ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 2, с. 244-250
ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^ СТАТЬИ
УДК 581.1
EFFECTS OF POTASSIUM DEFICIENCY AND REPLACEMENT OF POTASSIUM BY SODIUM ON SUGAR BEET PLANTS1
© 2014 Z. Pi*, P. Stevanato**, L. H. Yv*, ***, G. Geng*, ***, X. L. Guo*, Y. Yang*,
C. X. Peng*, X. S. Kong*
*Key Laboratory of Sugar Beet Genetic Breeding of Heilongjiang Province, Heilongjiang University, Harbin, China **Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente, Université degli Studi di Padova, Legnaro, Italy ***Chinese Academy of Agricultural Science Institute of Sugar Beet, Harbin, China
Received November 27, 2012
Two sugar beet (Beta vulgaris L.) genotypes were cultivated at different K+/Na+ concentration nutrient solutions (mM, 3.00/0 (control groups), 0.03/2.97 (K-Na replacement groups), and 0.03/0 (K deficiency groups)) to investigate the effects of potassium deficiency and replacement of potassium by sodium on plant growth and to explore how sodium can compensate for a lack of potassium. After 22 days of growth were determined: (i) dry weights of leaves, stems, and roots, (ii) the Na+ and K+ contents, (iii) MDA level, (iv) the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), and (v) the level of free amino acids. Potassium deficit inhibited plant growth, decreased the K+ content in leaves and roots, activated GPX and SOD, suppressed CAT activity, and increased the content of most amino acids. In K-Na replacement groups, the effects of K+ deficiency, including changes in the MDA level, antioxidant enzyme activities, and the level of free amino acids, were alleviated, but the degree of recovery did not reach the values characteristic for the control groups. Based on these results, we concluded that low potassium could lead to the inhibition of seedling growth, oxidative damage, and amino acid accumulation. While sodium was able to substitute potassium to a large extent, it cannot fulfil potassium fundamental role as an essential nutrient in sugar beet.
Keywords: Beta vulgaris - potassium-sodium replacement - potassium deficiency - sugar beet - antioxidant enzymes - free amino acids
DOI: 10.7868/S0015330314020110
INTRODUCTION
Potassium is involved in enzyme activation, osmoregulation, photosynthesis and protein metabolism [1-3]. Hence, sufficient K supply is essential for good crop growth and a key factor for high yield. Nevertheless, K deficiency occurs frequently due to continuous cropping and demands in particular crop growth stages. Various abiotic stresses, such as salt and drought stresses, can also lead to K deficiency in some crop tissues [4]. These problems may be resolved by applying K fertilizer, but a less expensive approach is required that could decrease the potassium requirements of crops.
1 This text was submitted by the authors in English.
Abbreviations'. CAT - catalase; GPX - glutathione peroxidase; NBT - nitro blue tetrazolium; PBS - phosphate-buffered saline; PVP - polyvinylpyrrolidone; TBA - thiobarbituric acid; SOD -superoxide dismutase.
Corresponding author. Gui Geng. Chinese Academy of Agricultural Science Institute of Sugar Beet, Harbin, 150080 China. Fax. +86 451 8660-8193; e-mail. genggui01@163.com
Sodium (Na) is also a monovalent cation. It is defined by Subbarao et al. [5] as a "functional element", which promotes plant growth to the maximal biomass or can reduce essential nutrient requirements by partial substitution. Several functions of cell organelles, such as the vacuoles and chloroplasts, were reported to be related to moderate sodium concentrations [6-9]. In some crops, including sugar beet, tomato, and rice, it was demonstrated that sodium can compensate for inadequate potassium [10-12]. In some cases, the biomass of sugar beet was not observably affected with about 50% potassium replacing by sodium and could normally develop even with 95% substitution in some genotypes [13]. More importantly, the sucrose content in sugar beet could be maintained when fertilizing with sodium and no potassium [14].
In this study, two sugar beet genotypes were cultivated in solutions with different potassium and sodium concentrations to investigate the effect of potassium deficiency as well as the replacement of potassium by sodium on plant growth, attempting to show how to
replace potassium by sodium and compensate for potassium deficiency in the cells.
MATERIALS AND METHODS
Plant materials and treatments. Two sugar beet (Beta vulgaris L.) genotypes: a salt-sensitive genotype 210 and a salt-tolerant genotype T510, were used in the study. The seeds were sterilized with 70% (v/v) etha-nol, 0.1% (w/w) mercuric chloride, and 0.2% (w/w) thiram, and germinated in vermiculite with daily watering. After six days of cultivation, the seedlings were transferred into a hydroponic container (40 x 40 x 15 cm) with 20 L of half-strength modified Hoagland solution. Plants were grown at three K/Na treatments (Kwas added as KCl, and Na as NaCl), where the K/Na concentration in the initial nutrient solution (mM) was: 0.03/0 (K deficiency groups), 0.03/2.97 (K-Na replacement groups), and 3.00/0 (control groups). Each treatment had 16 seedlings (8 seedlings of 210 and 8 seedlings of T510) and three replicates. To ensure cultivation of 210 and T510 under the same conditions, seedlings of both genotypes were planted equally into each half of the three containers. The plants were grown in a controlled environment chamber with a 13-h photoperiod, 25/20°C day/night temperature, quantum flux density of 450 ^mol/(m2 s) at the top of the canopy, and 70% relative humidity. The K+ concentration in the nutrient solution was determined and refilled to 0.03 mM once a day in the first ten days after transplanting and twice a day in the last six days before harvesting. 12 days after transplanting, the nutrient solution in all containers was renewed.
Harvest and the measurement of dry weight. Plants were harvested on the 16th day after transplanting (a total of 22 days). The fresh weight was measured of a sample of leaves, which were then stored at —80°C for further experiments. The other leaves, stems, and roots of the plants were dried at 70° C to constant weight; then the weight of leaves, stems, and roots was determined.
Determination of K+ and Na+ contents in leaves and roots. The dry matter of leaves and roots was passed through a 100 mesh sieve. A sample of 0.3 g was collected and digested in 10 mL of 98% sulfuric acid over 5 h. During digesting, 30% H2O2 was added in drops essentially until digest fading [15]. The Na+ and K+ contents were determined with a flame photometer.
MDA content and activities of antioxidant enzymes in the leaves. A portion of the frozen sample (0.5 g fr wt) was ground with an ice-cooled mortar and pestle for homogenization and then extracted with 5 mL of ice-cold 50 mM PBS, pH 7.8, containing 0.2 mM EDTA and 2% (w/v) PVP. The homogenate was then centri-fuged at 12 000 g for 20 min at 4°C. The supernatant collected after centrifugation was used for measurements [16].
MDA content was determined by the thiobarbitu-ric acid (TBA) reaction. 2.0 mL of enzyme extract and 2.0 mL of TBA reagent (0.5% (w/v) TBA dissolved in 10% (w/v) TCA) were mixed and heated for 30 min in a boiling water bath and then cooled on ice to stop the reaction. The concentration of MDA was calculated as follows:
6.45(^532 - ^60c) - 0.56A450.
The inhibition of the photochemical reduction of nitro blue tetrazolium (NBT) was used for assaying SOD activity. The reaction mixture contained 0.05 mL of the enzyme extract, 1.5 mL of 26 mM Met, 0.3 mL of 0.75 mM NBT 0.15 mL of 2.0 ^M EDTA, 0.15 mL of 0.04 mM riboflavin, and 0.85 mL of 50 mM PBS. After 20-min illumination from a 7 ^mol/(m2 s) fluorescent lamp, the absorbance of the mixture was measured at 560 nm and blanks were run the same way without illumination. One unit of SOD activity was defined as the amount of enzyme producing a 50% inhibition of NBT reduction at 560 nm.
GPX activity was measured by monitoring the increase in the guaiacol oxidation. The reaction medium comprised 0.05 mL of the enzyme extract, 2.55 mL of 100 mM PBS, pH 7.0, (containing 0.1 mM EDTA), 0.2 mL of 1% guaiacol, and 0.3 mL of 10 mM H2O2. An absorbance change in 0.01 unit/min at 470 nm was defined as one unit of GPX activity.
1.5 mL of 200 mM PBS, pH 7.8 (containing 1% PVP), 1.0 mL of H2O, 0.4 mL of 100 mM H2O2, and 0.2 mL of the enzyme extract were mixed; the decrease in the absorbance of the mixture at 240 nm reflecting the H2O2 content was monitored. CAT activity was defined as an absorbance change in 0.1 unit/min at 240 nm.
Content of free amino acids in the leaves. The frozen leaves (0.500 g fr wt) were homogenized with an ice-cooled mortar and pestle, and the homogenate was extracted using 40 mL of the extractant (mixed methanol, chloroform, and water, 12 : 5 : 3, v/v/v) for 3 days at 4°C. Centrifugation at 2000 g was then carried out for 5 min, and the supernatant was collected. The precipitate was extracted with 40 mL of the extractant and centrifuged again. Two supernatants were combined (80 mL) and shaken with 20 mL of chloroform followed by 30 mL of water. After standing, the resulting separated aqueous phase was evaporated to dryness in the rotary evaporator. This material was dissolved in 5 mL of deionized water. An appropriate volume was taken for the preparation of o-phthaldialdehyde derivatives prior to separation and analysis by reverse-phase HPLC as described by Martino et al. [17].
Statistical analyses. IBM SPSS 20.0 was used for all statistical analyses. The results were subjected to two-way analysis ofvariance. Means were compared at P < 0.05, and the values are given as means ± standard errors of three replications.
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