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 2007). 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. 2000, Orme et al. 2005). 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. 2011). Most culture mediums are basic formulations such as the MS medium formulated by Murashige and Skoog (1962), which is the most commonly used for in vitro culture (Aranda-Peres et al. 2009, Werner et al. 2010, Lima et al. 2020). Among the nutrients, that compose the culture medium, the nitrogen (N) is required in a major amount by most plants (Marschner & Marschner 2012, Buchanan et al. 2015, Esteban et al. 2016).

Ammonium (NH₄⁺) and nitrate (NO₃^-) ions are the most available N sources in the soil solution to be absorbed by plant roots (Epstein & Bloom 2006, Marschner & Marschner 2012, Esteban et al. 2016, Hachiya & Sakakibara, 2017). The roots absorb these two ions by high- (HATS) and low- (LATS) affinity transporters that are specific for each N form (Miller et al. 2007, Kraiser et al. 2011, Xu et al. 2012). 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. 2010, Braun et al. 2013). 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 2012, Taiz & Zeiger 2013). 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 2012, Taiz & Zeiger 2013, Buchanan et al. 2015, Liu & Wirén 2019). 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. 2006, Andrews et al. 2013, Luo et al. 2015). 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 2012, Andrews et al. 2013). Although the process of N assimilation in plants is well-known, much information is still lacking to better understand this metabolic activity (Krapp 2015).

After in vitro production, plants need to be acclimatized before their transfer to the field (Grattapaglia & Machado 1998, Berilli et al. 2011). 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. 2002, Hartmann et al. 2002, Albert 2004). Sudden changes in these conditions might be limiting for the propagation of some species (Grattapaglia & Machado 1998, Bregonci et al. 2008, Berilli et al. 2011), and therefore, highlight the importance of acclimatization studies.

Some studies showed that the joint use of NH₄⁺ and NO₃^- promotes plant growth (Hachiya & Sakakibara, 2017), including some bromeliad species, such as Ananas comosus (L.) Merr., Vriesea fosteriana L.B.Sm., Tillandsia pohliana Mez, and Nidularium minutum Mez (Endres & Mercier 2001, Nievola et al. 2001, Tamaki & Mercier 2007, Silva et al. 2010, Andrade & Tamaki 2016). 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. 2008, Dias et al. 2010, Dias et al. 2011a, b, Carvalho et al. 2012, Silva & Tamaki 2012, Dias et al. 2013, Silva et al. 2017). 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). Posteriorly, the seedlings were transferred to 250 mL glass flasks containing 30 mL of modified Murashige & Skoog (1962) (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). 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) 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 1967, MCcullough 1967) 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) 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 1971 modified by Nievola et al. 2001). 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). 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) 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 2007, Marschner & Marschner 2012, Esteban et al. 2016, Ji et al. 2019). However, some aspects of the mechanism of the NH₄⁺ toxicity are yet not fully understood (Britto & Kronzucker 2002, Li et al. 2014). Nevertheless, the tissue toxicity of the NH₄⁺ excess can be attributed to uncoupling of photophosphorylation, disturbance in pH balance (Li et al. 2011, Li et al. 2014), high carbon consumption and accumulation of abscisic acid and free putrescine in the plant tissues (Garnica et al. 2010). Furthermore, Andrews et al. (2013) 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 2007) and Triticum aestivum L.cv Amarok (Garnica et al. 2010). 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 2012, Liu & Wirén 2019). 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 1995, Miller et al. 2007, Krapp 2015), and the cytokinin synthesis, which is responsible for regulating cell growth (division and multiplication) and development (expansion) in higher plants (Garnica et al. 2010, Andrews et al. 2013, Buchanan et al. 2015). 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) 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. 2008, Kempinski et al. 2013). 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. 2012). 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. 2010, Andrews et al. 2013). 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. 2009, Zhang et al. 2011, Zhang et al. 2013). 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 2015). Studies have reported that plants supplemented with non-toxic concentrations of NH₄⁺ present higher chlorophyll content per leaf unit area (Raab & Tarry 1994, Claussen & Lenz 1999). 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), 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. 1999, Krapp 2015). The NO₃^- stored is important for the cellular osmotic balance, besides being a nitrogen reserve source for N-shortage periods (Chen et al. 2004, Miller et al. 2007, Krapp 2015).

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 1991), Vaccinium corymbosum L., Rubus idaeus L., and Fragaria ananassa Duch. (Claussen & Lenz 1999). However, plants of Catasetum fimbriatum (Morren) Lindl. (Majerowicz et al. 2000) and Solanum lycopersicum L. cv. Rio Grande (Horchani et al. 2010) 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 2002, Bernard & Habash 2009, Rennenberg et al. 2010, Xu et al. 2012, Krapp 2015), 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 1992, Chen et al. 1997) 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. 1999, Parry et al. 2003, Kato et al. 2005, Masclaux-Daubresse et al. 2010).

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 2003, Forde & Lea 2007). 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 2002, Lea & Miflin 2003). 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 1998, Hartmann et al. 2002, Bregonci et al. 2008, Berilli et al. 2011). 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) 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) 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. 2011, Vidal et al. 2013). 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. 2006, Tamaki & Mercier 2007, Tian et al. 2008). 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 2009, Ma et al. 2014).

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 NH₄NO_3. 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).

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Publication Dates

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

History

• Received
  15 July 2020
• Accepted
  11 Oct 2020