Research articleWhat does the RuBisCO activity tell us about a C3-CAM plant?
Introduction
Water stress is the primary environmental factor responsible for the worldwide loss of food crops, and global climate change indicates the expansion of desertification and semi-arid regions in the world (IPCC, 2014; Ming et al., 2015). In addition to the climatic changes often associated with anthropic actions, about 100 million tons of fertilisers are applied globally per year in crop areas, and large amounts of these fertilisers are leached, ultimately polluting the soils, rivers and oceans (Garnett et al., 2015). Therefore, numerous studies have focused on the development of techniques and strategies to increase global productivity, ensuring food security with less harmful environmental impacts on the natural environment.
Considering the above described scenarios, studies of plants that perform the Crassulacean Acid Metabolism (CAM) showed an alternative metabolic pathway for increasing crop productivity, since CAM metabolism can increase water and nitrogen use efficiencies of plants (Borland et al., 2014; Ming et al., 2015). In CAM plants, atmospheric CO2 is obtained overnight with low transpiration cost and fixed into oxaloacetate, which, in turn, is mainly converted into malic acid and then it is stored in the vacuoles (Ranson and Thomas, 1960; Lüttge et al., 1986). During the day, when the stomata are closed, the malic acid is remobilised from the vacuoles and decarboxylated (Casati et al., 1999). Afterward, the CO2 is fixed by ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO), with the subsequent production of carbohydrates (Cushman and Borland, 2002; Lüttge, 2004).
The content and activity of RuBisCO have already been questioned as being limiting to photosynthesis (Stitt and Schulze, 1994; Maxwell, 2002). More recently, photosynthesis has been described to be limited by the reduction of CO2 diffusion from the atmosphere to the sites of carboxylation, which has been associated with decreases both in stomatal conductance and mesophyll conductance to CO2, especially under dry conditions (Chaves and Oliveira, 2004; Flexas et al., 2012). However, in some plants with CO2-concentrating mechanisms, such as CAM plants, the diffusion of CO2 may not limit photosynthesis. In CAM plants, studies evaluating the efficiency of photosynthesis and the activity of RuBisCO in drought conditions are rare (Maxwell et al., 1999; Maxwell, 2002), but even with most of the studies evaluating C3 plants (i.e., plants that assimilate atmospheric CO2 during the day when stomata are open), our understanding of how RuBisCO behaves during drought is still rather poor. For example, Flexas et al. (2006) have suggested that RuBisCO can limit photosynthesis under severe or long-term water deficit, while other studies with C3 species have shown a reduction or no inhibition of RuBisCO activity under drought stress (Pelloux et al., 2001; Parry et al., 2002; Zhou et al., 2007). These discrepancies might be a consequence of stress duration and intensity, in addition to being species-dependent.
The RuBisCO content differs between CAM and C3 plants (Maxwell, 2002). In C3 plants, RuBisCO comprises 30–50% of total leaf protein, and its high concentration in leaves balances the photorespiration processes (Woodrow and Berry, 1988; Quick et al., 1991). In contrast, CAM plants are described as having less RuBisCO content, although this does not necessarily reflect either lower enzyme activity or a lower photosynthetic capacity (Maxwell, 2002). Concerning plants that display the facultative C3-CAM metabolism, one may hypothesize that they may not exhibit large variations in RuBisCO content (Maxwell, 2002), as their photosynthetic metabolism may vary rapidly according to environmental conditions. In addition, as RuBisCO corresponds to a large portion of leaf proteins, it is considered the major nitrogen storage in plants and therefore plays a relevant role in storing nitrogen in nutrient-poor environments compared to their nitrogen-rich counterparts (Millard and Thomson, 1989; Chapin III et al., 1990). Epiphytic plants, which are adapted to nitrogen-poor habitats, use RuBisCO to store nitrogen in their leaves despite their lower RuBisCO content (Maxwell, 2002). Besides RuBisCO, photosynthetic pigments are also considered a nitrogen stock, and their production is directly related to photosynthesis and RuBisCO activity (Evans, 1989).
The epiphytic bromeliad Guzmania monostachia (Tillandsioideae) displays the facultative C3-CAM metabolism, which uses the C3 pathway to maximise its vegetative growth under favourable environmental conditions while performing CAM in stressful or in dynamic conditions (Lüttge, 2010; Rodrigues et al., 2014). Furthermore, G. monostachia can express CAM idling, in which the stomata remain closed during day and night, presenting little nocturnal acidification due to the CO2 recycled from respiration (Cushman, 2001; Freschi et al., 2010; Pikart et al., 2018). Recently, Pikart et al. (2018) have shown that G. monostachia performs CAM idling after 4 days of drought and, besides the slight acid accumulation during the night, malic acid contributed to the carbon balance of these plants, maintaining the integrity of the photosystem II (PSII). In another C3-CAM reversible species, Mesembryanthemum crystallinum, was observed that the roles of chloroplasts and the performance of PSII differ between the C3 and CAM plants, in which chloroplasts could provide malate and keep photochemical activity in CAM plants (Kuźniak et al., 2016; Matsuoka et al., 2018). Additionally, M. crystallinum showed a rapid reversible response of the PEPC1 expression (i.e., the CAM-specific gene of the enzyme phosphoenolpyruvate carboxylase, PEPC) regarding abiotic environmental conditions (Nosek et al., 2018). The variation of the nutritional status of C3-CAM species, e.g., ammonium nutrition and nitrogen-deficient plants, coupled with water deficit can also intensify the CAM pathway (Pereira et al., 2018). Combining these studies, G. monostachia can manifest CAM idling under water deficit, and this photosynthetic pathway can be intensified when there is a combination with nutritional deficiency. Nevertheless, it is still unclear whether RuBisCO activity and the efficiency of photosynthesis can shift during the transition from C3 to CAM in facultative plants subjected to water and nutrient variation.
In this study, we conducted an experiment submitting G. monostachia to a factorial combination of availability and deficiency of water and nutrients to investigate how such variations affect the RuBisCO activity, its activation state and the efficiency of photosynthesis during the transition from C3 to CAM. Specifically, we submitted bromeliads to both water and nutritional deficits to investigate (1) whether bromeliads expressing CAM, evaluated by the nocturnal malic acid accumulation, can increase their RuBisCO activity and RuBisCO activation state in order to keep their photosynthetic efficiency; (2) whether the efficiency in the electron flux through the PSII is preserved; and (3) as a consequence of the maintenance of photosynthesis, whether the production of sugars, carotenoids and chlorophylls are kept. With this experimental design, we expected to observe an intensification in CAM induction in G. monostachia when the plants were submitted to both deficits (Pereira et al., 2018); therefore, bromeliads submitted to them were our focus in the evaluation of RuBisCO and the efficiency of photosynthesis during CAM expression.
Section snippets
Plant material, growth conditions and experimental design
Plants of Guzmania monostachia (L.) Rusby ex Mez var. monostachia were obtained as described in Rodrigues et al. (2016). Adult plants were cultivated in pots (14 cm in diameter, 11 cm high) containing a substrate mixture of Pinus bark and a commercial organic substrate (Tropstrato®) for about 2.5 years in a glasshouse at the Department of Botany at the University of São Paulo, Brazil. After this period, plants with the same substrate mentioned before were transferred and maintained in a growth
Results
Our results showed that the concentration of malic acid varied in leaves of Guzmania monostachia submitted to different treatments, especially on the fourth experimental day, when bromeliads gradually increased malic acid contents with the imposition of deficits: well-watered and nourished bromeliads (WN) showed the lowest nocturnal malic acid accumulation, bromeliads submitted to water (df.W) and nutritional (df.N) deficits presented intermediate levels of malic acid, while bromeliads
Discussion
Our results indicate that besides an increase in CAM activity, showed by the increment of malic acid in Guzmania monostachia plants submitted to both impositions of water and nutritional deficits, the initial and total activities of RuBisCO did not change. Interestingly, the bromeliads under either deficiencies or only nutritional deficit showed approximately three-fold higher RAS than bromeliads submitted to water deficit or well-watered and nourished bromeliads. The highest RAS may have
Author contributions
AZG and SL contributed equally to the production of this manuscript.
AZG conceived, designed and performed the experiments, analysed the data and drafted the manuscript.
SL conceived, designed and performed the experiments, analysed the data and drafted the manuscript.
KCD provided technical and theoretical assistance to perform the RuBisCO activation state analysis.
MAM conceived the experiment and performed the PSII quantum yield analysis.
MPMA conceived the experiment and performed the PSII
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We are grateful to Prof. Dr. Nadja Cristhina de Souza Pinto (Instituto de Química, Universidade de São Paulo) for the helpful comments and essential help in the RuBisCO essay calculations. This work was partly funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001 to SL, by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, n° 2011/50637-0 and 2018/12667-3) to HM, n° 2016/09699-5 to AZG, and by the Conselho Nacional de
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