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Feb 5, 2013 - ... β-strands, β-propeller and His residues coordinating catalytic sites of SoNCED were highly conserved as in the NCEDs from other plants.
Genes Genom (2013) 35:101–109 DOI 10.1007/s13258-013-0065-9

RESEARCH ARTICLE

Molecular cloning and characterization of SoNCED, a novel gene encoding 9-cis-epoxycarotenoid dioxygenase from sugarcane (Saccharum officinarum L.) Chang-Ning Li • Manoj-Kumar Srivastava Qian Nong • Li-Tao Yang • Yang-Rui Li

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Received: 8 March 2012 / Accepted: 13 August 2012 / Published online: 5 February 2013 Ó The Genetics Society of Korea 2013

Abstract Abscisic acid (ABA) plays important roles in adaptive responses to various environmental stresses. The rate-limiting step in ABA biosynthesis is the oxidative cleavage of cis-epoxycarotenoids, which is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). In this experiment, a full-length cDNA encoding NCED gene was cloned by RT-PCR and RACE from sugarcane (Saccharum officinarum L.). The full-length of SoNCED is 2,521 bp with 1,827 bp open reading frame, encoding a peptide of 608 amino acids. The calculated molecular weight of protein was 65.9 kDa with isoelectric point of 6.04. Conserved domains prediction indicated a chloroplast-targeting peptide located at N-terminus of SoNCED. Phylogenetic tree, constructed by Neighbor-Joining method indicated that SoNCED shared high identity with the NCEDs reported from other plant species. Sequence alignment revealed that the basic secondary structure including a-helices, b-strands, b-propeller and His residues coordinating catalytic sites of SoNCED were highly conserved as in the NCEDs from other plants. Tissue specific expression analysis using quantitative real-time PCR C.-N. Li  Q. Nong  L.-T. Yang  Y.-R. Li (&) College of Agriculture, State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, Guangxi, People’s Republic of China e-mail: [email protected] C.-N. Li e-mail: [email protected] C.-N. Li  M.-K. Srivastava  L.-T. Yang  Y.-R. Li Guangxi Crop Genetic Improvement and Biotechnology Lab, Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, P. R. China, Guangxi Key Laboratory of Sugarcane Genetic Improvement, Guangxi Academy of Agricultural Sciences, #174, East Da Xue Road, Nanning 530007, Guangxi, People’s Republic of China

showed a significant increase in SoNCED mRNA level and its correlation with O2– production rate and ABA accumulation in leaves and roots of sugarcane variety GT21 when exposed to water stress. Further, the stimulation of SoNCED mRNA level, O2– production rate and ABA content after exogenous application of ABA (100 lMol l-1) proved its involvement in pathways providing tolerance to drought stress. Keywords Sugarcane

Cloning  Gene expression  NCED  ROS 

Introduction Drought is one of the most important abiotic stresses affecting plant’s growth on over half of the earth’s surface area (Kogan 1997). Drought affected the crop production which was predicted to increase during the coming years (Marris 2008; Battisti and Naylor 2009). Sugarcane is the most important sugar crop growing in tropical and subtropical areas throughout the world, and is vulnerable to climatic changes, especially to abiotic stresses such as drought and low temperature. During the course of development, while facing disasters of changing environmental conditions, plants have developed a number of mechanisms to enhance tolerance to stress conditions by changing gene expression profiles leading to adaptive responses at cellular or systemic level. One of the mechanisms in the series of developments was the alteration in internal growth regulation system, and the most important phytohormone mediator that responds as alteration in gene expression of plants was abscisic acid (ABA). In addition to regulation of growth and developmental processes such as seed development, dormancy, shoot and root growth in plants (Finkelstein et al. 2008; Qiu

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and Yu 2009), ABA has been shown to mediate plant responses not only to drought, but also to numerous environmental stresses such as cold, salinity, water logging etc. Under drought stress conditions, plant ABA content increases significantly, which leads to restructuring of the cell cytoskeleton and stimulating stomata closure, biosynthesis of osmo-compatible solutes and activation of numerous stress-responsive genes to increase the capacity for coping with stresses (Shinozaki and YamaguchiShinozaki 2007; Cutler et al. 2010; Kim et al. 2010). The important role of ABA in plant growth and development makes it an attractive target in genetic modifications by controlling its levels in turn to control ABA regulated processes and improve the plant performance and productivity under stress conditions. Biochemical and genetic evidences indicated that the rate-limiting step in ABA biosynthesis was the catalytic breakdown of 90 -cis-neoxanthin or 9-cis-violaxanthin by a 9-cis-epoxycarotenoid dioxygenase (NCED) in order to generate xanthoxin, the direct C15 precursor of ABA, which was oxidized and converted through two subsequent steps to the biologically active ABA (Schwartz et al. 1997, 2003). Since its first isolation and characterization in ABA deficient Vp14 mutant of maize (Tan et al. 1997), the NCED genes have been cloned in a variety of plants (Chernys and Zeevaart 2000; Rodrigo et al. 2006; Yang and Guo 2007; Qin et al. 2008; Wang et al. 2009), and its catalytic activity in many plants has also been demonstrated. In some plant species, ABA biosynthesis is controlled by a NCED-like genes family, while only one subgroup involved in stress responses and regulation of ABA biosynthesis (Rodrigo et al. 2006; Qin et al. 2008), the expression analysis of these genes strongly indicated a major involvement of NCED in response to environmental stress of different tissues of plants, and a close correlation between NCED expression and ABA accumulation has also been reported (Qin and Zeevaart 1999). Previous researches indicated that transgenic plants constitutively expressing the NCED gene accumulated higher amount of ABA compared to the wild type and enhanced water stress resistance (Qin and Zeevaart 2002). In the present study, a NCED gene (SoNCED) was isolated from Saccharum officinarum L. using RT-PCR and RACE (Rapid amplification of cDNA ends) technology. The SoNCED features and basic secondary structures were characterized, and its interrelationship with NCEDs from other genera has been discussed. The interrelationship between O2– production rate, ABA content and mRNA levels of SoNCED in sugarcane leaves and roots have also been investigated under drought conditions which will provide great help in functional analysis of SoNCED gene.

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Materials and methods Plant materials and treatments Greenhouse experiments were conducted at Sugarcane Research Institute, Guangxi University (Nanning, China). The single bud sets of sugarcane variety GT21 were initially raised in sand culture. Three week old plants were transplanted in plastic pots (21 cm in diameter and 19 cm high) containing 5 l modified Hoagland solution [4.02 mM Ca(NO3)24H2O, 1.99 mM Mg2SO47H2O, 6.03 mM KNO3, 1.75 mM (NH4)2SO4, 1.03 mM KH2PO4, 0.15 mM EDTAFe, 10-3 mM H3BO3, 10-3 mM MnCl24H2O, 10-3 mM ZnSO47H2O, 10-4 mM CuSO45H2O, 5.0 9 10-6 mM (NH4)6MoO7O244H2O]. Solution of each pot containing four plants was aerated using indigenous air pump and the solutions were changed every 3 d in order to remove the deposition of toxic metabolites and root debris. The seedlings were maintained to grow for 5 weeks and treatments were given, which included C (control, normal growth), CA (control ? foliar application of 100 lM ABA), D (drought), and DA (drought ? foliar application of 100 lM ABA). Drought was imposed in solution culture by adding 25 % (w/v) polyethylene glycol (PEG 6000) into Hoagland solution. The ABA (98 % pure) used in the experiment was procured from Bio Basic Inc. (Toronto, Canada). Leaf and root samples were collected at 0, 3, 6, 9, 12 and 24 h after treatment, immediately frozen in liquid nitrogen and stored at -80 °C for further analysis. ABA content and superoxide anion radical measurement For ABA extraction, 1.0 g frozen samples were homogenized in chilled pestle and mortar using 80 % methanol (v/v). The homogenate was kept at 4 °C for 24 h and centrifuged at 10, 0009g for 20 min. The supernatant was collected and eluted through a Sep-Pak C18 cartridge (Waters, USA) for removing the polar compounds. ABA content was determined by using ELISA kit (Bei Nong Wei Tian Biotech Co. Ltd., Beijing, China) following the procedure described by Li et al. (2012). Superoxide anion radical (O–) 2 production rate was measured by the method described by Elstner and Heupel (1976) through determining nitrite formation from hydroxylamine in the presence of O2– with some modifications. Frozen samples 500 mg were homogenized with 4 ml of 65 mM potassium phosphate buffer (pH 7.8) and centrifuged at 10, 0009g for 15 min. The reaction mixture containing 0.5 ml of 65 mM phosphate buffer (pH 7.8), 0.1 ml of 10 mM hydroxylamine hydrochloride and 1 ml of the supernatant was incubated at 25 °C for 10 min. Then 1 ml 58 mM sulfanilamide and 1 ml 7 mM a-naphthylamine were added to

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the incubation mixture respectively, and again incubated at 25 °C for 30 min. Equal volume of chloroform was added and mixed after incubation and the mixture was centrifuged at 10, 0009g for 5 min. The absorbance of aqueous supernatant was recorded at 530 nm, a control reaction without hydroxylamine hydrochloride (using distilled water) was performed to subtract the pigment interference at the same time. Standard curve was prepared by using different concentrations of NO2– following the same procedure, and used to calculate the O2– production rate. RNA isolation and first strand cDNA synthesis Total RNA was isolated by using TRIzol reagent kit (Invitrogen, USA) according to the procedure described in supplier’s manual. RNA quality was checked on 1.2 % agarose gel (w/v). For amplification of SoNCED gene fragment, first strand cDNA was synthesized from 1 lg total RNA using M-MuLV reverse transcriptase (Fermentas, USA). For getting full-length of SoNCED, cDNA was amplified using SMARTerTM RACE cDNA Amplification Kit (Clontech, USA) according to manual procedure. Amplification of SoNCED gene Degenerated primers were designed based on the highly homologous regions among the NCED sequences of maize (AAB62181.2), sorghum (EER93751.1), rice (AAW21317.1) and barley (ABB71583.1) using a web primer designing software CODEHOP (http://bioinformatics.weizmann.ac.il/ blocks/codehop.html). SoNCED gene fragment was amplified using forward (50 -TGGGCTGCGCCATGATHGCNCAY CC-30 ) and reverse (50 -GCCTCCAGCCGCATGTCNGCN GCRTT-30 ) primers under following PCR conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. 30 -RACE primer (50 -CCC CGCCGACTCCATCTTCA-30 ) and 50 -RACE primer (50 -TT GCGGTTCACCATGCCCAC-30 ) were designed from SoNCED fragment sequence using Primer Premier 5.0 software, combined with the primer UPM (Universal Primer A Mix) supplied by the Kit. 30 and 50 ends of SoNCED were amplified using thermal cycle conditions as 95 °C for 10 min, followed by 40 cycles of 95 °C 30 s, 64 °C 30 s, 72 °C 2 min. A 103 bp length overlap between 30 -RACE and 50 -RACE products was found suitable for full-length assessment of SoNCED gene. All the reactions were performed in a total volume of 50 ll containing 25 ll 29GoldStar Best MasterMix (CWBIO, China), 2 ll cDNA, 1 ll of each 10 lM primer (for 30 -RACE and 50 -RACE, 5 ll UPM was added instead of reverse and forward primers, respectively), and RNase-free sterile water was added to make volume 50 ll. All PCR products were extracted and purified from gel (Bioer, China), cloned into pMD18-T vector (TakaRa,

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Japan), and sent to Sangon Biotech (Shanghai, China) for sequencing. Quantitative real-time PCR analysis Quantitative real-time PCR (qRT-PCR) was performed using SYBR All-in-OneTM qPCR Mix (GeneCopoeia, USA) in iQ5 Real-Time PCR Detection System (Bio-Rad, USA). Reactions were prepared in a total volume of 20 ll containing 10 ll 29 All-in-OneTMqPCR Mix, 2 ll cDNA, 1 ll of each 4 lM primer and 6 ll RNase-free sterile water. qRT-PCR primers of SoNCED (forward 50 -TGCTG GACAAGGAGAAGACG-30 , reverse 50 -AGGTGGAAGC AGAAGCAGTC-30 ) and control GAPDH gene of sugarcane (Accession number CA254672.1) (forward 50 -TGGT GCTGACTATGTCGTGGA-30 , reverse 50 -CATGGGTGC ATCTTTGCTTG-30 ) were designed by Primer Premier 5.0 software. PCR reactions were performed at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 20 s. Melting curve analysis was conducted for each reaction to confirm the specificity of the reaction, all cDNA samples were analyzed in triplicate. Data were analyzed by the software come with the instrument. Relative expression quantification of SoNCED to control GAPDH was analyzed using 2-DDCt method (Livak and Schmittgen 2001). Bioinformatics analyses Sequence homology of SoNCED gene was verified by NCBI database searches using blastx algorithm (http://blast.ncbi.nlm. nih.gov/). The molecular weigh and isoelectric point (pI) of protein (deduced amino acid sequences) were predicted using ExPASy (http://expasy.org/tools/). Protein location was found out by iPSORT (http://ipsort.hgc.jp/). Phylogenetic tree was constructed by using MEGA 5.0 (http://www.mega software.net/) (Tamura et al. 2011). The Multiple sequence alignments were performed using ClustalW2 (http://www. ebi.ac.uk/Tools/clustalw2/index.html) and colored with ESPript (http://espript.ibcp.fr/ESPript/ESPript/) according to maize VP14 protein structure. Protein secondary structure was worked out using SWISS-MODEL (http://swissmodel.expasy. org/) and edited by Swiss-PdbViewer (http://spdbv.vital-it. ch/TheMolecularLevel/SPVTut/).

Results RNA isolation and SoNCED amplification The quality of RNA was measured on 1.2 % agarose gel stained with ethidium bromide. 28S, 18S and 5S RNA fragments owing clean banding pattern in proper

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Fig. 1 Total RNA and electrophoresis results of amplified products a Total RNA isolated from leaves of sugarcane variety GT21; b 859 bp SoNCED gene fragment amplified using degenerate primers; c, d 30 -and 50 -RACE-PCR products of SoNCED gene, respectively

proportion (Fig. 1a) indicated high quality and integrity of total RNA. SoNCED fragment with 859 bp length (Fig. 1b) was amplified by degenerated primers. Sequence analysis of amplified SoNCED fragment showed its 90 % homology with the NCED from maize, sorghum, rice and wheat, so its sequence was used to generate gene specific primers (GSP) to obtain full-length cDNA of SoNCED. Using UPM and GSPs, 1,090 bp (Fig. 1c) and 1,637 bp (Fig. 1d) length products were amplified through 30 - and 50 -RACE-PCR reaction. After sequence assembling, the adapter and overlapping parts were removed, and a 2,521 bp full-length cDNA of SoNCED was constructed. Sequence analysis indicated that 50 -ends of the sequence contained initiating codon and the features fitted for the Kozak (1987) rules and there was a poly (A) signal at the 30 -end. The open reading frame (ORF), starting from 119 bp and measured 1,827 bp, encoded a putative protein of 608 amino acids. The SoNCED gene sequence has been registered in NCBI database (accession number JQ314108).

a-helical inserts and shaped an a-helical domain on top of a b-propeller which was formed by seven-blades (Fig. 4a, b), and four to five anti-parallel strands were included in each blade. Just like in other b-propeller structures, a long tunnel was surrounded by all blades which run through the center from one end to the other (Fig. 4b). His residues in SoNCED (His-298, 347, 412, 590) were predicted to be coordinated with catalytic iron for dioxygenase activity located inside the b-propeller as in other NCEDs secondary structures (Fig. 2; Messing et al. 2010). Surface characteristic analysis of a1-helice and a3-helice revealed a hydrophobic patch composed of 19 hydrophobic residues (Ala-95, Ala-96, Ala-97, Ala-98, Ala-99, Leu-100, Ala102, Val-108, Ala-109, Val-111, Leu-112, Ile-229, Ala230, Leu-233, Ala-234, Leu-235, Ala-237, Ala- 239 and Ala-240) (Fig. 2) which were predicted to penetrate the plastid membrane beyond the head groups of the membrane phospholipids and interact with fatty acid residues (Tan et al. 2001).

Structural analysis of SoNCED protein

Effect of drought and ABA treatments on O2- levels

The predicted molecular weight (MW) and pI of SoNCED protein were 65.9 kD and 6.04, respectively. The negative grand average of hydropathicity (GRAVY) of predicted protein (-0.172) indicated its hydrophilic nature. The SoNCED has been predicted to be located at chloroplast with a chloroplast-targeting peptide at the N-terminus (Fig. 2). Based on the deduced amino acid sequence alignment with the sequences of other crop plants, the SoNCED showed high similarity to the NCEDs of Zea mays, Oryza sativa, Citrus clementina and Solanum lycopersicum (Fig. 2). For assessing the interrelationship amongst NCEDs of different plants, a phylogenetic tree was constructed from aligned amino acid sequences using MEGA5.0 software. The results indicated the clustering of SoNCED with SbNCED1 (EER93751.1) and ZmVP14 (AAB62181.2) in a small branch owing to C4 plant species (Fig. 3). Secondary structure of the protein analyzed by using SWISS-MODEL indicated that the SoNCED folded as four

Effect of different treatments on O2- levels in leaves and roots of sugarcane variety GT21 seedlings was shown in Fig. 5. All the three treatments caused a significant increase in levels of O2- in both leaves (Fig. 5a) and roots (Fig. 5b) compared to the control, however, the increasing trends differed in different treatments. The treatment CA increased the O2- levels up to 12 h followed by a slight decrease at 24 h, though it was 37.1 % higher compared to the control. In the treatment D, the O2- levels continuously followed an increasing trend which reached its maximum at 24 h (82.4 % higher than the control) in leaves. In the treatment DA, the O2- levels were enhanced by 49.1, 44.6, 59.7, 55.4 and 62.0 % compared to the control at 3, 6, 9, 12 and 24 h after treatment, respectively, nevertheless, they were significantly lower than those in the treatment D at each time point. In roots, the treatment CA enhanced the O2- levels until 6 h before decreased it, though it was significantly higher than the control. The treatment D enhanced the O2- levels by 87.3, 113, 119.5, 138.8 and

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Fig. 2 Sequence alignment of SoNCED and NCED proteins from several crop plants SoNCED is aligned with NCED proteins from Zea mays (Zm), Oryza sativa (Os), Citrus clementina (Cc) and Solanum lycopersicum (Sl). Dark backgrounds represent identical sequences, the underlined residues reflect the predicted chloroplast transit

peptide of SoNCED, the number 1 under C410 and C430 indicates a di-sulfide bond, the triangles below the residues show identity for the His residues coordinated with the catalytic iron, and the alignment was performed using ClustalW2 and ESPript software

149.8 % at 3, 6, 9, 12 and 24 h in roots, while the increase was only 80.5, 98.1, 105.6, 117.3, and 128.1 % in the treatment DA, respectively.

6 h in roots, which later decreased and did not showed any specific trend, however, the enhancement in gene expression was higher at all time compared to the control. Both the treatments D and DA also caused significantly increased expressions of SoNCED, but the ABA contents were found higher in the treatment D and the highest in the treatment DA. The ABA contents were increased by 30.3, 23.0, 30.4, 18.2 and 18.0 % in leaves while 12.7, 25.4, 32.4, 51.8 and 66.0 % in roots in the treatment D compared to the treatment CA at 3, 6, 9, 12 and 24 h, respectively, while increased by 28.4, 23.7, 9.9, 15.2 and 28.7 % in leaves and 4.7, 14.4, 12.3, 21 and 13.3 % in roots in the treatment DA compared to the treatment D at 3, 6, 9, 12 and 24 h, respectively.

Effect of drought and ABA treatments on SoNCED expression and ABA content The treatments CA, D and DA significantly increased the expression of SoNCED and ABA content in leaves (Fig. 6a, c) and roots (Fig. 6b, d) compared to the control. The treatment CA caused a continuous increase in SoNCED expression and ABA content up to 12 h and remained almost constant until 24 h in leaves, while the enhancement kept for

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Fig. 3 Phylogenetic tree of NCED proteins from Saccharum officinarum L. and other crop plant species. The phylogenetic tree was constructed by MEGA5.0 software using a Neighborhood Joining Bootstrap method (Bootstrap analysis with 1,000 replicates). The numeric values in each node represent percentage of homology amongst NCEDs, verified by bootstrap, and the accession number of each protein is listed in the bracket. The first two characters before the

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protein names indicate abbreviations of plant genera and species as follows: Ah Arachis hypogaea; At Arabidopsis thaliana; Bn Brassica napus; Cc Citrus clementina; Cm Chrysanthemum x morifolium; Cs Crocus sativus; Dc Daucus carota; Hv Hordeum vulgare; Lf Lilium formosanum; Os Oryza sativa; Pa Persea americana; Rc Ricinus communis; Sb Sorghum bicolor; Sl Solanum lycopersicum; So Saccharum officinarum; Vv Vitis vinifera; Zm Zea mays

Fig. 4 The predicted secondary structure of SoNCED protein. a Side view of SoNCED with b-strands, a-helices and b-propeller; b Top view rotated 90° towards the viewer from ‘a’, showing seven-blades of the propeller

Discussion ABA plays an important role in plant’s growth and development processes (Finkelstein et al. 2008; Qiu and Yu 2009). NCED catalyzes the rate-limiting step in ABA biosynthesis (Tan et al. 1997; Qin and Zeevaart 1999) and a key regulator which determines endogenous ABA levels under different soil and environmental conditions (Schwartz et al.

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2003). In the present study, a full-length cDNA encoding NCED from sugarcane (SoNCED) was cloned by using RTPCR and RACE-PCR for the first time. The results of sequencing and deduced amino acids homology BLAST showed high similarity to comparable molecules from other plant species. The results of clustering patterns via phylogenetic tree analysis showed the SoNCED with SbNCED1 and ZmVP14 belonged to C4 plant of the same family. This

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Fig. 5 Production rate of superoxide anion radical (O2-) in leaves (a) and roots (b) of seedlings in sugarcane variety GT 21 grown under different treatments (CA Control?ABA; D Drought; DA Drought?ABA) at 0 (Control), 3, 6, 9, 12 and 24 h, respectively. Bars superscripted by a different letter are significantly different at the 0.05 probability at the same time

Fig. 6 Relative expression of SoNCED gene and accumulation of ABA content in leaves (a, c) and roots (b, d) of seedlings in sugarcane variety GT 21 grown under different treatments (CA Control?ABA; D Drought; DA Drought?ABA) at 0 (Control), 3, 6, 9, 12 and 24 h, respectively. Bars superscripted by a different letter are significantly different at the 0.05 probability at the same time

revealed its close evolutionary relationship within these plant species. The secondary structure of SoNCED constructed using SWISS-MODEL revealed that, similar to ZmVP14 (Messing et al. 2010), SoNCED also has a seven-bladepropeller domain and comprised largely of a-helices. The b-propeller portion structure, presented in the SoNCED, was suggested to be a conserved characteristic throughout the carotenoid cleavage dioxygenases family of enzymes

(Kloer and Schulz 2006) while the a-helices which contained patches of hydrophobic residues, were predicted to help in penetrating the plastid membrane beyond the head groups of the membrane phospholipids and interact with fatty acid residues, because the first helix deletion or disruption resulted in loss of interaction of NCED with the membrane and ABA biosynthesis ability (Tan et al. 2001). Previous study showed requirement of Fe2? for dioxygenase activity which is bound with four His residues in

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ZmVP14 (Messing et al. 2010), and the results of sequence alignment analysis showed that His residues coordinated with the catalytic iron for dioxygenase activity were conserved in SoNCED as in other NCEDs. As a perceptive signal under water stress conditions (Suhita et al. 2004), or on foliar application of ABA (Jiang and Zhang 2001, 2002), the plant hormone ABA increases in roots and leaves and plays an important role in regulation of plant responses from the whole plant to cellular level. O2- and other ROS (Reactive oxygen species) such as hydroxyl radical (OH.), perhydroxy radical (HO.2), alkoxy radicals (RO.), hydrogen peroxide (H2O2) and singlet oxygen (1O2) are produced continuously as byproducts of various metabolic pathways that are localized in different cellular compartments including mitochondria, chloroplast and peroxisomes (del Rı´o et al. 2006; Navrot et al. 2007). Under steady state conditions, the ROS can be removed by means of antioxidants or antioxidative enzymes and stay equilibrium (Gill and Tuteja 2010). However, metabolic pathways in plant cells are sensitive to environmental stress such as drought, salinity, chilling, heat shock, heavy metals, pathogen attack and high light stress, metabolic imbalances can induce an oxidative stress in cells by promoting a dramatically increase and accumulation of ROS, this may result in increased oxidation of cellular components, hindering metabolic activities and destroying organelle integrity. As a striking common component in response to abiotic stresses (Jaspers and Kangasja¨rvi 2010; Vaahtera and Brosche´ 2011), ROS also act as signaling molecules in the regulation of stress adaptation in contrast to their role as simply damaging agents in plant cells (Miller et al. 2008; Jaspers and Kangasja¨rvi 2010). Increasing evidences indicated that abiotic stresses induced ABA accumulation triggers the increased generation of ROS in plant tissues (Jiang and Zhang 2001, 2002; Hu et al. 2006). The present experimental results showed that the D treatment could cause significantly enhanced SoNCED expression and ABA accumulation, followed by a significant increase in O2- levels in both leaves as well as in roots of sugarcane seedlings as compared to the control. Even under normal conditions, exogenous application of ABA could also cause the increase of endogenous ABA and ROS (Jiang and Zhang 2001, 2002), just as the higher ABA and O2- levels followed by a higher expression of SoNCED in the CA treatment than the control in the present research. It has been documented that ROS is involved in cellular signaling process as the second messengers in ABA signaling, and play an important role in avoiding deleterious leaf dehydration by closing stomata (Ikegami et al. 2009), decreasing transpiration rate (Endo et al. 2008) and controlling root water uptake and/or plant water status via root growth and root hydraulic conductivity (Maurel et al. 2008) under stress conditions. Previous

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studies also showed that the expression of antioxidant genes encoding SOD (Kaminaka et al. 1999), CAT (Guan et al. 2000; Lu et al. 2009) could be induced by ROS which was triggered by ABA. Further research indicated that this kind of antioxidant defense system and ABA content could be further improved by foliar application of ABA, and enhanced the ability of ROS scavenged (Jiang and Zhang 2002; Zhang et al. 2009). This may well explain the higher ABA accumulation and lower O2- levels in the DA treatment and the close relationship among the ABA content, SoNCED expression and O2- levels in the present study. In summary, a 9-cis-epoxycarotenoid dioxygenase gene from Saccharum officinarum L., SoNCED, has been identified and characterized, and it was shown that the SoNCED gene expression plays an important role in regulation of ABA level in plants during drought stress and exogenous ABA application. A close correlation was found between O2- levels, SoNCED expression and endogenous ABA content in the plants, suggesting that SoNCED plays a primary role in the biosynthesis of ABA and modulation of oxidative stress responses in sugarcane. Acknowledgments This present study was supported by International Scientific Exchange Program projects (2008DFA30600, 2009DFA30820), Guangxi R & D Research Program projects (Gui Ke Neng 0815011, Gui Ke Chan 1123008-1), Guangxi Natural Science Foundation project (2011GXNSFF018002) and Guangxi Academy of Agricultural Sciences research fund (2011YT01).

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