ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 4, с. 459-465
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
PsbS EXPRESSION ANALYSIS IN TWO ECOTYPES OF Sedum alfredii AND THE ROLE OF SaPsbS IN Cd TOLERANCE1 © 2014 M. Zhang, X. E. Yang
College of Environmental & Resource Sciences, Zhejiang University, Zijingang Campus, Hangzhou, China
Received April 10, 2013
The effects of Cd on the chlorophyll fluorescence parameters of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of Sedum alfredii Hance were studied. The photosystem II (PSII) photochemical efficiency of HE plants was not affected, and the non-photochemical quenching (NPQ) value was significantly increased under Cd treatment. In the NHE plants, Cd treatment caused significant loss in PSII photochemical efficiency and NPQ value. Gene expression analysis showed that both ecotypes of S. alfredii accumulated more than twofold higher PsbS (PSII subunit S) transcript levels than control plants after exposure to Cd. Overexpression of PsbS gene isolated from HE plants (SaPsbS) enhanced tobacco growth. The transgenic tobacco possessed greater NPQ capacity in the presence of 100 ^M Cd and accumulated the significantly higher Cd content in shoots than the wild-type plants. These data indicate that the SaPsbS may function in NPQ capacity and protect the PSII reaction center in HE plants.
Keywords: Sedum alfredii - Nicotiana tabacum - hyperaccumulator - cadmium non-photochemical quenching photosystem IIsubunit S (PsbS)
DOI: 10.7868/S0015330314040216
INTRODUCTION
Cadmium, a bivalent cation, is one of the most toxic heavy metals and it is taken up by roots, probably in competition with other bivalent ions [1]. The inhibition of photosynthesis is a main effect of Cd, which has been already observed in most plants. Either an indirect action of Cd on plant water relations, stomatal conductance, and CO2 availability [2, 3] or a more direct effect on chloroplast organization, chlorophyll biosynthesis, and electron transport contribute to the inhibition. Studies investigated Cd toxicity on chloroplast functionality and electron transport have suggested that photosystem II (PSII) is the main target of Cd, and Cd inhibits photoactivation of PSII by competitive binding to the essential Ca2+ site [4-7].
1 This text was submitted by the authors in English.
Abbreviations'. cDNA-AFLP - cDNA-amplified fragment length polymorphism; Fv — maximum variable chlorophyll fluorescence yield in the dark-adapted state; F0 — minimum chlorophyll fluorescence yield in the dark-adapted state; Fm — maximum chlorophyll fluorescence yield in the dark-adapted state; — maximum chlorophyll fluorescence yield in the light-adapted state; HE — hyperaccumulating ecotype; NHE — non-hyperaccumulat-ing ecotype; NPQ — non-photochemical quenching; PSII — photosystem II; PsbS — photosystem II subunit S; SaPsbS — PsbS gene from HE plants.
Corresponding author. Xiao E. Yang. College of Environmental & Resource Sciences, Zhejiang University, Zijingang Campus, Hangzhou 310058, China. Fax. +86-571-8898-2907; e-mail. milyzhang84@hotmail.com
Hyperaccumulating ecotype (HE) of Sedum alfredii Hance (Crassulaceae) is a Zn/Cd hyperaccumulator, which is found in an old Pb/Zn-mined area [8, 9]. HE plants are tolerant to Cd and can accumulate 9 g Cd/kg leaf dry wt [9]. By contrast, the non-hyper-accumulating ecotype (NHE) of Sedum, which is found in tea gardens, cannot accumulate Cd in shoot. A greatly enhanced root-to-shoot translocation expressed in the HE plants is one of the metal detoxification mechanisms [10]. Disorganization of chloroplasts is more common when the NHE plants are exposed to 10-40 ^M Cd in hydroponic culture. In contrast, the chloroplast and mitochondria of HE plants are still relatively in the better state under 100 ^M Cd, which indicates the high Cd tolerance of HE plants [11]. However, in which way Cd accumulation heterogeneity under stress is coupled to heterogeneity in physiology, for example, mesophyll photosynthesis remained unknown. Chlorophyll fluorescence kinetic measurement is a powerful way to assess the physiological status of plants [12]. But only a few papers report on fluorescence characteristics in hyperaccumulators during heavy metal-induced stress and acclimation [13, 14].
Non-photochemical quenching (NPQ) is a potential photoprotective process, which can reduce energy delivery to the PSII reaction center and minimize the generation of singlet oxygen in the LHCII (light harvesting complex of PSII) by deactivating singlet excited chlorophylls through thermal dissipation of energy
[15]. Cd treatment has different effects on NPQ value in different plants. Under Cd treatment, the NPQ value was increased in Phragmites australis [16]. However, NPQ is less affected by Cd treatment than photochemical quenching in Zn/Cd hyperaccumulator Thlaspi caerulescens; the NPQ of non-hyperaccumu-lator Thlaspi fendleri in the light-acclimated state is up to double compared with the control during the initial period of inhibition [14]. However, the mechanism by which Cd affects the capacity of NPQ remains unknown. The PsbS protein is associated with the PSII core complex and plays a central role in NPQ [17, 18]. A recent study has shown that PsbS specifically controls the association between LHCII and the PSII core [19]. PsbS gene of HE plants was also found to be up-regulated after subjected 100 ^M Cd (M. Zhang et al., unpublished data). However, no data are available on the expression of PsbS gene in hyperaccumulators and its role in heavy metal acclimation.
The objectives of this study were: (1) to identify the effects of Cd on PsbS gene transcript level and NPQ value between HE and NHE plants; (2) to study the effect of SaPsbS overexpression on Cd tolerance and accumulation in tobacco. To analyze the function of PsbS gene, the full length cDNA of this gene was isolated from HE plants and transgenic tobacco lines overexpressing PsbS were generated.
MATERIALS AND METHODS
Plant materials and growth conditions. The HE
Sedum alfredii Hance plants were collected from an old Pb/Zn-mined area in Quzhou city of Zhejiang province, P.R. China, and the NHE Sedum plants were from tea gardens of Hangzhou, Zhejiang province. Healthy and equally-sized shoots were chosen and grown in distilled water for new roots initiation. After rooting, plants were grown in nutrient solution as described in [9]. The plants were grown in a greenhouse, and the nutrient solution was continuously aerated with an aquarium air pump. The nutrient solution was renewed every four days and pH value was adjusted to 5.5 with 0.1 M HCl or 0.1 M NaOH.
Experimental treatments. In order to compare the PsbS expression pattern between two ecotypes under Cd treatment for short time (24 h), both the HE and NHE plants were treated with 2, 20, or 200 ^M CdCl2. To determine the gene expression level and chlorophyll fluorescence during Cd acclimation, HE and NHE plants were treated with 2 ^M CdCl2 for 8 days, and untreated plants were used as control. For each treatment, nine plants were divided into three individual replications. After plants were harvested, portions of fresh leaves were immediately frozen in liquid nitrogen and stored at -80°C for RNA extraction. The upper leaves of control and Cd-treated plants were used for chlorophyll fluorescence analysis; the Cd concen-
trations in plants were determined as described by Yang et al. [9].
Chlorophyll fluorescence analysis of HE and NHE plants. After the leaves were dark-adapted for 1 h, chlorophyll fluorescence emission of both Cd-treated and control plants was measured using a PAM-2000 modulated fluorometer ("Walz", Germany) as described by Rorat et al. [20]. The initial level (F0) of chlorophyll fluorescence was excited by a dim red light modulated at 600 Hz and was determined after a 2-s illumination with far-red light. The maximal level of chlorophyll fluorescence (Fm) was induced by a 800-ms
pulse of intense white light; F'm was the maximal and steady-state levels of chlorophyll fluorescence. The leaves were kept at their natural angle during measurements. The maximal quantum yield of PSII photochemistry was calculated as (Fm - F0)/Fm = Fv/Fm;
NPQ was calculated as (Fm - F^) /F^. The measurements were performed on three different plants of each ecotype.
Isolation of full length PsbS cDNAs from HE and NHE plants. A PCR-based cloning strategy was used to isolate full-length cDNA encoding PsbS from HE plants (SaPsbS); primers were designed for 5'-RACE and 3'-RACE based on the sequence of transcript derived fragment (TDF) isolated from cDNA-AFLP analysis (Zhang et al., unpublished data). The full-length PsbS from NHE plants (SnPsbS) was amplified according to the sequence similarity between SaPsbS and SnPsbS.
Expression analysis of PsbS in two ecotypes of Sedum. Total RNA from leaves of HE and NHE plants with or without heavy metal treatments were prepared using Trizol reagent ("Invitrogen", United States). Five micrograms of total RNA was used to synthesize cDNA with SuperScript Reverse Transcriptase ("Invitrogen") and oligo(dT) as a primer ("Invitrogen"). Common primers (PsbS-F: 5-TTGTTGGCCGT-GTTGCTATGAT-3', PsbS-R: 5'-ATGGACCTCCT-TCGCTTAGACC- 3') for real-time RT-PCR were designed according to the sequence similarity between SaPsbS and SnPsbS. Actin primers were designed according to published actin sequence [21]. The sizes of the amplified fragments were confirmed by gel elec-trophoresis and sequencing. The real-time PCR was performed in Eppendorf Mastercycler® Realplex2 (Germany) with SYBR green I and Ex Taq (Perfect Real Time) reagent ("TaKaRa").
Construction of transgenic plants. To generate 35S-SaPsbS vector, the amplified ORF of SaPsbS was subcloned into pENTR/D-TOPO ("Invitrogen") and sequenced for validation. The pENTR/D-TOPO entry vector containing the SaPsbS was designated as pENTR-SaPsbS. Subsequent attL and attR (LR) substrate recombination reaction ("Invitrog
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