Osmotic stress induced by polyethylene-glycol alters macronutrient concentrations in sugarcane (Saccharum spp.) plants in vitro

El estrés osmótico inducido por polietilenglicol altera las concentraciones de macronutrimentos en plantas de caña de azúcar (Saccharum spp.) in vitro

Odón Castañeda-Castro^1,2, Fernando C. Gómez-Merino^2*, Libia I. Trejo-Téllez³, Miriam C. Pastelín-Solano¹

¹ Universidad Veracruzana. Facultad de Ciencias Químicas. Prolongación de Oriente 6 No. 1009. 94340. Orizaba, Veracruz. (odcastaneca@uv.mx), (mpastelin@uv.mx).

³ Edafología. Campus Montecillo, Colegio de Postgraduados. 56230. Montecillo, Estado de México. (tlibia@colpos.mx).

Received: January, 2015.
Approved: September, 2015.

Abstract

The potential capacity of plant roots to absorb water and nutrients generally declines in osmotic-stressed plants, presumably because of a reduction in the nutrient element demand, and such decrease varies among plant genotypes. In order to investigate the effect of the osmotic stress caused by polyethylene-glycol 6000 (PEG) on the macronutrient concentration in sugarcane (Saccharum spp.) in vitro, we established a factorial experiment with a completely randomized distribution. Study factors were variety (Mex 69-290 and CP 72-2086) and PEG in the nutrient medium (0, 3, 6 and 9 %, to generate osmotic potential corresponding to -0.18, -0.45, -0.65 and -0.80 MPa, respectively), which resulted in eight treatments, with five replicates each. The experimental unit consisted of a 500 mL flask, with 50 mL liquid MS medium, and three sugarcane plantlets each. Results were analyzed using an analysis of variance and means were compared using the Tukey test (p≤0.05). Plants were harvested 20 and 30 d after treatment (dat). In both varieties at 20 dat no effect on K, Ca, Mg and S concentrations was observed; however, N was lower in CP plants and P diminished in Mex plants. A different response was observed 30 dat with N, K, Ca, Mg and S concentrations higher in Mex plants, whereas P showed no differences between varieties due to the osmotic stress imposed by PEG. As for the osmotic stress, since PEG concentrations decreased in the nutrient medium, N and Ca concentrations diminished 20 dat, whereas N and K concentrations were lower 30 dat. Interaction between variety and osmotic stress showed highly significant effects on most minerals, with N and Ca being the most affected. In general, Mex 69-290 plants showed higher nutrient concentrations than CP 72-2086 plants under our experimental conditions.

Keywords: Plant nutrition, Poaceae, PEG, nitrogen, phosphorus, potassium.

La capacidad potencial de las raíces de las plantas para absorber agua y nutrimentos generalmente disminuye cuando éstas son sometidas a estrés osmótico, probablemente debido a un decremento en la demanda nutrimental, y tal disminución varía entre genotipos. A fin de investigar el efecto del estrés osmótico ocasionado por polietilenglicol (PEG) en la concentración de macronutrimentos en caña de azúcar (Saccharum spp.) in vitro, se estableció un experimento factorial con distribución completamente al azar. Los factores de estudio fueron variedad (Mex 69-290 y CP 72-2086) y PEG en el medio de cultivo (0, 3, 6 y 9 %, para generar potenciales osmóticos de - 0.18, - 0.45, - 0.65 y - 0.80 MPa, respectivamente), con lo que se obtuvieron ocho tratamientos, con cinco repeticiones cada uno. La unidad experimental consistió en un frasco de 500 mL de capacidad, con 50 mL de medio de cultivo MS líquido, y tres plántulas de caña de azúcar cada uno. Los resultados se analizaron con un análisis de varianza y prueba de comparación de medias por Tukey (p≤ 0.05). Las plantas se cosecharon 20 y 30 días después de aplicados los tratamientos (dat). En ambas variedades no se observaron efectos de los tratamientos en las concentraciones de K, Ca, Mg ni S; 20 dat pero la concentración de N fue menor en plantas CP, y la de P disminuyó en plantas Mex. Una respuesta diferente se observó 30 dat con concentraciones mayores de N, K, Ca, Mg y S en plantas Mex, en tanto que la concentración de P no mostró diferencias entre variedades debidas al estrés osmótico inducido por PEG. En cuanto al estrés osmótico, al disminuir las concentraciones de PEG en el cultivo, las concentraciones de N y Ca disminuyeron 20 dat, mientras que las de N y K se redujeron 30 dat. La interacción entre variedad y estrés osmótico mostró efectos altamente significativos para la mayoría de los nutrimentos, N y Ca fueron los más afectados. En general, las plantas de Mex 69-290 mostraron concentraciones mayores de nutrimentos que las de CP 72-2086, en las condiciones experimentales aquí probadas.

Palabras clave: Nutrición vegetal, Poaceae, nitrógeno, fósforo, potasio.

INTRODUCTION

The availability of water for crops plays an important role in regulating plant growth and obtaining good yields. Globally, water deficits limit crop productivity more than any other environmental factor, especially for crops such as sugarcane, which demand large amounts of water and fertilizers.

Mexico is ranked as the sixth largest sugarcane producer in the world and has shown significant increments in yield from fields and factories (FAO, 2015; CONADESUCA, 2015). However, in environmental terms, the effects of global climate change on agricultural production in Mexico could reduce national agricultural production in 25.7 % (Moyer, 2010) if relevant strategic measures are not taken to address this global phenomenon. Under climate change, the water footprint of sugarcane might increase 2 % by 2020 and 3-4 % by 2050, whereas the available water is predicted to fall 4-7 % by 2020, and 6-8 % by 2050, with negative effects on crop yields (Haro et al., 2013).

In fact, drought in Mexico, which was worsened by climate change, causes 80 % of all agricultural losses, which is noteworthy since 62 % of sugarcane acreage develops under rain-fed conditions (Sentíes-Herrera et al., 2014). In these sugarcane production areas, episodes of water deficiency and hydric stress are becoming frequent, which hinder the genetic potential of sugarcane varieties to express their highest yields. As water availability is the major limiting factor for sugarcane productivity (Tammisola, 2010), studies that lead to an increase in sugarcane drought tolerance are needed to provide tools to allow sugarcane plantations in drier regions (da Silva et al. 2012; Vargas et al., 2014). This is one of the greatest challenges for the sustainable expansion of sugarcane production that is carried out worldwide (Rudorff et al., 2010; Sentíes-Herrera et al., 2014).

Drought causes several effects in sugarcane plants, including significant reduction in stomatal conductance, transpiration, and net photosynthesis (Smith et al., 1999; Medeiros et al., 2013; Gentile et al., 2015), whereas leaf temperature increases (Rodrigues et al., 2009; Graça et al., 2010). Almost all sugarcane cultivars show decreased yields under drought, but some are more affected than others (Gentile et al., 2015). Interestingly, a cultivar considered as sensitive to drought (i.e. reduced yield under drought stress) may be considered a useful cultivar. For example, Ribeiro et al. (2013) found that cultivar IACSP86-2042 had a 50 % reduction in stalk yield under drought stress, much higher than IACSP94-2094 (29 % reduction) and SP87-365 (no reduction); however, the absolute stalk yields of these cultivars were similar under drought conditions, as IACSP86-2042 had a much higher productivity under non-stressful conditions. Losses due to drought are not unusual and almost every year some sugarcane growing regions suffer mild to severe water shortages (Gentile et al., 2015). Therefore, drought can cause major economic losses for sugarcane growers. Nevertheless, mild drought stress can have a positive impact on sugarcane yield. In irrigated fields, a period of drying off (water withheld) at the end of the season is a common practice. Apart from saving water and therefore costs associated with irrigation, this practice reduces soil compaction during harvest and may even increase sucrose content, as growth is more affected than photosynthesis and, as a result, assimilated CO₂ can be diverted from leaf and culm growth to sucrose accumulation in the culm (Singels et al., 2000; Inman-Bamber, 2004). Therefore, the regulation of sugarcane responses to drought will show differences with those observed in other crops and model plants.

Water constitutes approximately 72 % of plant weight, as is the case with sugarcane, and is essential for physiological processes involved in conveying metabolites and nutrients (Cavali et al., 2010). Therefore, in the event of water deficiency, nutrient imbalances can be observed in plant tissues. Although general mechanisms are identified for drought tolerance in plants, there are differences among species and cultivars as to how they respond to this type of stress, and responses vary according to plant developmental stage and stress duration and intensity. Sugarcane has a high demand for fertilizers (Sentíes-Herrera et al., 2014), and little is known about the effects of osmotic stress on the absorption and concentration of nutrients in its tissues under water deficit. In order to modify the osmotic potential of a nutrient solution and thereby induce water stress in a controlled manner, it is common to use polyethylene-glycol (PEG), especially in protocols for hydroponics experiments. Because PEG has a 6000 or 8000 molecular size and does not penetrate plant tissues, it is ideal for generating osmotic stress in nutrient solutions (Lagerwerff et al., 1961; Money, 1989). Therefore, the objective of our study was to analyze the concentrations of N, P, K, Ca, Mg and S in the stems of sugarcane varieties Mex 69-290 (Mex) and CP 72-2086 (CP), in response to water stress caused by the application of PEG 6000 in nutrient solution in vitro.

MATERIALS AND METHODS

Plant material and growing medium

Seedlings from varieties Mex 69-290 and CP 72-2086, the two most cultivated in Mexico (Sentíes-Herrera et al., 2014), were used for in vitro culture on MS medium (Murashige and Skoog, 1962) at 100 %, and supplemented with 2 % sucrose (w/v), myoinositol (100 mg L^-1), thiamine (50 mg L^-1), pyridoxine (100 mg L^-1), niacin (50 mg L^-1), glycine (300 mg L^-1), biotin (100 mg L^-1), arginine (50 mg L^-1) and ascorbic acid (50 mg L^-1). The pH of the medium was adjusted to 5.7±0.1 and remained in a liquid state throughout the experiment. The liquid culture medium was placed in 500 mL flasks which were sterilized in an autoclave (Lab-Tech model LAC5060s; Namyangju, South Korea) at 120 °C for 20 min.

Sugarcane varieties Mex 69-290 and CP 72-2086 were generated under different environmental conditions and from different genotypes and breeding programs. Mex 69-290 resulted from the hybridization between the Mexican varieties Mex 56-476 and Mex 53-142 under rain-fed conditions in Córdoba, Veracruz, Mexico (Gómez-Merino and Sentíes-Herrera, 2015), whereas the American variety CP 72-2086 was produced by the crossing between the American varieties CP 62-374 and CP 63-588 from Canal Point, Florida (USA), under irrigated conditions (Schueneman et al., 2008).

Experimental conditions, experimental design and statistical analysis

Seedlings were maintained in growth chambers under controlled conditions of 16 h light, 23 °C and 75 % relative humidity.

The study had a factorial design with a completely randomized distribution. The factors were: 1) Sugarcane variety (VAR; Mex69-290 and CP 72-2086), and 2) Peg 6000 in the culture medium (PEG). Four levels of polyethylene-glycol (Sigma-Aldrich Chmie GmbH, Steinheim, Germany) were tested as causative agent of water stress in the culture medium: 0, 3 6 and 9 % for generating osmotic potentials equivalent to -0.18, -0.45, -0.65 and -0.80 MPa, respectively. The osmotic potential were monitored with a cryoscopic osmometer (Osmomat-030, Gonotec GmbH, Berlin, Germany). Treatments had five replicates and each replicate consisted of a 500 mL flask with 50 mL of culture medium, culture medium and three seedlings of each sugarcane variety.

The data were analyzed using ANOVA and Tukey means comparison test (p≤0.05) with SAS (2011).

Nutrient analyses

RESULTS AND DISCUSSION

Since its discovery as an efficient osmotic stressor (Lagerwerff et al., 1961), PEG is used to induce controlled water deficits in plants under appropriate experimental protocols (Labanowska et al., 2013). Its immobility and lack of toxicity to plant cells make PEG an osmolite suitable for plant physiological studies (Al-Bahrany, 2002). Furthermore, as it does not react with other chemical and biological substances, PEG is the most applicable substance for experimentally inducing biological osmotic pressure (Macar et al., 2009). In our study, PEG altered osmotic pressure in the liquid nutrient medium, dropping it from -0.18 MPa in the control to -0.80 MPa in containers with 9 % PEG 6000. This decrease in osmotic potential resulted in altered nutrient uptake and concentration in sugarcane plants.

Effect of individual factors and their interaction on plant nutrient concentrations

Significant effects from PEG concentrations, sugarcane varieties and their interactions on N concentrations were observed in sugarcane plants 30 d after treatment (dat). Furthermore, significant effects from PEG concentrations were observed on Ca and K concentrations in sugarcane plants 20 and 30 dat, respectively. Sugarcane variety significantly affected N, Mg and S concentrations 20 dat, and the concentrations of N and Ca 30 dat. The interactions between PEG concentration and sugarcane variety were significant for P and K at 20 dat, and for N at 30 dat (Table 1).

Effect of varieties on plant nutrient oncentrations

Mex 69-290 had higher macronutrient concentrations than CP 72-2086 (Table 2). Nitrogen and magnesium concentrations were higher in the former variety at 20 and 30 dat, whereas K, Ca and S changed only at 30 dat. Variety CP 72-2086 showed higher P concentrations at 20 dat, although at 30 dat both varieties displayed the same P concentrations.

Effect of PEG on plant nutrient concentrations

The analysis of the effect of PEG concentration as an independent factor showed that N concentrations decreased in sugarcane plants at 20 and 30 dat with increased PEG concentration in the nutrient medium (Table 3). Besides, K concentrations in plants decreased with concentrations of PEG in the nutrient medium at 30 dat, whereas for Ca it was at 20 dat. All other nutrient concentrations did not change in response to the treatments tested.

The analysis of the effect of the interaction PEG x VAR on micronutrient concentrations in plant tissues, showed that differences were most evident at 30 dat, especially for K, Mg and S concentrations (Figure 1).

Nitrogen concentrations were significantly different at 20 and 30 dat, but were most evident at 30 dat, especially for CP 72-2086. No significant effects were observed for P at 20 or 30 dat. Potassium concentrations were not affected at 20 dat, but were significantly reduced 30 dat. Calcium was significantly reduced at 20 dat in both varieties, whereas the variety CP 72-2086 showed significant effects at 30 dat. Similarly, Mg concentrations were significantly reduced at 30 dat, and the variety CP 72-2086 was more affected. Finally, S concentrations did not change in response to osmotic potentials in either variety at 20 dat, although a reduction was evident at 30 dat in the variety CP 72-2086.

To our understanding, this is the first study reporting the effects of osmotic stress induced by PEG on sugarcane macronutrient concentrations in vitro. Importantly, N and Ca concentrations were significantly affected by PEG treatments under our experimental conditions. Nitrogen is a primary plant nutrient that is highly important in achieving maximum crop productivity and sugarcane absorbs N in greater amounts than any other essential nutrient because this mineral element is a crucial component of all enzymes and other vital molecules such as chlorophylls and nucleic acids (McCray et al., 2013). Thus, N is necessary for plant growth, development and performance. As N uptake, biomass production, yield and sugar production are strongly correlated, the N requirement of sugarcane is large and must be in balance with other nutrients, especially if plants are exposed to environmental stressors such as drought (Bäzinger et al., 2000). According to Villar-Salvador et al. (2013), maintaining a low N content allows plants experiencing water stress to overcome such events. Nitrogen, as ammonium (NH₄⁺) and nitrate (NO₃^-), has different effects on gas exchange parameters (Guo et al., 2007). Zhang et al. (2011) reported that increased NO₃^- nutrition plays a favored anti-oxidative metabolic role, compared with NH₄⁺ nutrition in plants, thereby increasing tolerance to drought-related stress. Such mechanisms are essential for tolerance to water stress, acting on N metabolism as well as helping to maintain or augment biomass. In our study, the MS medium contained higher concentrations of nitrate than ammonium (i.e., 39.5 mM of NO₃^- and 20.5 mM of NH₄⁺) (Bensaddek et al., 2001). However, sugarcane strongly prefers ammonium over nitrate (Robinson et al., 2011), which could explain the lower N contents observed in our sugarcane plants (an average of 11.6 g kg^-1 DW) in comparison to a sufficient leaf N range reported by McCray and Mylavarapu (2013) of 20 to 26 g kg^-1 DW.

Calcium plays an essential role in the structural and functional integrity of plant membranes and other cellular. In drought-stressed plants there is a decrease of nearly 50 % in leaf Ca²⁺ (Lisar et al. 2012), whereas soil water shortages can decrease root hydraulic conductivity and affect Ca uptake and movement throughout the plant (Wu et al., 2012). Osmotic stress induced by 10 % PEG 6000 significantly decreased cortical cell volume, and application of additional Ca²⁺ regulated the expression and activity levels of aquaporins according to water availability, which contributed to optimized water use (Wu et al., 2012). Abdalla and El-Khoshiban (2007) reported reduction in calcium content in wheat plants under drought-related stress, as did Akhondi et al. (2006) for shoots of Medicago sativa, and Hu et al. (2007) in maize, which is consistent with our results.

Under stress conditions, one of the first responses is the transient increase in cytosolic calcium, derived from the influx of apoplastic reserve or the release from internal compartments such as the vacuole and endoplasmic reticulum, which contain higher levels of Ca than the cell cytosol (Dedemo et al., 2013). Therefore, sugarcane genotypes capable of maintaining high Ca contents may display better performance under stress due to water deficit, as was the case of the Mex 69-290 variety.

Though no differences were observed among treatments for P concentrations 30 dat (Table 1; Figure 1), a drastic drop in the concentration of this macronutrient was observed when comparing it to that displayed by plants 20 dat (i.e. 3.0 g kg^-1 P 20 dat vs. 1.1 g kg^-1 P 30 dat). This decrease was indeed more evident in the CP 72-2086 variety (Figure 1). Jin et al. (2014) reported that phosphorus application and elevated CO₂ interactively enhanced periodic drought tolerance in field pea as a result of decreased stomatal conductance, deeper rooting and high phosphate availability for carbon assimilation in leaves. Phosphate is essential to photosynthetic processes and adjustments in the relationships among the leaf gas exchange network support the high drought tolerance of the sugarcane. Such adjustments enabled the homeostasis of both photosynthesis and plant growth under water deficit. Moreover, phosphate supply improves the sugarcane acclimation capacity by affecting plant characteristics related to water status and photosynthetic performance and causing network modulation under water deficit (Sato et al., 2010). Hence, the relative higher level of phosphate in the Mex 69-290 variety showed a better capacity of this genotype to overcome water deficit.

Of the mineral nutrients, potassium plays a pivotal role in improving plant tolerance to water stress conditions. It modulates many physiological processes such as activation of enzymes, photosynthesis, maintenance of turgescence, and translocation of photosynthates (Yadov, 2006). However, water deficit may inhibit K uptake and the extent of such inhibition is genotype dependent (Kirnak et al., 2001). For instance, between contrasting sugar beet varieties grown under water deficit, K increased in one, and decreased in the other (Alam, 1999). Our results demonstrate that 30 dat, both sugarcane varieties evaluated displayed a dramatic reduction of K concentration, though the decrease was more pronounced in CP 72-2086.

Under field conditions, both varieties Mex 69-290 and CP 72-2086 are drought tolerant (Salgado-García et al., 2012). Nevertheless, in laboratory and greenhouse studies, the variety Mex 69-290 exhibits better performance under osmotic stress than the variety CP 72-2086 does (Castañeda-Castro et al., 2014). This fact may explain, at least in part, the different behaviors displayed by these varieties under our experimental conditions.

Osmotic stress induced by PEG differentially affects macronutrient concentrations in sugarcane in vitro, with N and Ca more affected than the other nutrients studied. Furthermore, we found different responses between varieties, with Mex 69-290 plants having higher nutrient concentrations and therefore better performance under our experimental conditions.

ACKNOWLEDGEMENTS

We are very grateful to the Colegio de Postgraduados Science and Technology Trust, the Priority Research Line 5 - Microbial, Plant and Animal Biotechnology and to the Universidad Veracruzana, for providing laboratory facilities and financial support for this study.

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