ФИЗИОЛОГИЯ РАСТЕНИИ, 2011, том 58, № 4, с. 502-508
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
Genotypic Differences in the Responses of Gas Exchange, Chlorophyll Fluorescence, and Antioxidant Enzymes to Aluminum Stress in Festuca arundinacea
© 2011 S. H. Jin*, X. Q. Li**, X. L. Jia*
* School of Forestry and Biotechnology, Zhejiang A & F University, Lin'an, Zhengjingprovince, P. R. China ** Tianmu college, Zhejiang A & F University, Lin'an, Zhengjing province, P. R. China
Received August 18, 2010
In this study, the gas exchange, chlorophyll fluorescence, and antioxidant activity in eight tall fescue (Festuca arundinacea) cultivars were investigated under aluminum stress. The results showed that the net photosyn-thetic rate (PN) and stomatal conductance (gs) were decreased, while the intercellular CO2 concentration (Ci) was stable or increased under Al stress conditions. The efficiency of excitation capture by open PSII reaction centers (F^ /F^), the maximum quantum yield of PSII photochemistry (Fv/Fm), the quantum yield of PSII electron transport (ФРШ), and the photochemical quenching (qP) were also decreased after Al stress, while the non-photochemical quenching (NPQ) was increased. Moreover, Al stress increased the antioxidant activities and MDA contents in each tall fescue cultivars. However, there was a lot genotype differences between the Al-tolerant and Al-sensitive cultivars. Cv. Barrington was the most sensitive cultivar and cv. Crossfire 2 was the most tolerant cultivar. The excessive excitation energy could not be dissipated efficiently by antenna pigments, and reactive oxygen species could not be scavenged efficiently, thereby resulting in membrane lipid peroxidation in cv. Barrington under Al stress conditions.
Keywords: Festuca arundinacea — aluminum stress — antioxidant enzymes — chlorophyll fluorescence
INTRODUCTION
Aluminum (Al) is one of the most abundant metals in the earth crust, occurring in the form of harmless and stable aluminosilicates. If the soil becomes acidic, more and more Al is solubilized into toxic form, and the excess Al3+ in soil results in Al stress on plants [1]. Aluminum toxicity is an important growth-limiting factor for plants on many acid soils. It is well known that Al toxicity injures plants in terms of destroying
Abbreviations'. APX — ascorbate peroxidase; CAT — catalase; Cj — intercellular CO2 concentration; CK — control; Fv/Fm — maximum quantum yield of the PSII photochemistry; F'y/ F^ — efficiency of excitation capture by open PSII reaction centers; gs — stomatal conductance; Fm — maximum chlorophyll fluorescence; F^ — maximum chlorophyll fluorescence during actinic light illumination; Fq — minimum chlorophyll fluorescence; F0 — minimum chlorophyll fluorescence during actinic light illumination; Fs — steady-state chlorophyll fluorescence; NPQ — non-photochemical quenching; PN — net photosynthetic rate; PPFD — photosynthetic photon flux density; PSII — photosystem II; qP — photochemical quenching; SOD — superoxide dismutase; TBA — thiobarbituric acid; TCA — trichloroacetic acid; ®psii — quantum yield of PSII electron transport.
Corresponding author. Song Heng Jin. School of Forestry and Biotechnology, Zhejiang A & F University, Huanbei Road 88, Lin'an, 311300, Zhengjing province, P. R. China. Fax. 86-571-63740809; e-mail. shjin@zafu.edu.cn
microcosmic structure and root structure, decreasing the efficiency of absorbing minerals [2, 3], hindering the growth of leaves, restraining the assimilation of nutrients, resulting in a disorder of metabolism, which hinders the synthesis of chlorophyll. Many studies have showed that Al inhibits CO2 assimilation in many plant species, including citrus [4, 5], Sorghum bicolor [6], Lycopersicon esculentum [7], and Zea mays [8]. However, it is now still a matter of debate how Al stress inhibits photosynthesis. Simon et al. [7] suggested that stomatal closure is at least partially responsible for the Al-induced decrease in the CO2 assimilation rate in tomato due to the lowering of intercellular CO2 concentration. On the other hand, Moustakas et al. [9] concluded that Al causes a decline in photosynthesis in wheat (Triticum aestivum) as a result of the closure of photosystem II (PSII) reaction centers and a reduction of PSII electron transport rate. Therefore, one aim of this study was to establish why the photosyn-thetic rate was decreased under Al stress.
Constructing lawn is one of the most important methods to increase afforested areas, beautify the environment, and purify the air. However, the soil erosion by acid rain becomes more and more serious recently, which makes the area of acid soil larger and larger; the acid soil accounted for about 21% of the soil in China. Tall fescue is one of the most common grass
species in the construction of lawn; thus, it is significant to screen grass genotypes resistant to Al stress in landscaping and greening and improving ecological environment. The grass resistant to Al stress can alleviate the Al toxicity of acid soil. Thus, this research used different genotypes of tall fescue to study physiological changes under Al stress, including the changes in the leaf gas exchange parameters, chlorophyll fluorescence parameters, antioxidant enzyme activities, and so on. Our second aim is to select genotypes of tall fescue, which are sufficiently resistant to Al stress, so that we can extend the tall fescue area in the south of China.
MATERIALS AND METHODS
Plant materials and treatments. Eight tall fescue (Festuca arundinacea) genotypes were used: Southern Gold, Pearl 2, Barrington, Millennium, Bonsai 2000, Triple A, Houndog 5, and Crossfire 2. Tall fescue seeds were surface-sterilized in a 5% sodium hypochlorite (NaClO) solution for 30 min, rinsed four times with distilled water, and germinated on moist filter paper in darkness at a temperature of 28°C for 7 days. The seedlings were transplanted individually into plastic pots (18 cm tall, 14 cm top diameter) filled with coarse sand 2—3 mm in diameter. A half-strength Hoagland solution was used as the basic nutrient solution. The seedlings were grown in a shaded greenhouse with natural sunlight during the day (maximum of 800 ^mol/(m2 s)) and constant relative humidity of approximately 70%. The mean daytime maximum and minimum temperatures in the greenhouse were 28 and 20°C, respectively. About one month later, plants were randomly separated into the two groups of 20 plants each. The first group received nutrient solution without AlCl3 (control, CK), while another group received the same nutrient solution containing 0.25 mM AlCl3. The pH of the nutrient solution was adjusted to 4.5.
Measurement of gas exchange parameters and chlorophyll fluorescence. After 7 days of treatment, the measurements were made on the second fully expanded leaf from the top of four randomly selected seedlings from each treatment. Leaf chamber temperature was 25°C. Other environmental conditions for the measurements were 1000 ^mol/(m2 s) photosynthetic photon flux density (PPFD) and 50% relative humidity in the sample chamber. Leaf gas exchange and chlorophyll fluorescence were measured simultaneously using a LiCor-6400 portable photosynthesis system ("LiCor", United States) with an integrated fluorescence fluorometer (Li-6400-40 leaf chamber fluorometer) under ambient CO2 concentrations and 21% O2. Actinic light supplied with light-emitting diodes (90% red light, 630 nm; 10% blue light, 470 nm) were used to record the steady-state chlorophyll fluorescence level (Fs), net photosynthetic rate (PN), sto-matal conductance (gs) and intercellular CO2 concen-
tration (Ci). The minimum chlorophyll fluorescence at the open PSII center (F0) and maximum chlorophyll fluorescence at the closed PSII center (Fm) were measured after 30 min of dark adaptation. Measurement light (630 nm, 1 ^mol/(m2 s)) was used to determine F0. An 800-ms saturating pulse (>6000 ^mol/(m2 s)) was applied to measure Fm in the dark or during actinic light illumination (F^). The minimum (F0') fluorescence of light-adapted leaves was determined according to Kramer et al. [10]. The maximum quantum yield of the PSII primary photochemistry (Fv/Fm) was calculated as (Fm — F0)/Fm. The quantum yield of PSII
electron transport (®PSn; (F' — Fs)/F^), the efficiency of excitation energy capture by open PSII reaction centers (Fj/F^ = (F' — F0)/F^), photochemical quenching (qP = (F'm — Fs)/( F'm — F0')) and the non-
photochemical quenching (NPQ; Fm/ F'm — 1) were calculated from measured parameters [11]. After measuring steady-state photosynthesis, the leaf at same position of the seedlings was freeze-clamped into liquid nitrogen and subsequently used for biochemical measurements.
Determination of antioxidant enzyme activities.
Leaf tissue (0.5 g) was ground in liquid nitrogen and homogenized in 3.5 ml of 50 mM K-phosphate buffer (pH 7.8) containing 0.2 mM EDTA, 4% PVP-40, and 1 mM ascorbic acid. Samples assayed for superoxide dismutase (SOD) activity were extracted in the same buffer without ascorbic acid. All enzyme extractions and centrifugations were carried out at 4°C. The enzyme activity of each sample was measured four times at 25°C. Activities of SOD, ascorbate peroxidase (APX), and cat-alase (CAT) were assayed with the methods previously described by Verma and Mishra [12].
Determination of lipid peroxidation. Lipid peroxi-dation was determined by estimating the MDA content according to the method of Dhindsa et al. [13]. The MDA content was determined by thiobarbituric acid (TBA) reaction. About 1 g of each sample was homogenized in 5 ml of 0.5% trichloroacetic acid (TCA). After centrifugation at 12000 g for 15 min, 1 ml of the extract was taken and 4 ml of 0.6% TBA in 20% TCA was added. The mixture was heated in a boiling water bath for 15 min and then cooled quickly in an ice bath. The resulting mixture was centrifuged at 12000 g for 15 min, and the specific absorbance of products and nonspecific bac
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