ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 681-687
^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
УДК 581.1
CHARACTERISTICS OF BIOENERGY GRASSES IMPORTANT FOR ENHANCED NaCl TOLERANCE POTENTIAL1
© 2014 P. P. Mirshad*, S. Chandran**, J. T. Puthur*
*Plant Physiology and Biochemistry Division, Department of Botany, University of Calicut, Kerala, India **Department of Botany, SN College Cherthala, Alapuzha, Kerala, India Received October 22, 2013
Growing bioenergy grasses can contribute to a great extent towards the production of biomass, and it can be a potential source of renewable energy. Such grasses, if suitable for marginal land, will solve better the problem of its competition with the cultivation of food crops in arable land. Four different potential bioenergy grasses, e.g., Saccharum arundinaceum Retz., hybrid Napier var. CO-3, Saccharum spontaneum L., and Arundo donax L. were selected based on our earlier study, and these perennial grasses were subjected to NaCl stress, a characteristic feature of marginal lands. Various measurements to assess the NaCl tolerance mechanism, e.g., MDA content, antioxidant enzyme activity, photosynthetic pigments composition, chlorophyll fluorescence and photosystem I (PSI) and photosystem II (PSII) activities were analyzed after imparting NaCl stress and compared with the control plants. Among the grasses studied, a lower maximum quantum yield of PSII (Fv/Fm) and PSI and PSII activities were recorded in S. spontaneum and Napier var. CO-3 than in S. arundinaceum and A. donax. The latter two grasses showed less degradation of total chlorophyll and low MDA content. The maintenance of a better water status of A. donax and S. arundinaceum is attributed to the maintenance of favorable osmotic balance by the accumulation of the higher levels of compatible solutes, such as total soluble sugars and proline. The better performance of S. arundinaceum and A. donax under high NaCl conditions was also facilitated by the higher free radical-scavenging potential in them, as represented by the increase in peroxidase activity. These results suggest that S. arundinaceum and A. donax are better adapted to NaCl stress than S. spontaneum and Napier var. CO-3. The high NaCl tolerance potential, exhibited by S. arundinaceum and A. donax, makes them an appropriate choice for marginal lands affected by high levels of NaCl.
Keywords: bioenergy grasses — antioxidants — biofuel — marginal land — osmotic potential—photosystems — proline
DOI: 10.7868/S001533031405011X
INTRODUCTION
The global demand for alternate energy is increasing, because the fossil fuels are unsustainable due to the carbon emission and limited supply. Moreover, the high price of fossil fuels and the environmental pollution caused by it have resulted in increased attention towards biofuels, a renewable energy source [1]. Bio-ethanol has been projected as a prime candidate for the next generation biofuels. A number of diverse plant
1 This text was submitted by the authors in English.
Abbreviations'. CAT — catalase; Chl — chlorophyll; DW — dry weight; Fm — maximal fluorescence; Fv/Fm — maximum photochemical quantum yield of photosystem II; F0 — minimal fluorescence; FW — fresh weight; GR — glutathione reductase; LEDs — light emitting diodes; PS — photosystem; PEA — plant efficiency analyzer; SOD — superoxide dismutase; TWC — tissue water content.
Corresponding author. Jos T. Puthur. Plant Physiology and Biochemistry Division, Department of Botany, University of Calicut, C.U. Campus P.O. Kerala-673635, India; fax. +91-494-2400269; e-mail. jtputhur@yahoo.com
species have been investigated in the search for identifying the best source of biomass for the production of sustainable bioethanol. Switch grass and many other high biomass-accumulating grass species are being used as sustainable energy crop for the production of bioethanol [2].
Interference of biomass production with the existing crop cultivation in arable land is a major problem, and hence the challenge is to identify the plant species that accumulate high biomass and at the same time can be grown on various types of degraded lands. Arundo donax, a perennial grass, which has the high biomass accumulation potential and thrives well in marginal lands, is used as a feedstock for the production of ligno cellulosic bioethanol [3]. In India, the identification of energy crops, which can be grown in marginal land affected by salinity, is getting more attention [4], as 6.73 million ha of the land is being salt-affected [5]. The utilization of marginal lands for the cultivation of high-biomass plants will solve better the
problem of its competition with the food crop plants for arable land.
Utilization of marginal lands is a challenge for the growth of the plants, as it is affected by various environmental factors, including biotic and abiotic stresses. The salinization of cultivable land is expected to have serious global effects, resulting in 30% land loss within the next 25 years and up to 50% — by the year 2050 [6]. Soil salinity reduces the ability of plants to take up water from soil and thereby suppresses plant growth. Excessive salinity lowers the soil osmotic potential and causes ionic stress and nutritional imbalance [7]. To counteract salinity stress, plants undergo various adjustments at the cellular and metabolic levels, such as an increase in the concentration of a variety of compatible solutes, such as betaines, proline, and sugar alcohols [8].
Salinity enhances ROS production, which causes damage to lipid membranes, proteins, and nucleic acids [9]. Plants alleviate the oxidative damage by producing various antioxidant enzymes. Non-enzymatic means of ROS detoxifying include several small molecules that are antioxidants in nature, such as quaternary ammonium compounds, polyamines, polyols, al-pha-tocopherol, ascorbic acid (vitamin C), and caro-tenoids [10].
Our earlier studies for screening ofvarious bioener-gy grasses of superior quality based on the biomass accumulation rate, photosynthetic performance, sugar accumulation potential, and cellulose content were helpful in identifying four potential bioenergy grasses [11]. In the present study the metabolism related to NaCl tolerance was analyzed in the four promising perennial grasses to identify the sustainable bioenergy grass(es), which suits for the cultivation in the NaCl-affected land.
MATERIALS AND METHODS
Plant materials. Four grass species, e.g., Arundo donax L., Saccharum arundinaceum Retz., Saccharum spontaneum L., and hybrid Napiervar. CO-3 (Pennise-tum purpureum x P. americanum) were selected for the present investigation. Samples were collected from different areas of Calicut, Malappuram, and Kannur districts of Kerala. Healthy stem cuttings 20—30 cm long were selected and planted in bags containing 5 kg of top layer soil. Plants were maintained in a greenhouse at a relative humidity of 60 ± 5% and a temperature of 28 ± 2°C. NaCl stress (500 mM NaCl) was imposed to one plant group (12 plants), and the other group (12 plants) was watered with tap water. Various analyses were done on the 6th day after treatments.
Determination of and tissue water status. The
Ts of leaves was determined using a vapor pressure osmometer (Wescor 5520, United States). The leaf discs of 5 mm in diameter collected from the middle parts of the leaves were tested for Ts, after a daily calibration
of the instrument. Calibration of the chamber was done using standard solutions (290 and 1000 mmol/kg) (Wescor 5520).
The tissue water status was determined by measuring the fresh and dry weights of the leaves. The dry weight was recorded after drying the tissue at 100°C in the hot air oven for 1 h and later transferring it into hot air oven maintained at 60°C. The dry weight was recorded on every alternate day until the weights became constant. Tissue water content percentage (TWC %) was calculated using the following equation [12]:
TWC % = [(FW - DW)/FW] x 100.
Measurement of the osmolyte content. The accumulation of osmolytes, such as proline, was determined according to Bates et al. [13]. 0.5 g of plant tissues were homogenized in 10 mL of 3% sulfosalicylic acid, using a glass mortar and pestle, and the homoge-nate was centrifuged at 3000 g for 10 min. The supernatant was treated with ninhydrin dissolved in acetic acid, boiled for 1 h, and then absorbance at 520 nm was recorded with L-proline as a standard.
Total soluble sugars were determined according to Montgomery [14].
Measurement of lipid peroxidation. The intensity of lipid peroxidation was measured in terms of MDA content as described by Heath and Packer [15].
Determination of antioxidant enzyme activities.
The activity of antioxidant enzyme peroxidase was estimated according to Gasper et al. [16].
Chlorophyll a fluorescence measurement. Chl a fluorescence emission was measured by a Handy PEA (Plant Efficiency Analyzer, "Hansatech", United Kingdom). Before the measurements, leaves were dark-adapted with a small, lightweight leaf clip for 30 min. Dark adaptation ensured that all the reaction centers were fully oxidized and are available for photochemistry. Illumination consisted of a 1-s pulse of continuous red light (peak at the wavelength of 650 nm, 3000 ^mol photons/(m2 s)) provided by an array of three light-emitting diodes (LEDs). The first reliably measured point of the fluorescence transient was at 20 ^s, which was taken as F0. The ratio of variable to maximum fluorescence (Fv/Fm) was calculated as maximum quantum yield of PSII photochemistry under dark conditions.
Analysis of chlorophyll content and efficiency of photosystems. Chl estimation was done according to Arnon [17]. Photochemical activities of isolated thyla-koids were assayed polarographically with a Clark-type oxygen electrode (DW1/AD, "Hansatech"), which was connected with a digital control box (OXYG1, "Hansatech"). The light-dependent O2 uptake/evolution was measured by irradiating the sample with sat
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