 60 and 90 mM) and were maintained in a culture room for seven months. After three months, the plants were transferred to a new culture medium with the same concentrations to avoid nutritional deficiencies and/or pH changes. After seven months, 24 hours before the harvest of the plant material, the plants were transferred to 350 mL glass flasks containing 30 mL modified MS medium with the different N sources and concentrations, to guarantee the N supply. Afterward, the shoot and root fresh and dry weights, the endogenous content of ammonium, nitrate and photosynthetic pigments, and the activity of nitrate reductase (NR), glutamine synthetase (GS), and NADH-dependent glutamate dehydrogenase (NADH-GDH) were evaluated. For the enzyme analysis, plants were harvested three hours after the beginning of the light period in the culture room. The dry weights of the shoots and roots were determined by drying the samples in an oven at 60 °C to constant weight.

Plant acclimatization- After seven months of in vitro culture with different N sources and concentrations, the plants were acclimatized in 600 mL plastic pots (10 plants per pot) containing washed sand as substrate. Leaves and roots were irrigated every 15 days with 40 mL of nutrient solution consisting of a modified MS formulation without sucrose and agar and different N sources (NH4+, NO3- and NH4NO3) and concentrations (15, 30, 60 and 90 mM). Plants were maintained in culture room for three months under the same controlled conditions described above. After this period, the shoot and root fresh and dry weight and the content of photosynthetic pigments were analysed. The shoot and root dry weights were determined by drying the samples at 60 °C in the oven to constant weight.

Determination of the photosynthetic pigments content - The analysis of photosynthetic pigments followed the method described by Munné-Bosch & Lalueza and (2007)Munné-Bosch, S. & Lalueza, P. 2007. Age-related changes in oxidative stress markers and abscisic acid levels in a drought-tolerant shrub, Cistus clusii grown under mediterranean field conditions. Planta 225: 1039-1049.. Triplicates of the samples were analyzed in a spectrophotometer. The wavelengths for spectrophotometer reading, as well as the equations used for calculating the content of photosynthetic pigments, were described in Lichtenthaler & Buschmann (2001)Lichtenthaler, H.K. & Buschmann, C. 2001. Chlorophylls and carotenoids: measurement and characterization by UV-VIS. Current Protocols in Food Analytical Chemistry F4.3.1-F4. 1-8. and expressed in milligram of pigment per gram of dry weight (mg g-1 DW).

Determination of endogenous ammonium and nitrate content - Aliquots of ca. 100 mg of frozen material obtained from 10 plants were ground to a powder with liquid nitrogen and homogenized with 1,000 µL of ultrapure water. Samples were then centrifuged at 22,000 g and 4 ºC for 10 min, and the supernatants recovered. The endogenous ammonium content was determined with the colorimetric method based on the phenol hypochlorite assay (Weatherburn 1967Weatherburn, M.W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry 39(8): 971-974., MCcullough 1967MCcullough, H. 1967. The determination of ammonia in whole blood by a direct colorimetric method. Clinica Chimica Acta 17(2): 297-304.) and ammonium concentration was expressed as µmol of NH4+ produced per gram of dry weight (µmol NH4+ g-1 MS). The endogenous nitrate content followed the method described by Cataldo et al. (1975)Cataldo, D.A., Maroon, M., Schrader, L.E. & Youngs, V.L. 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Communications in Soil Science and Plant Analysis 6(1): 71-80. and the nitrate concentration was expressed as µmol of NO3- produced per gram of dry weight (µmol NO3- g-1 DW).

Analysis of the NR activity - Nitrate reduction was measured using an in vivo assay (Jaworski 1971Jaworski, E. 1971. Nitrate reductase assay in intact plant tissues. Biochemical and Biophysical Research Communications 43(6): 1274-1279. modified by Nievola et al. 2001Nievola, C.C., Mercier, H. & Majerowisz, N. 2001. Levels of nitrogen assimilation in bromeliads with different growth habits. Journal of Plant Nutrition 24(9): 1387-1387.). Aliquots of ca. 350 mg of fresh material were weighed and incubated in 5 mL of 0.1 M phosphate buffer (pH 7.5) containing 3% propanol and 0.1 M KNO3. Tubes containing the samples were vacuum infiltrated three times for 1 min and incubated in the dark at 30 ºC for 30 min. Aliquots of 1 mL were taken from the incubating medium at both the beginning and the end of the assay. The nitrite produced was determined by adding 0.3 mL of 1% sulphanilamide, which was prepared with 3 M HCl and 0.3 mL of 0.2% N-(1-naphthyl) ethylene-diamine. All assays were performed in triplicate. Absorbance was read at 540 nm after 30 min. Nitrate reductase activity was expressed as µmol nitrite produced per hour per gram of dry weight (µmol NO2- h-1 g-1 DW).

Extraction and analysis of GS and NADH-GDH activity - Aliquots of ca. 250 mg of frozen material obtained from 10 plants were ground to a powder with liquid nitrogen and homogenized with 1.5 mL of an extraction medium that consisted of 0.05 M imidazole buffer (pH 7.9) and 5 mM DTT. These samples were centrifuged at 21,000 g and 4 ºC for 60 min, and the supernatant was used to measure the activity of both enzymes. All assays were performed in triplicate.

Analysis of the GS activity - The GS activity was determined based on the in vitro method proposed by Elliott (1955) apud Farnden & Robertson (1980)Farnden, K.J.S. & Robertson, J.G. 1980. Methods for studying enzyme involved in metabolism related to nitrogenase. In: F.J. Bergsen (ed.). Methods for evaluating biological nitrogen fixation. Jonh Wiley & Sons, West Sussex/UK, pp. 279-286.. The total volume of the enzymatic reaction was 0.5 mL and consisted of 0.1 M imidazole buffer, pH 7.5, 49 mM hydroxylamine, 40 mM MgCl2, 160 mM glutamate, and 35 mM ATP (pH 7.5). The reaction started with the addition of 50 µL of the plant extract followed by incubation of the tubes at 35 °C. After 60 min, the reaction was interrupted with the addition of 1mL of a solution containing 0.123 M ferric chloride, 0.25 M HCl, and 0.1225 M trichloroacetic acid. The absorbance was read in a spectrophotometer at 540 nm. The GS activity was expressed in mmol Ɣ-glutamyl-hydroxamate produced per hour per gram of dry weight (mmol ƔGH h-1 g-1 DW).

Analysis of the NADH-GDH activity - The method described by Bullen (1956)Bullen, W.A. 1956. The isolation and characterization of glutamic dehydrogenase from corn leaves. Archives Biochemistry and Biophysics 62: 173-183. was used for the determination of the NADH-GDH activity. Firstly, an aliquot of the plant extract was desalted through in the Sephadex PD10/G-25 column (Amersham Pharmacia Biotech) and used in the GDH assay. The NADH-GDH activity was quantified through the amount of NADH consumed in the reaction. The total volume of the reaction was 1 mL and was composed of 0.1 M Tris buffer (pH 8.2), 13.3 mM 2-oxoglutarate, 0.1 M (NH4)2SO4, and 0.16 mM NADH. All reagents were maintained at 30 °C for 10 minutes before starting the enzymatic reaction. The reaction started with the addition of 0.25 mL of plant extract and instantly read in a spectrophotometer at 340 nm. The slope was calculated using the linear absorbance range recorded within a period of five minutes. The enzymatic activity was calculated using the molar extinction coefficient (ԑ 340) of 6.22 × 106 mmol -1.cm -1 and expressed as µmol of NADH consumed per minute per gram of dry weight (µmol NADH min-1.g-1 DW).

Statistical analyses and experimental design - The experiments followed a completely randomized design in a factorial arrangement (3×4). The experiment had 12 treatments and two harvesting times (seven months of in vitro growth and three months of ex vitro acclimatization) with three independent replicates. For in vitro growth, each experimental unit consisted of three glass flasks with five plants, whereas for the ex vitro acclimatization, each experimental unit had three pots with 10 plants. Data were subjected to a two-way analysis of variance (two-way ANOVA), and the differences among means were compared by Tukey´s test (P < 0.05).

Results and Discussion

Plants cultivated for seven months with NO3- and NH4NO3 presented 100% survival with all concentrations used and showed a healthier appearance compared to plants grown with NH4+, which had chlorotic leaves and atrophied appearance (figure 1). In addition, plants cultivated at 15, 30, 60, and 90 mM NH4+ showed a 55, 56, 57, and 65% survival, respectively. Furthermore, shoots of plants grown with different N sources and concentrations showed a biomass decrease when comparing the values of fresh and dry weights (figure 2 a and b). Several studies have described the toxicity of NH4+ for most higher plants when supplied as a single N source (Roosta & Schjoerring 2007Roosta, H.R. & Schjoerring, J.K. 2007. Effects of ammonium toxicity on nitrogen metabolism and elemental profile of cucumber plants. Journal of Plant Nutrition 30:1933-1951, Marschner & Marschner 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Esteban et al. 2016Esteban, R., Ariz, I., Cruz, C. & Moran, J.F. 2016. Review: Mechanisms of ammonium toxicity and the quest for tolerance. Plant Science 248: 92-101., Ji et al. 2019Ji, Y., Li, Q., Liu, G., Selvaraj, G., Zheng, Z., Zou, J. & Wei, Y. 2019. Roles of cytosolic glutamine synthetases in Arabidopsis development and stress responses. Plant and Cell Physiology 60: 657-671.). However, some aspects of the mechanism of the NH4+ toxicity are yet not fully understood (Britto & Kronzucker 2002Britto, D.T. & Kronzucker, H.J. 2002. NH4+ toxicity in higher plants: a critical review. Journal of Plant Physiology 159: 567-584., Li et al. 2014Li, B., Li, G., Kronzucker, H.J., Balusca, F. & Shi, W. 2014. Ammonium stress in Arabidopsis: Signaling, genetic loci, and physiological targets. Trends in Plant Science 19(2): 107-114.). Nevertheless, the tissue toxicity of the NH4+ excess can be attributed to uncoupling of photophosphorylation, disturbance in pH balance (Li et al. 2011Li, B., Shi, W. & Su, Y. 2011. The differing responses of two Arabidopsis ecotypes to ammonium are modulated by the photoperiod regime. Acta Physiologiae Plantarum 33: 325-334., Li et al. 2014Li, B., Li, G., Kronzucker, H.J., Balusca, F. & Shi, W. 2014. Ammonium stress in Arabidopsis: Signaling, genetic loci, and physiological targets. Trends in Plant Science 19(2): 107-114.), high carbon consumption and accumulation of abscisic acid and free putrescine in the plant tissues (Garnica et al. 2010Garnica, M., Houdusse, F., Zamarreno, A.M. & Garcia-Mina, J.M. 2010. The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. Journal of Plant Physiology 167: 1264-1272.). Furthermore, Andrews et al. (2013)Andrews, M., Raven, J.A. & Lea, P.J. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Annals of Applied Biology 163(2): 1-26. observed that NO3- is required to maintain the biosynthesis and flux of cytokinin at a level enough to mediate normal morphogenesis.

Figure 1
General view of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultivated in vitro with MS-1962 modified medium with different sources (NH4-, NO3-, and NH4NO3) and N concentrations (15, 30, 60, and 90 mM) for seven months. a-d. Plants grown with 15 mM, 30 mM, 60 mM, and 90 mM of N as NH4-, respectively. e-h. Plants grown with 15 mM, 30 mM, 60 mM, and 90 mM of N as NO3-, respectively. i-l. Plants grown with 15 mM, 30 mM, 60 mM, and 90 mM of N as NH4NO3, respectively. Bars = 1 cm. Photos from Priscila P. A. Silva.

Figure 2
Fresh (FW) and dry weight (DW) of shoot and root of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultured in vitro for seven months with 15, 30, 60, and 90 mM of N in the forms of NH4+, NO3- or NH4NO3. Different letters indicate significant statistical differences (p ≤ 0.05).

Plants cultivated with NO3- showed the highest means of fresh and dry weights of shoots and roots (figure 2 a and b). This increase in dry weight of plants cultivated with NO3- was also observed for other species, such as Cucumis sativus L. (Roosta & Schjoerring 2007Roosta, H.R. & Schjoerring, J.K. 2007. Effects of ammonium toxicity on nitrogen metabolism and elemental profile of cucumber plants. Journal of Plant Nutrition 30:1933-1951) and Triticum aestivum L.cv Amarok (Garnica et al. 2010Garnica, M., Houdusse, F., Zamarreno, A.M. & Garcia-Mina, J.M. 2010. The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. Journal of Plant Physiology 167: 1264-1272.). The NO3- and NH4+ ions are the most available sources of N for root uptake in most soils, however, the absorption of one particular ion or both of them will depend on the plant species and its physiological characteristics, as well as the ion availability in the soil or substrate (Marschners & Marschners 2012Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London., Liu & Wirén 2019Liu, Y. & Wirén, N.von. 2017. Ammonium as a signal for physiological and morphological responses in plants. Journal of Experimental Botany 68: 2581-2592.). It is well-known that NO3- plays a key role in the cell signalling that activates the gene expression related to uptake, transport and N assimilation (Crawford 1995Crawford, N. 1995. Nitrate: nutrient and signal for plant growth. The Plant Cell 7: 859-868., Miller et al. 2007Miller, A.J., Fan, X., Orsel, M., Smith, S.J. & Wells, D.M. 2007. Nitrate transport and signaling Journal of Experimental Botany 58(9): 2297-2306., Krapp 2015Krapp, A. 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Current Opinion in Plant Biology 25: 115-122.), and the cytokinin synthesis, which is responsible for regulating cell growth (division and multiplication) and development (expansion) in higher plants (Garnica et al. 2010Garnica, M., Houdusse, F., Zamarreno, A.M. & Garcia-Mina, J.M. 2010. The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. Journal of Plant Physiology 167: 1264-1272., Andrews et al. 2013Andrews, M., Raven, J.A. & Lea, P.J. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Annals of Applied Biology 163(2): 1-26., Buchanan et al. 2015Buchanan, B.B., Gruissem, W. & Jones, R.L. 2015. Biochemistry and Molecular Biology of Plants. 2nd ed. John Wiley & Sons, West Sussex/UK.). This could be the reason why we found a greater accumulation of biomass in plants grown with NO3 -.

With regard to the dry and fresh weight of roots, the lowest values were also obtained for plants grown with NH4+ (figure 2 c and d). This reduction can be attributed to several biochemical and physiological factors previously mentioned for shoots, but which could also affect roots. In addition, Barth et al. (2010)Barth, C., Gouzd, Z.A., Steelt, H.P. & Imperio, R.M. 2010. A mutation in GDP-mannose pyrophosphorylase causes conditional hypersen- sitivity to ammonium, resulting in Arabidopsis root growth inhibition, altered ammonium metabolism, and hormone homeo-stasis. Journal of Experimental Botany 61: 379-394. reported that NH4+ inhibit the activity of the enzymes GDP-mannose pyrophosphorylase and GDP-mannose, which are required for the biosynthesis of L-ascorbic acid and N glycoproteins. Failure in the N-glycosylation process causes membrane and cell wall deformations, compromising the cell cycle and leading to cell death, consequently inhibiting root growth (Qin et al. 2008Qin, C., Qian, W., Wang, W., Wu, Y., Yu, C., Jiang, X., Wang, D. & Wu, P. 2008. GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 105: 18308-18313., Kempinski et al. 2013Kempinski, C.F., Crowell, S.V., Smeeth, C. & Barth, C. 2013. The novel Arabidopsis thaliana svt2 suppressor of the ascorbic acid-deficient mutant vtc1-1 exhibits phenotypic and genotypic instability. F1000 Research 1: 2-6.). Furthermore, it has been observed that the NH4+ excess decreases root gravitropism in Arabidopsis sp. by the redistribution of auxin, which implies a decrease in the root length (Zou et al. 2012Zou, N., Li, B., Dong, G., Kronzucker, H.J. & Shi, W.M. 2012. Ammonium-induced loss of root gravitropism is related to auxin distribution and TRH1 function, and is uncoupled from the inhibition of root elongation in Arabidopsis. Journal of Experimental Botany 63: 3777-3788.). Thus, this disturbance negatively influenced root growth in plants cultivated with NH4+.

Moreover, both root fresh and dry weights gradually increased in plants cultivated with 15, 30, and 60 mM of N as NO3- compared to the other two nitrogen sources (figure 2 c and d). As previously mentioned, NO3- is a signalling molecule for several cell functions, including cytokinin biosynthesis (Garnica et al. 2010Garnica, M., Houdusse, F., Zamarreno, A.M. & Garcia-Mina, J.M. 2010. The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. Journal of Plant Physiology 167: 1264-1272., Andrews et al. 2013Andrews, M., Raven, J.A. & Lea, P.J. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Annals of Applied Biology 163(2): 1-26.). Some authors have observed that the cytokinin regulates auxin distribution in the root apical meristem and promotes cell division in the quiescent centre of the root apical meristem, which is usually mitotically inactive (Ruzicka et al. 2009Ruzicka, K., Simaskova, M., Duclercq, J., Petrasek, J., Zazımalova, E., Simon, S., Friml, J., Van Montagu, M.C.E., & Benkova, E. 2009. Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proceedings of the National Academy of Sciences 106: 4284-4289., Zhang et al. 2011Zhang, W., To, J.P.C., Cheng, C.Y., Schaller, G.E. & Kieber, J.J. 2011. Type-A response regulators are required for proper root apical meristem function through post-transcriptional regulation of PIN auxin efflux carriers. Plant Journal 68: 1-10., Zhang et al. 2013Zhang, W., Swarup, R., Bennett, M., Schaller, G.E. & Kieber, J.J. 2013. Cytokinin induces cell division in the quiescent center of the Arabidopsis root apical meristem. Current Biology 23: 1979-1989.). This increase in cell division induced by the cytokinin might explain the increase in root biomass in plants grown with NO3-.

With regard to the photosynthetic pigments in plants cultivated in vitro, the highest values were obtained in plants grown at all NH4NO3 concentrations, with the highest mean achieved in those grown with 60 mM of N as NH4NO3 (figure 3). The content of chlorophyll a was more than twice of the chlorophyll b content and three times higher than of carotenoids (figure 3). This increase in photosynthetic pigments and the same differences in the quantities of each pigment were observed for the bromeliad Alcantarea imperialis (Carrière) Harms cultivated with the same N sources and concentrations (Kurita 2015Kurita, F.M.K. 2015. Assimilation of different nitrogen sources in the induction of the mutiplication of bromeliad Alcantarea imperialis (Carrière) Harms cultivated in vitro. Thesis from Instituto de Botânica, São Paulo, Brazil.). Studies have reported that plants supplemented with non-toxic concentrations of NH4+ present higher chlorophyll content per leaf unit area (Raab & Tarry 1994Raab, T.K. & Terry, N. 1994 Nitrogen-source regulation of growth and photosynthesis in Beta vulgaris L. Plant Physiology 105: 1159-1166., Claussen & Lenz 1999Claussen, W. & Lenz, F. 1999. Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant Soil 208(1) 95-102.). This explains the higher amount of pigments in plants grown with different NH4NO3 concentrations, compared to plants cultivated with NO3- concentrations. However, there are no published studies that clarify this increase in photosynthetic pigments associated with the supplementation of different sources of inorganic N. According to Raab & Tarry (1994)Raab, T.K. & Terry, N. 1994 Nitrogen-source regulation of growth and photosynthesis in Beta vulgaris L. Plant Physiology 105: 1159-1166., the carbohydrate requirement for NH4+ assimilation might influence the NH4+ toxicity in plants, since the faster the NH4+ absorbed is assimilated, the less is the damage caused by toxicity. Therefore, the increase in photosynthetic pigments in plants of A. comosus var. ananassoides grown with different NH4NO3 concentrations seems to be related to the need for a supply of carbon skeletons for the assimilation of the NH4+ absorbed in the roots.

Figure 3
Photosynthetic pigments content in leaves of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultured in vitro for seven months with 15, 30, 60, and 90 mM of N in the forms of NH4+, NO3- or NH4NO3. a. Chlorophyll a and b. b. Carotenoids. Different letters indicate significant statistical differences (p ≤ 0.05)

With regard to nitrogen assimilation, both NR activity (NR) and endogenous NO3- content (ENC) were below the detection levels of the method in plants grown with different NH4+ concentrations, and therefore, it was not possible to quantify them. In these treatments, little growth was observed.

With regard to the NR activity, the highest means were obtained in plants cultivated with 90 mM of N as NH4NO3 compared to those cultivated with the same concentrations of NO3- (figure 4 a), which might be related to higher ENC in plants grown with 90 mM of N as NO3- (figure 4 b). This is because in angiosperms, the NO3- absorbed can be either reduced immediately in the roots or transported via the xylem to the shoot, where it is stored in the vacuoles for later reduction according to the plant necessity (Cárdenas-Navarro et al. 1999Cárdenas-Navarro, R., Adamowicz, S. & Robin, P. 1999. Nitrate accumulation in plants: a role for water. Journal of Experimental Botany 50: 613-624., Krapp 2015Krapp, A. 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Current Opinion in Plant Biology 25: 115-122.). The NO3- stored is important for the cellular osmotic balance, besides being a nitrogen reserve source for N-shortage periods (Chen et al. 2004Chen, B.M., Wang, Z.H., Li, S.X., Wang, G.X., Song, H.X. & Wang, X.N. 2004. Effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. Plant Science 167: 635-643., Miller et al. 2007Miller, A.J., Fan, X., Orsel, M., Smith, S.J. & Wells, D.M. 2007. Nitrate transport and signaling Journal of Experimental Botany 58(9): 2297-2306., Krapp 2015Krapp, A. 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Current Opinion in Plant Biology 25: 115-122.).

Figure 4
Nitrate reductase activity (a) and endogenous NO3- content (b) in leaves of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultured in vitro for seven months with 15, 30, 60, and 90 mM of N in the forms of NH4+, NO3- or NH4NO3. Different letters indicate significant statistical differences (p ≤ 0.05).

As shown in figure 5 a, plants cultivated with 15, 30, 60, and 90 mM of N as NH4NO3 showed the highest glutamine synthetase activity (GS) when compared to plants grown with the other two nitrogen sources (NH4+ and NO3-). Some studies have reported that GS is not affected by different N supplies in Solanum lycopersicum L. and Zea mays L. (Magalhães & Huber 1991Magalhães, J.R. & Huber, D.M. 1991. Response of ammonium assimilation enzymes to nitrogen form treatments in different plant species. Journal of Plant Nutrition 14: 175-185.), Vaccinium corymbosum L., Rubus idaeus L., and Fragaria ananassa Duch. (Claussen & Lenz 1999Claussen, W. & Lenz, F. 1999. Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant Soil 208(1) 95-102.). However, plants of Catasetum fimbriatum (Morren) Lindl. (Majerowicz et al. 2000Majerowicz, N., Kerbauy, G.B., Nievola, C.C. & Suzuki, R.M. 2000. Growth and nitrogen metabolism of Catasetum fimbriatum (Orchidaceae) grown with different nitrogen sources. Environmental and Experimental Botany 44: 195-206.) and Solanum lycopersicum L. cv. Rio Grande (Horchani et al. 2010Horchani, F., Aschi-Smiti, S. & Brouquisse, R. 2010. Involvement of nitrate reductase in the tolerance of tomato (Solanum lycopersicum L.) plants to prolonged root hypoxia. Acta Physiologiae Plantarum 32: 1113-1123.) grown with NH4NO3 or NH4+ showed a higher GS activity than those grown with NO3-. Therefore, the GS response to the nitrogen source provided seems to vary according to the plant species.

Figure 5
Glutamine synthetase activity (a), glutamate dehydrogenase activity (b) and endogenous NH4+ content (c) in leaves of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultured in vitro for seven months with 15, 30, 60, and 90 mM of N in the forms of NH4+, NO3- or NH4NO3. Different letters indicate significant statistical differences (p ≤ 0.05).

The NH4+ assimilation into glutamine and glutamate performed by the enzymes glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) is considered the main pathway of N assimilation (Miflin & Habash 2002Miflin, B.J. & Habash, D.Z. 2002. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. Journal of Experimental Botany 53(370): 979-987., Bernard & Habash 2009Bernard, S.M. & Habash, D.Z. 2009. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytologist 182: 608-620., Rennenberg et al. 2010Rennenberg, H., Wildhagen, H. & Ehlting, B. 2010. Nitrogen nutrition of pop- lar trees. Plant Biology 12:275-291., Xu et al. 2012Xu, G., Fan, X. & Miller, A.J. 2012. Plant nitrogen assimilation and use efficiency. Annual Review of Plant Biology 63: 153-182., Krapp 2015Krapp, A. 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Current Opinion in Plant Biology 25: 115-122.), and NH4+ detoxification under normal conditions of plant growth. Thus, the higher GS activity observed in A. comosus var. ananassoides cultivated with NH4NO3 might be a mechanism to avoid the toxicity of the NH4+ absorbed.

The lower GS activity in plants grown with different NH4+ concentrations (figure 5 a), associated with the other parameters previously analysed (fresh and dry weights, and content of photosynthetic pigments) and the appearance of the plants, indicated that they were going through the senescence process. Some authors have verified that during the senescence process, leaves show low GS activity (Peeters & Van Laere 1992Peeters, K.M.U. & Van Laere, A.J. 1992. Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark. Physiologia Plantarum 84: 243-249., Chen et al. 1997Chen, S., Hung, K. & Kao, C. 1997. Ammonium accumulation is associated with senescence of rice leaves. Plant Growth Regulation 21: 195-201.) due to the high degradation of proteins such as ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo), plastid GS and plastid GOGAT in the chloroplast stroma (Palatnik et al. 1999Palatnik, J.F., Carillo, N. & Valle, E.M. 1999. The role of photosynthetic electron transport in the oxidative degradation of chloroplastic glutamine synthetase. Plant Physiology 121: 471-478., Parry et al. 2003Parry, M.A., Andralojc, P.J., Mitchell, R.A., Madgwick, P.J. & Keys, A.J. 2003. Manipulation of Rubisco: the amount, activity, function and regulation. Journal of Experimental Botany 54: 1321-33., Kato et al. 2005Kato, Y., Yamamoto, Y., Murakami, S. & Sato, F. 2005. Post-translational regulation of CND41 protease activity in senescent tobacco leaves. Planta 222: 643-651., Masclaux-Daubresse et al. 2010Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gaufichon, L. & Suzuki, A. 2010. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany 105: 1141-1158.).

In addition, plants cultivated with NH4+ had the highest glutamate dehydrogenase activity (GDH) and endogenous NH4+ content (EAC) (figure 5 b and c). The NH4+ accumulation is due to the high proteolysis that occurs during the senescence process, which releases large amounts of amino acids that are catabolized by GDH and asparaginases (Bernad & Habash 2009). The GDH can catalyse both the amination of 2-oxoglutarate (2-OG) and deamination of glutamate (Glu) (Lea & Miflin 2003Lea, P.J. & Miflin, B.J. 2003. Glutamate synthase and the synthesis of glutamate in plants. Plant Physiology and Biochemistry 41: 555-564., Forde & Lea 2007Forde, B.G. & Lea, P.J. 2007. Glutamate in plants: metabolism, regulation and signalling. Journal of Experimental Botany 58: 2339-2358.). These processes occur as a deviation to ensure that N-assimilation does not excessively decrease the 2-OG amount in the mitochondria since this is an intermediate in the tricarboxylic acid cycle (Robison et al. 1991, Miflin & Habash 2002Miflin, B.J. & Habash, D.Z. 2002. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. Journal of Experimental Botany 53(370): 979-987., Lea & Miflin 2003Lea, P.J. & Miflin, B.J. 2003. Glutamate synthase and the synthesis of glutamate in plants. Plant Physiology and Biochemistry 41: 555-564.). Therefore, the higher GDH in plants of A. comosus var. ananassoides cultivated with NH4+ can be related to the Glu catabolism to generate both 2-OG for the tricarboxylic acid cycle and NADH for other metabolic cell processes.

Three months after being transferred to ex vitro conditions, plants acclimatized with different NO3- and NH4NO3 concentrations showed no mortality and presented a healthy appearance in all concentrations used. On the contrary, plants grown with different NH4+ concentrations had 100% mortality. Changes in some factors, such as increases in transpiration rate and luminosity, replacement of a heterotrophic condition to autotrophic condition, among others, during the transition from in vitro culture to ex vitro conditions, might be limiting for the propagation of some species (Grattapaglia & Machado 1998Grattapaglia, D. & Machado, M.A. 1998. Micropropagação. In: A.C. Torres, L.S. Caldas & J.A. Buso (eds.). Cultura de tecidos e transformação genética de plantas. 1 ed. Embrapa-CNPH, Brasília, pp.183-260., Hartmann et al. 2002Hartmann, H.T., Kester, D.E., Davis Júnior, F.T. & Geneve, R.L. 2002. Plant propagation: principles and practices. Englewood Clipps, New York., Bregonci et al. 2008Bregonci, S.I.; Reis, E.S., Almeida, G.D., Brum, V.J. & Zucoloto, M. 2008. Avaliação do crescimento foliar e radicular de mudas micropropagadas do abacaxizeiro cv. gold em aclimatação. Idesia 26(3): 87-96., Berilli et al. 2011Berilli, S.S., Carvalho, A.J.C., Freitas, S.J., Faria, D.C. & Marinho., C.S. 2011. Avaliação do desenvolvimento de diferentes tamanhos de mudas micropropagadas de abacaxizeiro, após aclimatação. Revista Brasileira de Fruticultura 33(1): 208-214.). Consequently, changes in these factors might have induced the death of the plants cultivated in vitro with NH4+.

Plants grown ex vitro with 30, 60, and 90 mM of N as NH4NO3 showed higher means in the fresh and dry weights of the shoots compared to the other treatments (figure 6 a and b). Up to date, there are no published studies that evaluate the acclimatization of plants cultivated in vitro with different N sources and concentrations. Nevertheless, Kurita (2015)Kurita, F.M.K. 2015. Assimilation of different nitrogen sources in the induction of the mutiplication of bromeliad Alcantarea imperialis (Carrière) Harms cultivated in vitro. Thesis from Instituto de Botânica, São Paulo, Brazil. verified that the bromeliad A. imperialis cultivated under controlled environmental conditions showed higher growth when cultivated with NO3- than when fertilized with NH4+ or NH4NO3. In the present study, opposite results were obtained for A. comosus var. ananassoides. In the case of roots, both the fresh and dry weights increased with 15 mM of N as NO3- (figure 6 c and d). Kiba et al. (2011)Kiba, T., Kudo, T., Kojima, M. & Sakakibara, H. 2011. Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin. Journal of Experimental Botany 62:1399-1409. observed that high N supplementation suppressed root growth in most plants, while N limitation accelerated their growth. This modulation in the root growth related to low or high N availability is mediated mainly by the signalling of the phytohormones auxin and abscisic acid (Kiba et al. 2011Kiba, T., Kudo, T., Kojima, M. & Sakakibara, H. 2011. Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin. Journal of Experimental Botany 62:1399-1409., Vidal et al. 2013Vidal, E.A., Moyano, T.C., Riveras, E., Contreras-Lopez, O. & Gutierrez, R.A. 2013. Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots. Proceedings of the National Academy of Sciences 10:12840-12845.). Some studies have shown that a decrease in N availability might promote auxin accumulation in the roots and, consequently, lead to root elongation (Walch-Liu et al. 2006Walch-Liu, P., Ivanov, I., Filleur, S., Gan, Y., Remans, T. & Forde, B.G. 2006. Nitrogen regulation of root branching. Annals of Botany 97: 875-881., Tamaki & Mercier 2007Tamaki, V., Mercier, H. & Nievola, C.C. 2007. Cultivo in vitro de clones de Ananas comosus (L.) Merril cultivar Smooth Cayene em diferentes concentrações de macronutrientes. Hoehnea 34(1): 67-73., Tian et al. 2008Tian, Q., Chen, F., Liu, J., Zhang, F. & Mi, G. 2008 Inhibition of maize root growth by high nitrate supply is correlated with reduced IAA levels in roots. Journal of Plant Physiology 165:942-951.). Furthermore, it was observed that auxins were also responsible for the induction of the lateral root development in response to low N supplementation (Fukaki & Tasaka 2009Fukaki, H. & Tasaka, M. 2009. Hormone interactions during lateral root formation. Plant Molecular Biology 69: 437-449., Ma et al. 2014Ma, W., Li, J., Qu, B., He, X., Zhao, X., Li, B., Fu, X. & Tong, Y. 2014. Auxin biosynthetic gene TAR2 is involved in low nitrogen-mediated reprogramming of root architecture in Arabidopsis. Plant Journal 78:70-79.).

Figure 6
Fresh (FW) and dry weight (DW) of shoot and root of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal ex vitro acclimatized for three months with 15, 30, 60, and 90 mM of N in the forms of NH4+, NO3- or NH4NO3. Different letters indicate significant statistical differences (p ≤ 0.05).

The content of photosynthetic pigments in plants acclimatized ex vitro was the highest when cultivated with different NH4NO3 concentrations, with the highest mean in those plants grown with 60 mM of N as NH4NO3 (figure 7 a and b). In all treatments, the content of chlorophyll a was more than twice the content of chlorophyll b and three times greater than the carotenoid content (figure 7), showing a similar tendency to plants grown in vitro.

Figure 7
Photosynthetic pigments content in leaves of Ananas comosus var. ananassoides (Baker) Coppens & F.Leal cultured ex vitro for seven months with 15, 30, 60, and 90 mM of N in the forms of NH4+, NO3- or NH4NO3. (A) Chlorophyll a and b; (B) Carotenoids. Different letters indicate significant statistical differences (p ≤ 0.05)

Both in vitro and ex vitro results indicated that the cultivation of A. comosus var. ananassoides with NH4+ as the only N source is not feasible. Our results indicated that the growth of plants with NO3- as the only N source is possible, but it is not favourable for photosynthetic pigments.

In addition, the enzymes NR and GS assimilated less N as NO3- than as NH4NO3. Therefore, the use of NH4NO3 for both in vitro and ex vitro cultivation seems to be the best option, since plants presented higher amounts of photosynthetic pigments in all analysed concentrations, higher biomass in both in vitro and ex vitro cultivation, and higher N uptake in vitro. In general, 60 mM of N as NH4NO3 seems to be the best NH4NO3 concentration, since it leads to the highest content of photosynthetic pigments in both in vitro and ex vitro cultivated plants. Furthermore, plants grown with this concentration showed higher biomass and GS activity, and assimilated more N than plants cultivated with the other NH4NO3 concentrations.

Conclusion

Our study showed that plants of A. comosus var. ananassoides are not able to grow with NH4+ as the sole source of N and that it is recommended to use 60 mM of N as NH4NO3 for both in vitro and ex vitro cultivation of this bromeliad.

  • 1
    Part of the Masters’ Dissertation of the first Author

Acknowledgments

This work is part of the Masters’ Dissertation of the first Author, carried out at Instituto de Botânica, São Paulo, SP, Brazil and had financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Literature cited

  • Andrade, S.V. & Tamaki, V. 2016. In vitro growth of Nidularium Minutum Mez ( Bromeliaceae ) in different concentrations of nitrogen, phosphorus, potassium and calcium. Journal of Plant Nutrition 39(11): 1634-1643.
  • Andrews, M., Raven, J.A. & Lea, P.J. 2013. Do plants need nitrate? The mechanisms by which nitrogen form affects plants. Annals of Applied Biology 163(2): 1-26.
  • Albert, L.H.B. 2004. Aspectos morfoanatômicos de mudas de abacaxizeiro ‘Smooth Cayenne’ micropropagadas. Tese Doutorado, Universidade Federal de Lavras, Lavras.
  • Aranda-Peres, A.N., Peres, L.E.P., Higashi, E.N. & Martinelli, A.P. 2009. Adjustment of mineral elements in the culture medium for the micropropagation of three Vriesea bromeliads from the brazilian atlantic forest: the importance of calcium. Hortscience 44: 106-112.
  • Araújo, N.A.B.de, Kurita, F.M.K. & Tamaki, V. 2012. Nutritional reserves in seeds of bromeliad species. Communications in Plant Sciences 2(3-4): 57-59.
  • Barth, C., Gouzd, Z.A., Steelt, H.P. & Imperio, R.M. 2010. A mutation in GDP-mannose pyrophosphorylase causes conditional hypersen- sitivity to ammonium, resulting in Arabidopsis root growth inhibition, altered ammonium metabolism, and hormone homeo-stasis. Journal of Experimental Botany 61: 379-394.
  • Bullen, W.A. 1956. The isolation and characterization of glutamic dehydrogenase from corn leaves. Archives Biochemistry and Biophysics 62: 173-183.
  • Berilli, S.S., Carvalho, A.J.C., Freitas, S.J., Faria, D.C. & Marinho., C.S. 2011. Avaliação do desenvolvimento de diferentes tamanhos de mudas micropropagadas de abacaxizeiro, após aclimatação. Revista Brasileira de Fruticultura 33(1): 208-214.
  • Bernard, S.M. & Habash, D.Z. 2009. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytologist 182: 608-620.
  • Braun, H., Coelho, S.F., Silva, M.C.C., Fontes, P.C.R., Cecon, P.R. & Busato, C. 2013. Absorção, metabolismo e diagnóstico do estado de nitrogênio em plantas de batata. Revista de Ciências Agrárias 56(3): 185-195.
  • Bregonci, S.I.; Reis, E.S., Almeida, G.D., Brum, V.J. & Zucoloto, M. 2008. Avaliação do crescimento foliar e radicular de mudas micropropagadas do abacaxizeiro cv. gold em aclimatação. Idesia 26(3): 87-96.
  • Britto, D.T. & Kronzucker, H.J. 2002. NH4+ toxicity in higher plants: a critical review. Journal of Plant Physiology 159: 567-584.
  • Buchanan, B.B., Gruissem, W. & Jones, R.L. 2015. Biochemistry and Molecular Biology of Plants. 2nd ed. John Wiley & Sons, West Sussex/UK.
  • Bunn, E., Turner, S.R. & Dixon, K. 2011. Biotechnology for saving rare and threatened florain a biodiversity hotspot. In Vitro Cellular & Developmental Biology Plant 47(1): 188-200.
  • Calvete, E.O., Azevedo, M., Bordignon, M.H. & Suzin, M. 2002. Análises anatômicas e da biomassa em plantas de morangueiro cultivadas in vitro e ex vitro Horticultura Brasileira 20(4): 649-653.
  • Cárdenas-Navarro, R., Adamowicz, S. & Robin, P. 1999. Nitrate accumulation in plants: a role for water. Journal of Experimental Botany 50: 613-624.
  • Carvalho, A.C.P.P.de, Pinheiro, M.V.M., Dias, G.deM.G. & Morais, J.P.S. 2012. Estiolamento in vitro de plantas: alternativa para a produção de mudas micropropagadas de abacaxizeiro ornamental. Embrapa Agroindústria Tropical, Circular Técnica 42: 1-12.
  • Cataldo, D.A., Maroon, M., Schrader, L.E. & Youngs, V.L. 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Communications in Soil Science and Plant Analysis 6(1): 71-80.
  • Chen, S., Hung, K. & Kao, C. 1997. Ammonium accumulation is associated with senescence of rice leaves. Plant Growth Regulation 21: 195-201.
  • Chen, B.M., Wang, Z.H., Li, S.X., Wang, G.X., Song, H.X. & Wang, X.N. 2004. Effects of nitrate supply on plant growth, nitrate accumulation, metabolic nitrate concentration and nitrate reductase activity in three leafy vegetables. Plant Science 167: 635-643.
  • Claussen, W. & Lenz, F. 1999. Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant Soil 208(1) 95-102.
  • Crawford, N. 1995. Nitrate: nutrient and signal for plant growth. The Plant Cell 7: 859-868.
  • Dias, G.deM.G., Carvalho, A.C.P.P.de, Pinheiro, M.V.M. & Morais, J.P.S. 2008. Micropropagação de abacaxi ornamental (Ananas comosus var. ananassoides) por estiolamento e regeneração de plântulas. Plant Cell Culture & Micropropagation 4(1): 1-7.
  • Dias, M.M., Pasqual M., Araújo, A.G., Santos, V.A.dos, Custódio T.N. & Costa, F.H.S.da. 2010. Enraizamento ex vitro e aclimatização de plantas micropropagadas de abacaxizeiro ornamental. Tecnologia & Ciência Agropecuária 4(2): 29-33.
  • Dias, M.M., Pasqual, M., Araújo, A.G.de & Santos, V.A. 2011a. Reguladores de crescimento na propagação in vitro de abacaxizeiro ornamental. Revista Brasileira de Ciências Agrárias 6(3): 383-390.
  • Dias, M.M., Pasqual, M., Araújo, A.G.de, Santos, V.A.dos, Oliveira, A.C.de & Rodrigues, V.A. 2011b. Concentrações de reguladores vegetais no estiolamento in vitro de ananás do campo. Semina: Ciências Agrárias 32(2): 513-520.
  • Dias, M.M., Nietsche, S. & Pereira, M.C.T. 2013. Concentrações de NPK no cultivo in vitro de abacaxizeiro ornamental. Tecnologia & Ciência Agropecuária 7(4): 1-6.
  • Endres, L. & Mercier, H. 2001. Ammonium and urea as nitrogen sources for bromeliads. Journal of Plant Physiology 158: 205-212.
  • Epstein, E. & Bloom, A.J. 2006. Nutrição mineral de plantas: princípios e perpectivas. 2 ed. Trad. Editora Planta, Londrina.
  • Esteban, R., Ariz, I., Cruz, C. & Moran, J.F. 2016. Review: Mechanisms of ammonium toxicity and the quest for tolerance. Plant Science 248: 92-101.
  • Farnden, K.J.S. & Robertson, J.G. 1980. Methods for studying enzyme involved in metabolism related to nitrogenase. In: F.J. Bergsen (ed.). Methods for evaluating biological nitrogen fixation. Jonh Wiley & Sons, West Sussex/UK, pp. 279-286.
  • Forde, B.G. & Lea, P.J. 2007. Glutamate in plants: metabolism, regulation and signalling. Journal of Experimental Botany 58: 2339-2358.
  • Fukaki, H. & Tasaka, M. 2009. Hormone interactions during lateral root formation. Plant Molecular Biology 69: 437-449.
  • Garnica, M., Houdusse, F., Zamarreno, A.M. & Garcia-Mina, J.M. 2010. The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. Journal of Plant Physiology 167: 1264-1272.
  • Grattapaglia, D. & Machado, M.A. 1998. Micropropagação. In: A.C. Torres, L.S. Caldas & J.A. Buso (eds.). Cultura de tecidos e transformação genética de plantas. 1 ed. Embrapa-CNPH, Brasília, pp.183-260.
  • Hachiya, T. & Sakakibara, H. 2017. Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. Journal of Experimental Botany 68(10): 2501-2512.
  • Hartmann, H.T., Kester, D.E., Davis Júnior, F.T. & Geneve, R.L. 2002. Plant propagation: principles and practices. Englewood Clipps, New York.
  • Horchani, F., Aschi-Smiti, S. & Brouquisse, R. 2010. Involvement of nitrate reductase in the tolerance of tomato (Solanum lycopersicum L.) plants to prolonged root hypoxia. Acta Physiologiae Plantarum 32: 1113-1123.
  • Jaworski, E. 1971. Nitrate reductase assay in intact plant tissues. Biochemical and Biophysical Research Communications 43(6): 1274-1279.
  • Ji, Y., Li, Q., Liu, G., Selvaraj, G., Zheng, Z., Zou, J. & Wei, Y. 2019. Roles of cytosolic glutamine synthetases in Arabidopsis development and stress responses. Plant and Cell Physiology 60: 657-671.
  • Kato, Y., Yamamoto, Y., Murakami, S. & Sato, F. 2005. Post-translational regulation of CND41 protease activity in senescent tobacco leaves. Planta 222: 643-651.
  • Kempinski, C.F., Crowell, S.V., Smeeth, C. & Barth, C. 2013. The novel Arabidopsis thaliana svt2 suppressor of the ascorbic acid-deficient mutant vtc1-1 exhibits phenotypic and genotypic instability. F1000 Research 1: 2-6.
  • Kiba, T., Kudo, T., Kojima, M. & Sakakibara, H. 2011. Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin. Journal of Experimental Botany 62:1399-1409.
  • Kurita, F.M.K. 2015. Assimilation of different nitrogen sources in the induction of the mutiplication of bromeliad Alcantarea imperialis (Carrière) Harms cultivated in vitro Thesis from Instituto de Botânica, São Paulo, Brazil.
  • Kraiser, T., Gras, D.E., Gutierrez, A.G., Gonzalez, B. & Gutierrez, R.A. 2011. A holistic view of nitrogen acquisition in plants. Journal of Experimental Botany 62: 1455-1466.
  • Krapp, A. 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Current Opinion in Plant Biology 25: 115-122.
  • Lea, P.J. & Miflin, B.J. 2003. Glutamate synthase and the synthesis of glutamate in plants. Plant Physiology and Biochemistry 41: 555-564.
  • Li, B., Li, G., Kronzucker, H.J., Balusca, F. & Shi, W. 2014. Ammonium stress in Arabidopsis: Signaling, genetic loci, and physiological targets. Trends in Plant Science 19(2): 107-114.
  • Li, B., Shi, W. & Su, Y. 2011. The differing responses of two Arabidopsis ecotypes to ammonium are modulated by the photoperiod regime. Acta Physiologiae Plantarum 33: 325-334.
  • Lichtenthaler, H.K. & Buschmann, C. 2001. Chlorophylls and carotenoids: measurement and characterization by UV-VIS. Current Protocols in Food Analytical Chemistry F4.3.1-F4. 1-8.
  • Lima, A.P.P.S., Brito, A.L. & Santana, J.R.F. 2020. Micropropagation of Chapada Diamantina ornamental bromeliad. Ciência Rural 50(2): e20180922.
  • Liu, Y. & Wirén, N.von. 2017. Ammonium as a signal for physiological and morphological responses in plants. Journal of Experimental Botany 68: 2581-2592.
  • Luo, J., Zhou, J., Li, H., Shi, W., Polle, A., Lu, M., Sun, X. & Luo, Z.B. 2015. Global poplar root and leaf transcriptomes reveal links between growth and stress responses under nitrogen starvation and excess. Tree Physiology 35: 1283-1302.
  • Magalhães, J.R. & Huber, D.M. 1991. Response of ammonium assimilation enzymes to nitrogen form treatments in different plant species. Journal of Plant Nutrition 14: 175-185.
  • Majerowicz, N., Kerbauy, G.B., Nievola, C.C. & Suzuki, R.M. 2000. Growth and nitrogen metabolism of Catasetum fimbriatum (Orchidaceae) grown with different nitrogen sources. Environmental and Experimental Botany 44: 195-206.
  • Marschner, H. & Marschner, P. 2012. Marschner’s mineral nutrition of higher plants. 3 ed. Academic Press, London.
  • Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gaufichon, L. & Suzuki, A. 2010. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany 105: 1141-1158.
  • Ma, W., Li, J., Qu, B., He, X., Zhao, X., Li, B., Fu, X. & Tong, Y. 2014. Auxin biosynthetic gene TAR2 is involved in low nitrogen-mediated reprogramming of root architecture in Arabidopsis Plant Journal 78:70-79.
  • MCcullough, H. 1967. The determination of ammonia in whole blood by a direct colorimetric method. Clinica Chimica Acta 17(2): 297-304.
  • Miflin, B.J. & Habash, D.Z. 2002. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. Journal of Experimental Botany 53(370): 979-987.
  • Miller, A.J., Fan, X., Orsel, M., Smith, S.J. & Wells, D.M. 2007. Nitrate transport and signaling Journal of Experimental Botany 58(9): 2297-2306.
  • Munné-Bosch, S. & Lalueza, P. 2007. Age-related changes in oxidative stress markers and abscisic acid levels in a drought-tolerant shrub, Cistus clusii grown under mediterranean field conditions. Planta 225: 1039-1049.
  • Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.
  • Myers, N., Mittermeier, R.A., Mittermeier C.G., Fonseca, G.A.B.da & Kent, J. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853-858.
  • Nievola, C.C., Mercier, H. & Majerowisz, N. 2001. Levels of nitrogen assimilation in bromeliads with different growth habits. Journal of Plant Nutrition 24(9): 1387-1387.
  • Orme, C.D.R., Davies, R.G., Burgess, M., Eigenbrod, F., Pickup, N., Olson, V.A., Webster, A.J., Ding, T-S., Rasmussen, P.C., Ridgely, R.S., Statterfield, A.J., Bennett, P.M., Blackburn, T.M., Gaston, K.J. & Owens, I.P.F. 2005. Global hotspots of species richness are not congruent with endemism or threat. Nature 436(7053): 1016-1019.
  • Palatnik, J.F., Carillo, N. & Valle, E.M. 1999. The role of photosynthetic electron transport in the oxidative degradation of chloroplastic glutamine synthetase. Plant Physiology 121: 471-478.
  • Parry, M.A., Andralojc, P.J., Mitchell, R.A., Madgwick, P.J. & Keys, A.J. 2003. Manipulation of Rubisco: the amount, activity, function and regulation. Journal of Experimental Botany 54: 1321-33.
  • Peeters, K.M.U. & Van Laere, A.J. 1992. Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark. Physiologia Plantarum 84: 243-249.
  • Proença, S.L. & Sajo, M.G. 2007. Anatomia foliar de bromélias ocorrentes em áreas de cerrado do Estado de São Paulo, Brasil. Acta Botanica Brasilica 21(3): 657-673.
  • Qin, C., Qian, W., Wang, W., Wu, Y., Yu, C., Jiang, X., Wang, D. & Wu, P. 2008. GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana Proceedings of the National Academy of Sciences 105: 18308-18313.
  • Raab, T.K. & Terry, N. 1994 Nitrogen-source regulation of growth and photosynthesis in Beta vulgaris L. Plant Physiology 105: 1159-1166.
  • Rennenberg, H., Wildhagen, H. & Ehlting, B. 2010. Nitrogen nutrition of pop- lar trees. Plant Biology 12:275-291.
  • Robinson, S.A., Slade, A.P., Fox, G.G., Phillips, R., Ratcliffe, R.G. & Stewart, G.R. 1991. The Role of Glutamate Dehydrogenase in Plant Nitrogen Metabolism. Plant Physiology 95: 509-516.
  • Roosta, H.R. & Schjoerring, J.K. 2007. Effects of ammonium toxicity on nitrogen metabolism and elemental profile of cucumber plants. Journal of Plant Nutrition 30:1933-1951
  • Ruzicka, K., Simaskova, M., Duclercq, J., Petrasek, J., Zazımalova, E., Simon, S., Friml, J., Van Montagu, M.C.E., & Benkova, E. 2009. Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proceedings of the National Academy of Sciences 106: 4284-4289.
  • Silva, P., Couto, J. & Santos, A. 2010. Absorção dos íons amônio e nitrato e seus efeitos no desenvolvimento do girassol em solução nutritiva. Revista de Biologia e Ciências da Terra 10(2): 97-104.
  • Silva, P.P.A., Kurita, F.M.K. & Tamaki, V. 2017. In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45(3): 313-320.
  • Silva, P.P.A. & Tamaki, V. 2012. Micropropagação da bromélia Ananas ananassoides (Baker) L.B. Sm. In: XVI Encontro de Iniciação Científica do Instituto de Botânica. Instituto de Botânica da Secretaria do Meio Ambiente, São Paulo, pp 22.
  • Skopelitis, D.S., Paranychianakis, N.V., Paschalidis, K.A., Plia- konis, E.D., Delis, I.D., Yakoumakis, D.I., Kouvarakis, A., Papa- dakis, A.K., Stephanou, E.G. e Roubelakis-Angelakisa, K.A. 2006. Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 18: 2767-2781.
  • Souza, F.V.D., Souza, A.daS., Serejo,J.A.dosS, Souza, E.H.de, Junghans, T.G. & Silva, M.deJ.de. 2009 Micropropagação do abacaxizeiro e outras bromeliáceas. In: T.G. Junghas & A.S. Souza (eds.). Aspectos práticos da micropropagação de plantas. Embrapa Mandioca e Fruticultura Tropical, Cruz das Almas, pp. 177-205.
  • Taiz, L & Zeiger, E. 2013. Fisiologia Vegetal. 5 ed. Artmed, Porto Alegre.
  • Tamaki, V., Mercier, H. & Nievola, C.C. 2007. Cultivo in vitro de clones de Ananas comosus (L.) Merril cultivar Smooth Cayene em diferentes concentrações de macronutrientes. Hoehnea 34(1): 67-73.
  • Tamaki, V. & Mercier, H. 2007. Cytokinins and auxin communicate nitrogen availability as long-distance signal molecules in pineapple (Ananas comosus). Journal of Plant Physiology 164(11): 1543-1547.
  • Tian, Q., Chen, F., Liu, J., Zhang, F. & Mi, G. 2008 Inhibition of maize root growth by high nitrate supply is correlated with reduced IAA levels in roots. Journal of Plant Physiology 165:942-951.
  • Walch-Liu, P., Ivanov, I., Filleur, S., Gan, Y., Remans, T. & Forde, B.G. 2006. Nitrogen regulation of root branching. Annals of Botany 97: 875-881.
  • Weatherburn, M.W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry 39(8): 971-974.
  • Werner, E.T., Milanez, C.R.D., Mengarda, L.H.G., Vendrame, W.A. & Cuzzuol, G.R.F. 2010. Meios de cultura, reguladores de crescimento e fontes de nitrogênio na regulação da calogênese do pau-brasil (Caesalpinia echinata Lam.). Acta Botanica Brasílica 24(4): 1046-1051.
  • Vidal, E.A., Moyano, T.C., Riveras, E., Contreras-Lopez, O. & Gutierrez, R.A. 2013. Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots. Proceedings of the National Academy of Sciences 10:12840-12845.
  • Xu, G., Fan, X. & Miller, A.J. 2012. Plant nitrogen assimilation and use efficiency. Annual Review of Plant Biology 63: 153-182.
  • Zhang, W., To, J.P.C., Cheng, C.Y., Schaller, G.E. & Kieber, J.J. 2011. Type-A response regulators are required for proper root apical meristem function through post-transcriptional regulation of PIN auxin efflux carriers. Plant Journal 68: 1-10.
  • Zhang, W., Swarup, R., Bennett, M., Schaller, G.E. & Kieber, J.J. 2013. Cytokinin induces cell division in the quiescent center of the Arabidopsis root apical meristem. Current Biology 23: 1979-1989.
  • Zou, N., Li, B., Dong, G., Kronzucker, H.J. & Shi, W.M. 2012. Ammonium-induced loss of root gravitropism is related to auxin distribution and TRH1 function, and is uncoupled from the inhibition of root elongation in Arabidopsis. Journal of Experimental Botany 63: 3777-3788.
  • Zoucchi, M.R., Silva, M.W., Sibov, S.T. & Pires, L.L. 2019. Ornamental and landscape potential of a bromeliad native to the Cerrado. Ornamental Horticulturae 25(4): 425-433.

Publication Dates

  • Publication in this collection
    11 Nov 2020
  • Date of issue
    2020

History

  • Received
    15 July 2020
  • Accepted
    11 Oct 2020
Instituto de Pesquisas Ambientais Av. Miguel Stefano, 3687 , 04301-902 São Paulo – SP / Brasil, Tel.: 55 11 5067-6057, Fax; 55 11 5073-3678 - São Paulo - SP - Brazil
E-mail: hoehneaibt@gmail.com