ACTA BOT. CROAT. 80 (1), 2021 29 Acta Bot. Croat. 80 (1), 29–34, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-005 ISSN 0365-0588 eISSN 1847-8476 Plant development, gas exchanges and pigments of Mesosphaerum suaveolens submitted to osmoconditioning and saline stress Francisco Romário Andrade Figueiredo1*, Jackson Silva Nóbrega2, Reynaldo Teodoro de Fátima3, Toshik Iarley da Silva4, Rodrigo Garcia da Silva Nascimento2, Maria de Fátima de Queiroz Lopes2, Thiago Jardelino Dias2, Riselane de Lucena Alcântara Bruno2 1 Federal Rural University of the Semi-Arid Region, Department of Agronomic and Forestry Sciences, Mossoró, Brazil 2 Federal University of Paraíba, Department of Plant Science and Environmental Sciences, Areia, Brazil 3 Academic Unit of Agricultural Engineering, Federal University of Campina Grande, Campina Grande, Brazil 4 Federal University of Viçosa, Department of Plant Science, Viçosa, Brazil Abstract – Salinity is one of the main plant abiotic stresses affecting the establishment and development of crops. It is thus a matter of prime importance to search for technologies that minimize the damage caused by salinity. The aim of the present work was to evaluate the effect of salinity stress and osmotic conditioning of seeds on the biomass, gas ex- changes and chlorophyll pigments in Mesosphaerum suaveolens (L.) Kuntze. The statistical design adopted was a ran- domized block design, combined according to the central composite design, referring to electrical conductivities of irrigation water and osmotic potentials, with minimum (–α) and maximum (α) values of 0.5 and 10.0 dS m–1 and 0.0 and –1.0 MPa, respectively, totaling nine combinations. The characteristics of dry biomass, gas exchange and chloro- phyll indices were evaluated at 45 days after the beginning of irrigation with saline water. The salinity of irrigation water severely affected the dry biomass and the gas exchanges of M. suaveolens. Irrigation water of electrical conduc- tivity above 3.2 dS m–1 caused reductions in chlorophyll a, b and total contents in M. suaveolens plants. Seed osmocon- ditioning did not attenuate the negative effects of saline stress on M. suaveolens plants. Keywords: bamburral, chlorophyll, plant growth, photosynthesis, salinity Introduction Mesosphaerum suaveolens (L.) Kuntze, also known as “bamburral” in the Brazilian Northeast, is a species belong- ing to the botanical family Lamiaceae, which presents sub- shrub growth habit and leaves with high aromatic potential (Sabóia et al. 2018). The species, besides being widely used by folk medicine in several regions of Brazil, is also rich in secondary compounds (alkaloids, flavonoids, tannins and others), which are used in the pharmaceutical and cosmet- ics industries (Alves et al. 2017, Bezerra et al. 2017). The chemical quality of irrigation water is one of the fac- tors that directly affect plants’ metabolic processes, espe- cially in semi-arid regions, where water is scarce and, in many cases, saline (Guimarães et al. 2019). In the semi-arid region of Brazil, salinity is among the abiotic stresses that most hinder the growth and production of crops (Sales et al. 2015). Salinity can affect plants in two ways: by reducing the soil osmotic potential, thus reducing water and nutrient up- take, and by accumulating specific ions (Na+ and Cl–), lead- ing to toxicity and nutritional imbalance (Taiz et al. 2017). Osmotic and ionic effects on crops can cause changes in the plants’ physiological and biochemical functions (Ouhad- dach et al. 2018). Therefore, it is necessary to search for tech- nologies that might attenuate these salinity stress negative effects on irrigated agricultural systems. In this sense, seed osmoconditioning is a practice that has been adopted with the purpose of reducing the germi- nation time, increasing the germinability, uniformity and * Corresponding author e-mail: romarioagroecologia@yahoo.com.br FIGUEIREDO F. R. A., NÓBREGA J. S., DE FÁTIMA R. T., DA SILVA T. Y., NASCIMENTO R. G. S., LOPES M. F. Q., DIAS T. J., BRUNO R. L. A. 30 ACTA BOT. CROAT. 80 (1), 2021 performed: pH = 7.8; organic matter (g kg–1) = 22.2; P (mg kg–3) = 85.3; K+ (mg kg–3) = 693.6; Ca+2 (cmolc dm–3) = 2.9; Mg+2 (cmolc dm–3) = 1.59; Na+ (cmolc dm–3) = 0.23; H++Al+3 (cmolc dm–3) = 0.0; Al+3 (cmolc dm–3) = 0.0; sum of bases (cmolc dm–3) = 6.5; cation exchange capacity (cmolc dm–3) = 6.5. The evaluations were carried out at 45 days after the ir- rigation with saline water started. To determine root and shoot dry mass, these plant parts were packed in kraft paper bags and dried in a forced air circulation oven at 65 °C until reaching constant weight. Subsequently, the material was weighed, with results expressed in g plant–1; the total dry mass was obtained from the sum of the dry masses of the root and shoot, with the results in g plant–1. Using the dry mass data, the shoot: root ratio was estimated (shoot dry mass/root dry mass). The gas exchange evaluations were performed on the fourth leaf, from apex to base, between 9:00 and 10:00 a.m., using the portable infrared gas analyzer IRGA (model LI- 6400XT, LI-COR®, Nebraska, USA) with 300 mL min–1 air- flow, 400 µmol CO2 m–2 s–1 and a coupled light source of 1200 μmol m–2 s–1. The variables assessed were: net CO2 as- similation rate (A) (μmol CO2 m–2 s–1), stomatal conduc- tance (gs) (mol H2O m–2 s–1), CO2 concentration in intercel- lular spaces (Ci) (μmol CO2 mol air–1), transpiration rate (E) (mmol H2O m–2 s–1) and foliar temperature (°C). From these variables, the water use efficiency (WUE: A/E), intrinsic wa- ter use efficiency (iWUE: A/gs) and instantaneous carbox- ylation efficiency (iCE: A/Ci) were calculated. The contents of chlorophyll a, b and total, as well as the chlorophyll a/b ratio, were also determined on the fourth leaf, from apex to base, between 9:00 and 10:00 am, by a non-destructive method using a portable chlorophyllometer (ClorofiLOG®, model CFL 1030, Porto Alegre, RS). The val- ues were expressed as the Falker chlorophyll index (FCI). The data were submitted to analysis of variance by the F-test (P < 0.05), and in the case of a significant effect, the data were submitted to regression analysis, using the statis- tical program SAS University (Cody 2015). Results There was no significant interaction between the stud- ied factors, seed osmoconditioning and salinity levels of ir- rigation water. For the shoot (SDM), root (RDM) and total (TDM) dry mass, a decreasing effect is observed, with re- ductions of 80.7%, 89.4% and 86.8%, respectively, at 10.00 dS m–1 salinity level compared to the control treatment (Fig. 1a-c). However, the SDM/RDM ratio presented an inverse behavior to the above-mentioned variables, increasing lin- early as a function of increases in salinity level of the irriga- tion water (Fig. 1d). Regarding the gas exchange variables, a decreasing lin- ear effect was observed for A. Plants irrigated with water at 10.00 dS m–1 salinity level presented a reduction of 54.9% in A when compared to the control treatment (Fig. 2a). How- vigor of the seedlings, which is advantageous in plants sub- jected to salt stress conditions (Cardoso et al. 2012). During physiological conditioning, seed hydration occurs slowly, which gives more time for the repair or reorganization of plasma membranes, allowing the formation of tissues in a more orderly manner, reducing the risk of damage to the embryonic axis (Windauer et al. 2007). Polyethylene glycol (PEG) has been widely used for the formulation of solutions with different osmotic potentials, simulating drought stress, which could induce secondary dormancy (Souza et al. 2011). Thus, the aim of the present work was to evaluate the ef- fect of salinity stress and osmotic conditioning of seeds on the biomass accumulation, gas exchanges and chlorophyll pigments in M. suaveolens plants. Materials and methods The research was carried out in a greenhouse belonging to the Department of Plant Science and Environmental Sciences at the Federal University of Paraíba, located in the city of Areia, Paraíba, Brazil. The statistical design adopted was a randomized block design, factorially combined according to the central com- posite matrix of Box, referring to electrical conductivities of irrigation water (ECw) and osmotic potentials, with mini- mum (–α) and maximum (α) values of 0.5 and 10.0 dS m–1 and 0.0 and –1.0 MPa, respectively, with four replicates con- sisting of two plants, totaling nine combinations (Mateus et al. 2001). Saline water was prepared by adding sodium chloride (NaCl) to the water of the supply system (ECw = 0.5 dS m–1) in the proportions required. The salinity level of the waters was measured using the Instrutherm® (model CD – 860) micro-processed portable conductivity meter. Irrigation was performed daily, with the application of saline water 10 days after sowing. The irrigation volume applied was estab- lished through drainage lysimetry, from the difference be- tween the amount applied and drained. Osmoconditioning was performed by soaking seeds in PEG 6000 (Dinâmica®, Brazil) solutions. PEG 6000 was di- luted in 200 mL of distilled water in the proportions re- quired for each osmotic potential. The seeds were soaked for 8 hours at 25 °C, in containers covered with aluminum foil, being the amounts of PEG 6000 established according to Villela et al. (1991). Subsequently, the seeds were washed with distilled water. The seeds used in the experiment were obtained from native plants found at the Novo Horizonte settlement, mu- nicipality of Várzea, Paraíba, Brazil. The seedlings were produced in 1.2 L capacity polyethylene bags, 10 seeds being sown per container. After seedling emergence, thinning was performed, keeping only the most vigorous plant in each container. The polyethylene bags were filled with a substrate com- posed of soil (Latosol type), washed sand and tanned cattle manure in a 3:1:1 ratio. A substrate chemical analyse was ECOPHYSIOLOGY OF M. SUAVEOLENS UNDER SALINE STRESS ACTA BOT. CROAT. 80 (1), 2021 31 ever, Ci presented an increasing linear effect as a function of the increase in salinity level, with an increase of 11.3% (Fig. 2b). For iCE there was a 58.7% reduction in ECw at 10.0 dS m–1 salinity level when compared to the control treat- ment (Fig. 2c). It was observed that increasing salinity levels in the irrigation water provided a linear reduction in the transpiration rate (E), decreasing by 33.8% at 10.0 dS m–1 when compared to the control (Fig. 2d). The plants tended to use water less efficiently with increasing salinity levels, decreasing by 36.6% (Fig. 2e). It can be observed that only from 3.2 dS m–1 salinity lev- el was there a reduction in the indexes of chlorophyll a, b and total, with decreases of 25.8%, 42.3% and 29.4%, respec- tively, at the highest salinity level when compared to the control treatment (Fig. 3A-C). Regarding the chlorophyll a/b ratio, the data show that there were increases in this variable from the ECw of 3.2 dS m–1 (Fig. 3d). Seed osmoconditioning with PEG did not provide sig- nificant differences for chlorophyll a. However, total chlo- rophyll and chlorophyll b were negatively affected by the increase in osmotic potential reduction of the solution, with decreases of 27.2 and 18.2%, respectively, when comparing the highest osmotic potential (–1.00 MPa) with the control (Fig. 4a,b). The reduction of the osmotic potential of the so- lution provided increases in the chlorophyll a/b ratio, with a 16.2% increase at the potential of –1.00 MPa (Fig. 4c). Discussion The results for the dry mass may be related to the exces- sive absorption of ions such as Na+ and Cl–, causing reduc- tions in the accumulation of photoassimilates due to a low- er photosynthetic rate. Also, there is an increase in energy expenditure by the plant due to the reduction of the osmot- ic potential, which makes it difficult to absorb water through the roots (Araújo et al. 2016). Similar results were verified by Bione et al. (2014) in salt-stressed basil plants (Ocimum basilicum L.) cultured in a hydroponic NFT system and Oliveira et al. (2016) in maize (Zea mays L.) submitted to saline stress and biostimulant treatment. These authors ver- ified that dry mass production was reduced with the in- crease of salinity. The reduction in A was possibly due to the damage caused by salts to the chloroplast, thus reducing CO2 fixa- tion by rubisco. High concentrations of Na+ and Cl– cause toxicity at different levels and may alter several biochemical and physiological processes, among them the fixation of carbon dioxide during the photosynthesis process (Roupha- el et al. 2012). The increase in Ci can be directly related to the photosynthetic rate reduction, which results in a lower CO2 consumption during the carboxylation process. Reduc- tions in the photosynthetic rate lower the ATP and NADPH availability as well as the substrate for rubisco, which is as- sociated with the reduction of intrinsic carboxylation effi- Fig. 1. Shoot dry mass (a), root dry mass (b), total dry mass (c) and shoot/root dry mass ratio (d) of Mesosphaerum suaveolens sub- jected to electrical conductivities of irrigation water (0.50, 1.45, 5.00, 8.55 and 10.00 dS m–1). FIGUEIREDO F. R. A., NÓBREGA J. S., DE FÁTIMA R. T., DA SILVA T. Y., NASCIMENTO R. G. S., LOPES M. F. Q., DIAS T. J., BRUNO R. L. A. 32 ACTA BOT. CROAT. 80 (1), 2021 ciency as a function of the increase in salinity (Silva et al. 2015). This decrease in E can usually be an adaptation mechanism in response to abiotic stresses, specifically sa- line and hydric, in order to reduce water loss. Several authors have observed reductions in net CO2 as- similation rate, transpiration rate and carboxylation effi- ciency as a function of saline stress, for example Huang et al. (2015) in Boehmeria nivea L., Yarami and Sepaskhah (2015) in Crocus sativus L., and Rouphael et al. (2016) in Cu- curbita pepo L. This reduction in chlorophyll indices may be related to several factors, such as: decreased chlorophyll a biosynthe- sis, increased chlorophyllase activity and instability of pro- tein complexes caused by saline stress effects (Houimli et al. 2010). Melo et al. (2017), studying the effect of irrigation with saline water in bell pepper plants, obtained similar re- sults, stating that chlorophyll a was the most sensitive vari- able to salinity. However, the increase in the chlorophyll a/b ratio, possibly, can be attributed to the greater reduction observed in chlorophyll b (42.3%) than in chlorophyll a (25.8%). Corroborating these results, Shimoda et al. (2012) report that the first step in the degradation of chlorophyll b is its conversion to chlorophyll a. These decreases in total chlorophyll and chlorophyll b can reflect a biochemical adaptation of the plant in response to the osmotic conditioning and the action of oxidative deg- Fig. 2. Net CO2 assimilation rate – A (a), internal CO2 concentration – Ci (b), instantaneous carboxylation efficiency – iCE (c), transpi- ration rate – E (d) and water use efficiency – WUE – A/E (e) of Mesosphaerum suaveolens subjected to electrical conductivities of irriga- tion water (0.50, 1.45, 5.00, 8.55 and 10.00 dS m–1). ECOPHYSIOLOGY OF M. SUAVEOLENS UNDER SALINE STRESS ACTA BOT. CROAT. 80 (1), 2021 33 Fig 3. Chlorophyll a (a), chlorophyll b (b), total chlorophyll (c) and chlorophyll a/b ratio (d) of Mesosphaerum suaveolens subjected to electrical conductivities of irrigation water (0.50, 1.45, 5.00, 8.55 and 10.00 dS m–1). Fig. 4. Chlorophyll b (a), total chlorophyll (b) and chlorophyll a/b ratio (c) of Mesosphaerum suaveolens subjected to osmoconditioning with PEG (–1.00, –0.85, –0.50, –0.15 and 0.00 MPa). FIGUEIREDO F. R. A., NÓBREGA J. S., DE FÁTIMA R. T., DA SILVA T. Y., NASCIMENTO R. G. S., LOPES M. F. Q., DIAS T. J., BRUNO R. L. A. 34 ACTA BOT. CROAT. 80 (1), 2021 radation agents (Cardoso et al. 2012). This increase in chlo- rophyll a/b ratio was possibly due to the reduction in chlo- rophyll b and unchanged chlorophyll a indices. However, in basil plants Santos et al. (2012) observed divergent results, where plants cultivated without water deficit showed a high- er chlorophyll a/b ratio, attributing this behavior to the fact that chlorophyll a degradation by oxidative damage occurs faster when compared to chlorophyll b. Conclusions Seed osmoconditioning did not attenuate the negative effects of saline stress on M. suaveolens plants. 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