ABSTRACT
(Daily temperature amplitude plays a key role in the metabolic adaptation to high-temperature stress in the rupicolous bromeliad Alcantarea imperialis (Carrière) Harms). Climatic variations predicted for a future scenario may influence nitrogen metabolism, affecting the survival of plants in the face of climate change. The objective of this work was to investigate the tolerance of plants of Alcantarea imperialis to different temperatures. This species is native from inselbergs where daily temperatures vary from 5 to 40 °C. Three-month-old plants were kept at 15 °C, 25 °C, 37 °C and 37 °C/15 °C (day/night) for 15 days. The activities of glutamine synthetase (GS), glutamate dehydrogenase (GDH), ammonium content, growth and photosynthetic parameters (pigments and Fv/Fm ratio) were measured. Only 40% of plants under constant 37 °C survived, showing an increase in the ammonium concentration and GDH activity while photosynthetic pigment content, Fv/Fm ratio and GS activity decreased. All plants under other temperatures survived without damage. This result showed that cool nights are necessary for the plant to recover from the stress of the high diurnal temperature. These insights are crucial to understand the response of plants to temperature increases in a climate change scenario, constituting essential knowledge for the formulation and implementation of biodiversity conservation policies.
Keywords: ammonium; climatic changes; nitrogen metabolism; thermal variations
RESUMO
(A amplitude diária da temperatura desempenha um papel fundamental na adaptação metabólica ao estresse de alta temperatura na bromélia rupícola Alcantarea imperialis (Carrière) Harms). Variações climáticas previstas para um cenário futuro podem influenciar o metabolismo do nitrogênio, afetando a sobrevivência das plantas frente às mudanças climáticas. O objetivo deste trabalho foi investigar a tolerância de plantas de Alcantarea imperialis a diferentes temperaturas. Esta espécie é nativa de inselbergs onde as temperaturas diárias variam de 5 a 40 °C. Plantas com três meses de idade foram mantidas a 15 °C, 25 °C, 37 °C e 37 °C/15 °C (dia/noite) durante 15 dias. Foram medidas as atividades da glutamina sintetase (GS), glutamato desidrogenase (GDH), teor de amônio, crescimento e parâmetros fotossintéticos (pigmentos e relação Fv/Fm). Apenas 40% das plantas sob temperatura constante de 37 °C sobreviveram, mostrando um aumento na concentração de amônio e na atividade de GDH, enquanto o conteúdo de pigmento fotossintético, a relação Fv/Fm e a atividade de GS diminuíram. Todas as plantas sob outras temperaturas sobreviveram sem danos. Este resultado mostrou que são necessárias noites frias para que a planta se recupere do estresse da alta temperatura diurna. Estes conhecimentos são cruciais para compreender a resposta das plantas aos aumentos de temperatura num cenário de alterações climáticas, constituindo conhecimento essencial para a formulação e implementação de políticas de conservação da biodiversidade.
Palavras-chave: amônio; metabolismo do nitrogênio; mudanças climáticas; variações térmicas
Introduction
According to the Intergovernmental Panel on Climate Change (IPCC), climate change could alter water availability and increase temperatures by up to 4.8 °C (IPCC 2014). Temperature changes will not occur linearly but will be different in each region of the planet and between day and night (Sullivan et al. 2020). Night temperatures are prone to increase faster than daytime temperatures in most locations leading to a decline in diurnal temperature amplitude in over 51% of the land area (Xia et al. 2014). Although in tropical regions the daily amplitude is about 10 degrees (Prasad et al. 2011), rupiculous environments, such as inselbergs, can reach 20 to 30 degrees of amplitude (Porembski, S. 2007). Rupiculous bromeliads species are exposed to large diurnal thermal amplitudes, ranging between 5 and 38 °C (Ibama 1989) and require adaptations and metabolic adjustments to survive in a wide temperature amplitude.
Cach-Pérez et al. (2014) proposed that bromeliads are suitable and reliable models for evaluating plant responses to global climate change scenarios. It was proposed that bromeliads could be good models for studying tolerance to thermal fluctuation (Duarte et al. 2019) over a few days or during the same day. Alcantarea imperialis (Carrière) Harms is a rupiculous bromeliad specie living in conditions of great variation in day/night temperature in its natural environment (Barbará et al. 2009, Versieux and Wanderley 2015) presenting biochemical adjustments to resist sudden temperature changes. According to Mollo et al. (2011), the best growth condition for this species is the alternating temperature (15 °C night /30 °C day), even better than constant 25 °C. However, Cheng-gang et al. (2013) reported that thermal amplitude has been decreasing as an effect of climate change. Nevertheless, warmer nights are associated with lower yields in crops (Vara Prasad & Djanaguiraman 2011). Recently, Bruhn et al. (2022) reported that constant temperature can reduce the respiration rate depending on the sensitivity of the plant and that this can affect plant survival on warmer nights. These effects are specific to each species and has not been evaluated in rupicolous bromeliads, which are considered resistant to different temperatures in their natural environment.
High temperatures stimulate plant proteolysis (Buchanan et al. 2015) and directly affect nitrogen metabolism (Giri et al. 2017), producing metabolites such as ammonium, which can become toxic at high accumulated levels (Steban et al. 2016, De La Haba et al. 2014). Elevated levels of this ion have been associated with uncoupled electron transport photophosphorylation, altered intracellular pH, reduced growth and perturbed osmotic balance (Gerendás et al. 1997). To reduce the toxic action of ammonium, its assimilation must be efficient and with the increase in temperature, there is a change in the activities of some nitrogen metabolism enzymes, such as glutamine synthetase (GS) which reduces its activity and glutamate dehydrogenase (GDH) which increases (De La Haba et al. 2014). Glutamate dehydrogenase (GDH) and glutamine synthetase (GS) are the primary enzymes responsible for ammonium reassimilation (Cui et al. 2006, Liang et al. 2011, Zhong et al. 2017).
Understanding the effects of temperature on the physiology plants is paramount to assess the effects of climate change on biodiversity. The exploration of the physiological responses of a rupicolous species in particular, as a model for thermal variation resistance, may elucidate the thresholds governing plant resistance to climatic changes. This study was proposed to increase the knowledge about the impact of elevated temperatures and changes in daily temperature amplitude on the metabolism of A. imperialis, by cultivating juvenile plants under different temperatures and analyzing key parameters of nitrogen metabolism.
Material and methods
Plant Material - Seeds of Alcantarea imperialis were disinfected and germinated on filter paper in Petri dishes. After one month, the seedlings were transferred to transparent trays containing 600 mL of Pinus bark and fertilized every two weeks with 40 mL of MS/2 (MS medium with 50% nutrient concentration, Murashige and Skoog 1962), without sucrose or agar. During both germination and post-transfer, the seeds and seedlings were maintained at 25±2 °C in a culture room with a 12h photoperiod and 30 μmol m−2.s−1 of photosynthetically active radiation.
This light intensity is routinely described in bromeliad physiology studies, such as Mollo et al. (2011) e Aoyama et al. (2012) kept in a growth room and subsequently submitted to germination chambers, as described below.
Temperature Treatments - Three months after the transference of seedlings, trays were placed in growth chambers at 15 °C, 25 °C, 37 °C and 37/15 °C (day/night). Samples were collected and analyzed after 1, 4, 7, 11, and 15 days.
For the biometric analyzes carried out, 10 plants were separated from each collection and measurements were taken (leaf number, root number, leaf size in cm, root size in cm and dead leaves). These measurements were carried out after 15 days of cultivation on plants maintained in different heat treatments.
Leaf temperatures were measured with a Testo infrared camera (Model #875-1i, Lenzkirch, Germany), as described previously (Liu Y et al. 2011). As shown in Figure 1, the leaf and growth chamber temperatures were consistent with one another.
Thermographic images of Alcantarea imperialis (Carrière) Harms at (a) 25 ºC; (b) alternating 15 ºC and 37 ºC; (c) 15 ºC and (d) 37 ºC.
Endogenous ammonium content and Soluble protein quantification - Fresh leaf mass (0.1 g) was ground in liquid nitrogen and transferred to microtubes. Then, 1 mL of ultra-purified water was added to the microtubes and centrifuged. The supernatant was collected, and the ammonium concentrations were measured using the phenol-hypochlorite method (Weatherburn 1967; McCullough 1967). In a 1.5 mL tube, 0.5 mL of Phenol solution was added to 200 μL of the plant extract, followed by 0.5 mL of sodium hypochlorite. The mixture was incubated for 30 minutes at 35 °C, and the absorbance was recorded at 625 nm. Commercially available ammonium sulfate was used as the standard.
Soluble protein content in the enzymatic extracts was assessed by mixing 30 µL of protein extract and 2.5 mL of Coomassie Brilliant Blue G250 (Bradford 1976). After 5 minutes, absorbance was measured at 595 nm. Bovine serum albumin (BSA) was used as the standard.
Enzyme analyses - GS and GDH enzymes were extracted from 0.2 g of fresh leaf mass, previously ground in liquid nitrogen and transferred to pre-cooled microtubes. Next, 1.5 mL of 0.05 M imidazole buffer, pH 7.9, containing 0.005 M dithiothreitol was added to the microtubes. The samples were centrifuged, and the supernatants were loaded onto a PD10 column (Sephadex G-25) for desalting. After isolation, the samples were maintained at 4 °C until the GS and GDH activities were determined.
According to an in vitro method described by Elliott (1955) and Farnden & Robertson (1980), the GS activity was determined. Briefly, the GS assay reactions were initiated by adding 50 μL of extract to 450 μL of Assay Buffer (0.1 mM imidazole buffer, pH 7.5, 49 mM hydroxylamine, 40 mM MgCl2, 160 mM glutamate, and 45 mM ATP). The reactions were monitored for 1 hour at 35 °C and terminated by adding 1 mL of the Stop Solution (0.123 M ferric chloride, 0.25 M HCl, and 0.1225 M trichloroacetic acid - TCA). The activity was calculated based on the absorbance at 540 nm.
The GDH activity was assessed using the method described by Bulen (1956), with modifications, and is based on the consumption of NADH during the reaction. Reactions were initiated by adding 0.25 mL of the extract to 0.75 mL of the Assay Buffer (0.1 M TRIS buffer, pH 8.2, with13.3 mM 2-oxoglutarate, 0.1 M (NH4)2SO4 and 0.16 mM NADH). All reagents were incubated for 10 minutes at 30 °C before starting the reaction. The enzymatic activity of GDH was calculated based on the absorbance at 340 nm after 3 minutes, using the molar extinction coefficient (ε) of 6.22 × 106 mmol−1 cm−1 for NADH.
Fluorescence - Fully-expanded leaves from 10 plants were dark-adapted for 30 minutes, and the fluorescence of the adaxial epidermis was measured in a Hansatech fluorimeter (City, State, Country). Quantum efficiency values (i.e., Fv/Fm ratio) of photosystem II (PSII) were determined for the thermal treatments and de-acclimation. Additionally, the initial (F0), maximum (Fm), and variable (Fv) fluorescence were determined for de-acclimation.
Content of photosynthetic pigments - Chlorophylls a and b and carotenoids were extracted from the leaves, according to Munné-Bosch & Lalueza (2007), with modifications. First, fresh leaf mass was ground in liquid nitrogen. Then, 0.05 g of this material was homogenized in 1 mL of 100% acetone. This suspension was placed in an ultrasonic bath for 30 minutes at 4 °C, centrifuged, and the supernatants were collected. This procedure was repeated using the pellet as the starting material. In the end, the two supernatants were combined and measured spectrophotometrically. The wavelengths and equations for the calculation of photosynthetic pigment concentrations were previously described by Lichtenthaler (1987).
Statistical analysis - The experiment was completely randomized. Data were subjected to analysis of variance (ANOVA) testing, and differences between means were compared using Tukey’s test. The level of significance was set at p ≤ 0.05.
Results
As shown in Table 1, all juvenile A. imperialis plants cultivated at 15 °C, 25 °C and 37 °C/15 °C (day/night) survived for the entire experimental period. In contrast, only 40% of the plants exposed to high constant temperature (37 °C) survived, despite the longer and more abundant roots. It is important to note that the measurements were only performed with the plants that survived. Consequently, some of the parameters do not reflect the detrimental effects of the high constant temperature.
Survival (%) and biometric parameters of leaves and roots from Alcantarea imperialis (Carrière) Harms grown at different temperatures for 15 days. Results are presented as the mean ± SEM/SD/SE. Statistically significant differences (p < 0.05) detected by ANOVA are indicated with (a), whereas significant differences detected by Tukey’s test are indicated with (b).
The dry mass of plants grown at 37 °C was significantly reduced compared to the other groups, reaching the lowest value after 15 days. When comparing the dry mass values of the 15 °C, 25 °C and 37 °C/15 °C treatments, no significant differences were detected. Plants grown at 25 °C had significantly more leaves (table 1).
In the A. imperialis plants cultivated at 15 °C, 25 °C and 37 °C/15 °C (day/night), the leaf ammonium concentrations remained relatively stable throughout the experimental period. However, the concentrations of this ion showed a noticeable increase after four days and a sharp rise from days 7 to 15 in plants cultivated at a constant 37 °C. After 15 days, ammonium concentrations in the plants grown at 37 °C were up to 30 times higher than those detected in the other groups (figure 2 a). As shown in Figure 2 b, we observed a reduction in soluble protein that coincided with the rise in ammonium levels after day 7. In comparison, the soluble protein content in the plants grown at 15 °C, 25 °C, and 37 °C/15 °C (day/night) was approximately ten times greater than the plants exposed to constant 37 °C after 15 days.
Concentrations of ammonium (a) and soluble protein (b) in plants of Alcantarea imperialis (Carrière) Harms grown at grown at 15 ºC, 25 ºC, 37 ºC and alternating 15 ºC and 37 ºC.
At the beginning of the temperature treatments, the GS activity decreased slightly in plants cultivated at a constant 15 °C (figure 3 a) and 25 °C (figure 3 a). However, it returned to the initial activity at the end of the experiment (15 days). This variation did not occur in the 37 °C/15 °C treatment (figure 3 a). At high constant temperature (37 °C), the activity decreased significantly and remained low until the end of the experiment (figure 3 a). On the other hand, GDH activity increased after 11 days when the plants were maintained at 37 °C (figure 3 b). The other treatments (15 °C, 25 °C and 37 °C/15 °C) showed no significant changes in the GDH activity.
Glutamine synthase (GS) activity (a) and Glutamate dehydrogenase (GDH) activity (b) in the above ground part of Alcantarea imperialis (Carrière) Harms grown at 15 ºC, 25 ºC, 37 ºC and alternating 15 ºC and 37 ºC.
As shown in Figure 4 a, the Fv/Fm ratio remained close to 0.8 for the 15 °C and 25 °C treatments throughout the experimental period. In plants cultivated at 37°/15 °C, the Fv/Fm ratio was decreased at days 7 and 11 but recovered on day 15. On the other hand, the Fv/Fm ratio of plants cultivated at 37 °C was attenuated on day seven and continued to fall until day 15, reaching approximately 0.3.
Maximum quantum efficiency of PSII (Fv/Fm ratio) (a); chlorophyll a (b); chlorophyll b (c); carotenoids in plants (d) of Alcantarea imperialis (Carrière) Harms grown at 15 ºC, 25 ºC, 37 ºC and alternating 15 ºC and 37 ºC.
The chlorophyll a (figure 4 b), chlorophyll b (figure 4 c) and carotenoid (figure 4 d) concentrations decreased after seven days in plants grown at a high constant temperature, reaching half of the initial values at the end of the experiment. In contrast, no significant changes were observed throughout the experiment in the 15 °C, 25 °C and 37 °C/15 °C treatments even though plants were exposed to 37 °C during the day.
Discussion
The increase in night temperature has been the subject of intense study in the last decade, and it is already known that this has a negative influence on many plant species (Peng et al. 2013). In the present study, A. imperialis showed a set of stress responses when the night temperature was high. Cheng-gang et al. 2013 pointed out that in climate change scenario could be reducing daytime thermal alternation, causing damage to cultivated plants such as rice, which was studied by, at temperatures of 35/30 °C. Our study showed nitrogen metabolism alterations, accompanied by a deleterious response in a plant considered resistant to temperature variations in rupiculous environment.
Plants of A. imperialis exposed to a high constant temperature (37 °C Day and night) for 15 days exhibited reduced growth and high mortality (60%) concomitantly with ammonium increase. In contrast, all plants exposed to 15 °C, 25 °C and 37 °C/15 °C (day/night) temperatures did not show these symptoms. The observed differences in growth between the constant 37 °C and alternating 37 °C/15 °C treatments indicate that thermal amplitude is essential to the recovery of the plants from the high-temperature effects during the day. Stressed plants presented similar stages of a kind set of symptoms that Vartapetian (2006) named metabolic syndrome: alarm, adaptation, and exhaustion stages. In our work, we considered responses to thermal stress.
Efficient free ammonium assimilation can occur if the GS-GOGAT cycle pathway is active (Buchanan et al. 2015). However, our results showed that GS activity reduced during the first days of incubation only at constant 37 °C, leading to failure in efficient ammonium assimilation, as the enzyme may have been compromised. Shan et al. (2016) also related a reduced GS activity at high temperatures, which interrupts NH4+ assimilation.
Considering that GDH plays an essential role in ammonium assimilation and reassimilation under extreme temperature stress (Cui et al. 2006), the increase in free ammonium in A. imperialis concomitant as well as with depletion in soluble protein content are expected to be deaminant GDH activity after seven days at 37 °C. De La Haba et al. (2014) verified that, under high temperature, sunflower leaves in the process of senescence show higher activity of glutamate dehydrogenase releasing ammonia from amino acids, during the remobilization of nitrogen. Similar decrease in proteins were observed in tomato plants exposed to high-temperature stress (Giri et al. 2017). Increased GDH activity might not be playing ammonium assimilation but glutamine anaplerosis to replenish alpha-ketoglutarate for deterioration of TCA cycle and photosynthesis (Robinson et al. 1991, Stewart et al. 1995, Diab & Limami 2016, Miyashita & Good 2008). Notably, plants subjected to high temperatures only during the day (alternating 37 °C/15 °C) did not display alterations in ammonium or soluble protein levels, showing the important role of cool nights in maintaining physiological functions.
High temperature in photosynthesis is related to the imbalance of plant homeostasis (Mathur & Jajoo 2014). It modifies the enzymatic activities and can cause the accumulation of other metabolites such as proline, glycine, soluble sugars and quaternary ammonium compounds (Mathur & Jajoo 2014). As photosynthesis is impaired, ammonium buildup can be a result of this stress (De La Haba et al. 2014). In our study, the deleterious effect of high night temperature could be caused by oxidative damage to A. imperialis leaves, leading to decreased photosynthesis (Prasad & Djanaguiraman 2011). This result reinforces that low temperature at night is necessary for the plant to recover from heat stress which occurs during the day. These results could be due to damage to photosynthesis. Although high temperatures accelerate metabolism (Pyl et al. 2012), an imbalance causing ammonium accumulation and reduced protein content are considered detrimental effects of the high constant temperature on photosynthesis (Neales 1972, Mathur et al. 2014). The reduced Fv/Fm ratio and photosynthetic pigment content observed here in A. imperialis can indicate photosynthesis disturb and stress response during higher night temperatures (Prasad & Djanaguiraman 2011). In our work pigments concentrations decreased soon after seven days in plants grown at a high constant temperature, reaching half of the initial values at the end of the experiment. High temperatures have been shown to modify the membrane composition and structure of the chloroplasts, resulting in thylakoid membrane disorganization and PSII inactivation (Prasad & Djanaguiraman 2011), including the dismantling of chloroplasts (De La Haba et al. 2014). It is plausible that chloroplast disassembly may explain the reduced Fv/Fm ratio and photosynthetic pigment content observed here with A. imperialis. Even though it is not possible to identify a single factor as a deleterious effect onA. imperialis, constant high temperatures and production of ammonium, can result in a set of irreversible effects.
Interestingly, constant cold (15ºC) did not induce damage in imperial bromeliad of this study. Responses as the Fv/Fm ratio remained close to 0.8 throughout the experimental period. On the other hand, the Fv/Fm ratio of plants cultivated at 37 °C was attenuated on day seven and continued to fall until day 15, reaching approximately 0.3. It was previously proposed that cool nights improve photosynthesis in bromeliads (Neales 1972), and constant low temperature (15 °C) does not constitute stress in this species (Mollo et al. 2011). Therefore, juvenile A. imperialis plants can tolerate temperatures of up to 37 °C as long as the nights are cooler. Under conditions in which a cooler period is not provided (e.g., constant 37 °C), the photosynthetic apparatus is damaged, and both the Fv/Fm ratio and photosynthetic pigment content are reduced. Therefore, in the natural environment of A. imperialis, cooler nights play a crucial role in survival, allowing the plants to recover from the stress caused by the high daytime temperatures. This metabolic performance likely favors their establishment in the rupicolous environment. We propose three stages related to stress responses to high-temperature constant: in the first 4 days there is a slight increase in ammonium while there is still some storage (alarm stage). This adapted metabolism sustains the plantlets for three more days (adaptation stage) and from this day to the fifteenth, there is an increase in free ammonium, associated with the fall of soluble proteins and the onset of a response that, if not reversed, will lead to the death of the plant (exhaustion). The description of the phenomenon as a series of discrete qualitative alterations of the metabolism creates a model that can be used to predict the response of plants to temperature stress that may have many applications to the study of growth and conservation of biodiversity.
Acknowledgments
Gabriela Maria Cabral Nascimento thanks the Conselho Nacional de Desenvolvimento Tecnológico e Científico, for the Doctoral fellowship (134022/2017-3). The authors would also like to thank the Fundação de Amparo à Pesquisa do Estado de São Paulo, for grant support (2017/50341-0).
Literature cited
- Aoyama, E.M., Versieux, L.M., Nievola, C.C. & Mazoni-Viveiros, S.C. 2012. Avaliação da eficiência da propagação de Alcantarea imperialis (Bromeliaceae) cultivada in vitro e ex vitro Evaluating the effectiveness of the propagation of Alcantarea imperialis (Bromeliaceae) cultivated in vitro and ex vitro. Rodriguésia 63(2): 321-331.
- Barbará, T., Lexer, C., Martinelli, G., Maayo, S., Fay, M.F. & Heuertz, M. 2008. Within-population spatial genetic structure in four naturally fragmented species of a neotropical inselberg radiation, Alcantarea imperialis, A. geniculata, A. glaziouana and A. regina (Bromeliaceae). Heredity 101: 285-296.
- Bradford, M.M. 1976. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72: 248-254.
- Bruhn, D., Newman, F., Hancock, M., Povlsen, P., Slot, M., Sitch, S., Drake, J., Weedon, G.P., Clark, D.B., Pagter, M., Ellis, R.J., Tjoelker, M.G., Andersen, K.M., Correa, Z.R., McGuire, P.C. & Mercado L.M. 2022. Nocturnal plant respiration is under strong non-temperature control. Nature Communications 13: 5650.
- Buchanan, B.B., Gruissem, W., Jones, R.L. 2015. Biochemistry and Molecular Biology of Plants. 2 ed. Wiley-Blackwell, Hoboken.
- Cach-Pérez, J., Andrade, M.J.L. & Reyes-García, C. 2014. La susceptibilidad de las bromeliáceas epífitas al cambio climático. Botanical Sciences 92: 157-168.
- Liang, C., Zhang, Q., Xu, G., Wang, Y., Ryu, O. & Li, T. 2013. High Temperature During Rice Grain Filling Enhances Aspartate Metabolism in Grains and Results in Accumulation of Aspartate-Family Amino Acids and Protein Components, Rice Science 20(5): 343-348.
- Cui, L., Cao, R., Li, J., Zang, L. & Wang, J. 2006. High temperature effects on ammonium assimilation in leaves of two Festuca arundinacea cultivars with different heat susceptibility. Plant Growth Regulation 49: 127-136.
- De la Haba, P., De la Mata, L., Molina, E. & Aguera, E. 2014. High temperature promotes early senescence in primary leaves of sunflower (Helianthus annuus L.) plants. Canadian Journal of Plant Science, 94(4): 659-669.
- Diab, H. & Limani, A.M. 2016. Reconfiguration of N Metabolism upon Hypoxia Stress and recovery: roles and aminotransferase (AlaAT) and glutamate dehydrogenase (GDH). Plants, 5(2): 25.
- Duarte, A.A., da Silva, C.J., Marques, A.R., Modolo, L.V. & Filho, J.P.L. 2019. Does oxidative stress determine the thermal limits of the regeneration niche of Vriesea friburgensis and Alcantarea imperialis (Bromeliaceae) seedlings? Journal of Thermal Biology 80: 150-157.
- Elliott, F. H. & Andrew V. Schally. 1955. Chromatography of steroids produced by rat adrenals in vitro. Canadian journal of biochemistry and physiology 33(2): 174-180.
- Farnden, K.J.S. & Robertson, J.G. 1980. Methods for studying enzyme involved in metabolism related to nitrogenase. In: Bergsen, F.J. (ed.), Methods for Evaluating Biological Nitrogen Fixation. Jonh Wiley & Sons Ltda, Nova Jersey, pp. 279-286.
- Gerendás, J., Zhu, Z., Bendixen, R., Rateliffe, R.G. & Sattelmacher, B. 1997. Physiologial and biochemical processes related to ammonium toxicity in higher plants. Journal of Plant Nutrition and Soil Science 160: 239-251.
- Giri, A., Heckathorn, S., Mishra, S. & Krause, C. 2017. Heat stress decreases levels of nutrient-uptake and assimilation proteins in tomato roots. Plants, 6(1): 6.
- IBAMA. 1989. Unidades de conservação do Brasil: parques nacionais e reservas biológicas. ed. Ministério do Interior, Brasília.
- IPCC. 2014. Climate Change 2014: Impacts, Adaptation, and Vulnerability. Part A: Global and Sectoral Aspects. Contribution of Working Group II to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge.
- Liang, C.G., Chen, L.P., Wang, Y., Liu, J., Xu, G.L. & Li, T. 2011. High temperature at grain-filling stage affects nitrogen metabolism enzyme activities in grains and grain nutritional quality in rice. Rice Science 18: 210-216.
- Lichtenthaler, H.K. 1987. Chlorophylls and Carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology 148: 350-382.
- Liu.Y, Subhas.C., Yan.J., Song.C., Zhao.J. & Li. J. 2012. Maize leaf temperature responses to drought: Thermal imaging and quantitative trait loci (QTL) mapping. Environmental and Experimental Botany 71(2): 158-165.
- Majerowicz, N. 2012. Fotossíntese. In: Kerbauy, G.B. Fisiologia Vegetal (eds.). Guanabara Koogan, Rio de Janeiro, pp. 117-121.
- Mathur, S., Agrawal, D. & Jajoo, A. 2014. Photosynthesis: response to high temperature stress. Journal of Photochemistry and Photobiology B Biology, 137: 116-126.
- McCullough, H. 1967. The determination of ammonia in whole blood by a direct colorimetric method. Clinica Chimica Acta 17: 297-304.
- Miyashita, Y. & Good, A.G. 2008. NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis Thailand duringdark-induced carbon starvation. Journal of Experimental Botany 59(3): 667-680.
- Mollo, L., Martins, M.M.C., Oliveira, V.F., Nievola, C.C. & Figueiredo-Ribeiro, R.C.L. 2011. Effects of low temperature on growth and non-structural carbohydrates in the Imperial Bromeliad Alcantarea imperialis cultured in vitro. Plant Cell Tissue and Organ Culture 107(1):141-149.
- 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: 473-497.
- Neales, T.F. 1972. Effect of night temperature on the assimilation of carbon dioxide by mature pineapple plants, Ananas comosus (L.) merr. Australian Journal of Biological Sciences 26 (3): 539-546.
- Peng, S., Piao, S., Ciais, P., Myneni, R.B., Chen, A., Chevallier, F., Dolman, A.J., Janssens, I.A., Peñuelas, J., Zhang, G., Vicca, S., Wan, S., Wang, S. & Zeng, H. 2013. Asymmetric effects of daytime and night-time warming on Northern Hemisphere vegetation. Nature 501: 88-92.
- Pyl, E.T., Piques, M., Ivakov, A., Schulze, W., Ishihara, H., Stitt, M. & Sulpice, R. 2012. Metabolism and Growth in Arabidopsis Depended on the Daytime Temperature but are Temperature-Compensated Against Cool Nights. The Plant Cell 24: 2443-2469.
- Porembski, S. 2007. Tropical inselbergs: habitat types, adaptive strategies and diversity patterns. Revista Brasileira de Botânica 30(4): 579-586.
- Prasad P.V. & Djanaguiraman, M. 2011. High night temperature decreases leaf photosynthesis and pollen function in grain sorghum. Functional Plant Biology 38: 993-1003.
- Robinson, S.A., Slade, A.P., Fox, G.G., Phillips, R., Ratcliff, R.G. & Stewart, G.R. 1991. The Role of Glutamat Dehydrogenaze in Plant Nitrogen Metabolism. Plant Physiology 95: 509-516.
- Shan, X., Zhou, H., Sang, T., Shu, S., Sun, J. & Gou, S. 2016. Effects of exogenous spermidine on carbon and nitrogen metabolism in tomato seedlings under high temperature. Journal of the American Society for Horticultural Science 141(4): 381-388.
- Steban, R., Ariz, I., Cruz, C. & Moran, J.S. 2016. Review: Mechanisms of ammonium toxicity and the quest for tolerance. Plant Science 248: 92-101.
- Stewart, G.R., Shatilov, V.R., Turnbull, M.H. & Robinson, G.R. 1995. Evidence that glutamate dehydrogenase plays a role in the oxidative deamination of glutamate in seedlings of Zea mays. Functional Plant Biology 22(5): 805-809.
- Sullivan, M.J.P., Lewis, S.L., Affum-Baffoe, K. Castilho, C., Costa, F., Sanchez, A.C., Ewango, C.E.N., Hubau, W., Marimon, B. & Monteagudo-Mendoza, A. 2020. Long-term thermal sensitivity of Earth’s tropical forests. Science 368(6493): 869-874.
- Vartapetian, B.B., Generosova, I.P., Zakhmylova, N.A. & Snkhchyan, A.G. 2006. Demonstration of plant adaptation syndrome in plants and possible molecular mechanisms of its realization under conditions of anaerobic stress. Russian Journal of Plant Physiology 53(5): 663-670.
- Versieux, L.M., & Wanderley, M.G.L. 2015. Bromélias gigantes do Brasil. Capim Macio & Offset Gráfica e Editora LTDA, Natal.
- Weatherburn, M.W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry 39: 971-974.
- Xia, J., Chen, J., Piao, S., Ciais, P., Luo, Y., & Wan, S. 2014. Terrestrial carbon cycle affected by non-uniform climate warming. Nature Geoscience 7(3): 173-180.
- Zhong, C. & Cao, X. 2017. Nitrogen Metabolism in Adaptation of Photosynthesis to Water Stress in Rice Grown under Different Nitrogen Levels. Frontier in Plant Science 8: 1-15.