Cloning and functional analysis of a novel ascorbate peroxidase (APX) gene from Anthurium andraeanum

Cloning and functional analysis of a novel ascorbate peroxidase (APX) gene from Anthurium andraeanum 1 2 first-author 2 2 2 2 1 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhjzhu@zju.edu.cn †E-mail:zhjzhu@zju.edu.cn zhjzhu@zju.edu.cn College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, China College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, China Research & Development Center of Flower, Zhejiang Academy of Agricultural Sciences, Hangzhou 311202, China Research & Development Center of Flower, Zhejiang Academy of Agricultural Sciences, Hangzhou 311202, China School of Agricultural and Food Science, Zhejiang A&F University, Lin’an 311300, China School of Agricultural and Food Science, Zhejiang A&F University, Lin’an 311300, China An 888-bp full-length ascorbate peroxidase (APX) complementary DNA (cDNA) gene was cloned from Anthurium andraeanum , and designated as AnAPX . It contains a 110-bp 5′-noncoding region, a 28-bp 3′-noncoding region, and a 750-bp open reading frame (ORF). This protein is hydrophilic with an aliphatic index of 81.64 and its structure consisting of α-helixes, β-turns, and random coils. The AnAPX protein showed 93%, 87%, 87%, 87%, and 86% similarities to the APX homologs from Zantedeschia aethiopica , Vitis pseudoreticulata , Gossypium hirsutum , Elaeis guineensis , and Zea mays , respectively. AnAPX gene transcript was measured non-significantly in roots, stems, leaves, spathes, and spadices by real-time polymerase chain reaction (RT-PCR) analysis. Interestingly, this gene expression was remarkably up-regulated in response to a cold stress under 6 °C, implying that AnAPX might play an important role in A. andraeanum tolerance to cold stress. To confirm this function we overexpressed AnAPX in tobacco plants by transformation with an AnAPX expression construct driven by CaMV 35 S promoter. The transformed tobacco seedlings under 4 °C showed less electrolyte leakage (EL) and malondialdehyde (MDA) content than the control. The content of MDA was correlated with chilling tolerance in these transgenic plants. These results show that AnAPX can prevent the chilling challenged plant from cell membrane damage and ultimately enhance the plant cold tolerance. AnAPX Gene expression Cold stress Anthurium andraeanum Introduction 1 Plants frequently encounter various unfavorable environmental conditions, such as extreme temperatures, salinity, drought, and heavy metals, which adversely affect plant growth and development. Anthurium andraeanum , originating from the tropic areas, is an indoor ornamental potted flower produced popularly in Zhejiang Province, China. In this area the production of this flowering plant in protected cultivation requires heating in winter as this plant can only grow well above 15 °C, and is vulnerable to low temperature below 12 °C. Therefore, the high energy cost especially in winter reduces its market profitability. A cultivar with a higher cold tolerance is thus highly desired for this flower production. Apart from traditional breeding methods, genetic engineering is now thought to be a successful approach to create new stress tolerant varieties (Bhatnagar-Mathur et al., 2008 2008 ). However, it remains very difficult to identify the target genes for genetic manipulation or modification, resulting in such tolerant plants. The reactive oxygen species (ROS) are ubiquitously generated in all of the aerobic organisms even under no stress conditions, but accumulated at a lower level which is fine regulated by their production and degradation (Mittova et al., 2004 2004 ). The hydroxyl radical (·OH), single oxygen ( 1 O 2 ), superoxide anion radical (O 2 − ·), and hydrogen peroxide (H 2 O 2 ) are four common ROS arising from many metabolic pathways such as β-oxidation, photorespiration, and purine metabolism. Among these ROS, H 2 O 2 is the most stable one. Its removal by the ascorbate-glutathione cycle (AGC) constitutes one of the major ROS scavenging systems, protecting plants against oxidative stress caused by H 2 O 2 surge (Asada, 1992 1992 ). The AGC can be constituted with many enzymes including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DAR), and glutathione reductase (GR). However, APX is the most generally employed in plants which can rapidly convert H 2 O 2 to water at the expense of ascorbate (AsA) consumption as its specific electron donor (AH 2 ). APX has several isoenzymes which are named according to their subcellular localizations, such as cytosolic APX (cAPX) in cytosolic, membrane-bound APX (mAPX) in glyoxysome and peroxisome, thylakoid membrane-bound APX (tAPX) and stromal APX (sAPX) in chloroplasts, and mitochondrial membrane-bound APX (mitAPX) in mitochondria (Shigeoka et al., 2002 2002 ). APX activity was up-regulated substantially when plants were subjected to environmental stresses, for instance, heavy metals, high salinity, drought, high temperature, and wounding (Shi et al., 2001 2001 ). APX has been cloned from many plant species such as pea (Mittler and Zilinskas, 1991 1991 ), Arabidopsis (Kubo et al., 1992 1992 ; Santos et al., 1996 1996 ; Maruta et al., 2012 2012 ), maize (van Breusegem et al., 1995 1995 ), spinach (Webb and Allen, 1995 1995 ; Yoshimura et al., 1999 1999 ), tobacco (Orvar and Ellis, 1995 1995 ), strawberry (Kim and Chung, 1998 1998 ), barley (Shi et al., 2001 2001 ), potato (Kawakami et al., 2002 2002 ), rice (Lu et al., 2005 2005 ), Vitis pseudoreticulata (Lin et al., 2006 2006 ), birch (Wang et al., 2009 2009 ), muskmelon (Cheng et al., 2009 2009 ), lily (Chen et al., 2010 2010 ), grain sorghum (Chen et al., 2011 2011 ), and Nelumbo nucifera (Dong et al., 2011 2011 ). In this work, an APX gene from A. andraeanum was cloned and its expression patterns were analyzed. The function of this gene was also investigated and proposed to be associated with the plant tolerance to cold. These results could provide information for APX gene as a target for genetic engineering to improve the production of this flower plant. Materials and methods 1 Plant and treatment 2 Plantlets of A. andraeanum ‘Alabama’ were grown in pots with the medium of turves:perlite (4:1, v/v) in a greenhouse. Different tissue samples of root, stem, leaf, spathe, and spadix were collected from one-year-old plants. The cold treatment was carried out by placing the pot-grown one-year-old plants in a phytotron with a 12-h light/dark photoperiod at 25 °C for 3 d pretreatment, and then at 6 °C for low temperature treatment. Samples of the first fully expanded leaves were taken at 0, 12, 24, 36, and 48 h, respectively, during treatment. All samples were immediately frozen in liquid nitrogen and then stored at −80 °C. Cloning of AnAPX gene 2 Based on a highly conserved region of plant APX , the primer pairs (pAPXF: 5′-AAGGAGCAGTTCCCCATCC-3′; pAPXR: 5′-GCAAAGAAMGCRTCCTCRTC-3′) were designed and used to clone a partial complementary DNA (cDNA) fragment of AnAPX through polymerase chain reaction (PCR). The 5′- and 3′-rapid amplification of cDNA ends (RACE) were used to obtain a full-length gene. The total RNA from young leaves was extracted with the RNA Extraction Kit (TaKaRa) and the reverse transcription was carried out with the 5′/3′-Full RACE Kit (TaKaRa). The 5′ specific primer (5′-CAGCAACGACACCAGCCAA-3′) and 3′ primer (5′-AGCGTTCTGGCTTTGAGGGA-3′) were designed based on AnAPX . After purification of the expected PCR products, they were cloned with PCR instrument (BIO-RAD S1000™ Thermal Cycler) and sequenced on an ABI-PRISM3730 sequencer. The putative 5′- and 3′-RACE products and the partial AnAPX fragment were matched to form a putative cDNA contig which contained the initiation codon, termination codon, and open reading frame (ORF). A pair of full-length primers (APXcomF: 5′-GGGCGAGATCGAGCTGCTAAG-3′; APXcomR: 5′-GGCGATCAGCATCACACATATTCCT-3′) was designed with the basis of this contig and then used to amplify the full-length cDNA sequence of AnAPX . This AnAPX sequence contained a 110-bp 5′-nocoding region, a 28-bp 3′-nocoding region, and a 750-bp ORF. Sequence analysis 2 The full-length AnAPX cDNA was translated and analyzed on the website ( http://www.expasy.ch/tools http://www.expasy.ch/tools ). The homologous sequences were searched based on the obtained amino acid sequence through BLASTP in NCBI. The secondary structure of putative AnAPX protein was predicted with DNASTAR. Multiple alignments between A. andraeanum and other plants were carried out using DNAMAN software (Lynnon Co., Canada). The cluster analysis was performed using MEGA5.1. Real-time PCR (RT-PCR) 2 The total RNA of A. andraeanum was extracted using RNA Extraction Kit (TaKaRa) from different tissues and the cold-treated (6 °C) leaves as described above. The reverse transcription was performed with M-MLV (TaKaRa) and the obtained cDNA templates were applied to RT-PCR. Primer pairs (Real-FP: 5′-CTCAGCCACGGGGCCAACAA-3′; Real-RP: 5′-GGGATCTCAGGCCCTCCGG-3′) were designed using Primer Express 2.0 (ABI). This pair of primers was used for PCR amplification and generated products with the size of 100–300 bp. We used Actin (Actin-FP: 5′-GCTGAGCGGGAAATTGTTCG-3′; Actin-RP: 5′-CAGGCAGCTCATAGGTCTTCTC-3′) as a reference gene, which spans a 121-bp sequence. RT-PCR was performed with ABI7900 Real-Time System, carried out with Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Three different RNA samples were used as three biological replicates, and each biological replicate has three technical replicates. The fold changes of each gene were calculated using the 2 −Δ C T method. Enzyme activity assay 2 The AnAPX activity was determined according to Dalton et al. ( 1987 1987 ) and Kong and Yi ( 2008 2008 ) with a modified protocol. For extraction of APX, the leaves (0.5 g) were homogenized with 50 mmol/L potassium phosphate buffer saline (PBS; pH 7.8) containing 0.3 mmol/L AsA in a cold mortar and pestle. The homogenate was quickly transferred to a test tube and centrifuged at 12 000 r/min for 15 min. The supernatant was transferred to another tube and used to determine the APX activity. The mixture containing 1700 μl PBS (25 mmol/L, pH 7.0), 100 μl AsA (5 mmol/L), 100 μl H 2 O 2 (20 mmol/L), and 100 μl supernatant was incubated in a quartz cuvette at 290 nm. We determined the decline in the optical density during 1 min and calculated the activity. Transformation and transgenic tobacco analysis 2 The AnAPX gene was cloned with primer pairs (APXcomF and APXcomR), digested with Sac I and pst I, and then inserted into the expression vector pCAMBIA2300. Tobacco plants were transformed using Agrobacterium tumefaciens mediated transformation method according to Horsch et al. ( 1985 1985 ). The transgenic tobacco plants survived in culture medium that contained 50 μg/ml kanamycin and 200 μg/ml cephalosporin were verified by PCR analysis. The controls were wild-type (WT) tobacco plants. Tobacco cold tolerance assays 2 For the cold tolerance assays, the following procedures were carried out. AnAPX transgenic tobacco seedlings and controls were sown on holed plates at 25 °C for one month. Then the seedlings were transferred to 4 °C for 12 h in a low-temperature incubator in continuous light (2 000 lux) and returned to the original growth conditions for 7 d. To study the chilling injury degree of transgenic seedlings, we used tender leaf (the first fully expanded leaf) derived from the transgenic and control seedlings subjecting to identical cold stress treatment as the whole plants. The percentage of electrolyte leakage (EL) and malondialdehyde (MDA) contents were assayed according to Hara et al. ( 2003 2003 ). Statistical analysis 2 Each experiment was repeated at least three times. All values were represented as mean±standard deviation (SD). The comparisons between values were evaluated within each tissue and treatment time by a single factor analysis of variance using Duncan’s method. The significance of difference was set at 0.05 in all such cases. Results 1 Cloning of full-length AnAPX gene 2 To amplify the sequences encoding APX from A. andraeanum , the first cDNA strands was used as the templates for PCR. PCR products of the predicted size were sequenced and identified. Based on the 446-bp partial A. andraeanum APX fragment, 5′- and 3′-RACE were performed to obtain full-length sequences of AnAPX . The 5′-RACE products of 757 bp and 3′-RACE products of 533 bp were obtained, and the resulting sequences were consistent with the putative APX partial fragments. These three fragments were then aligned and a 1160-bp contig that contained 116-bp of 5′ noncoding region, 294-bp of 3′ noncoding region, and 750-bp of ORF was obtained. For the purpose of validating the sequence correctness of this contig, the full-length primers (APXcomF and APXcomR) were used for PCR, producing an 888-bp sequence, named as AnAPX (GenBank accession No. JQ838071) (Fig. 1a 1a ). (a) The deduced sequence of amino acid residues was aligned based on the A. andraeanum APX cDNA; (b) The secondary structure of putative AnAPX protein was predicted with DNASTAR Sequence analysis of AnAPX showed that the AnAPX sequence was perfectly associated with that of contig, indicating that the sequence is precise and accurate. The ORF of AnAPX encodes a 250-amino acid protein. The molecular mass of AnAPX protein is 27.4 kD and the isoelectric point (pI) is 5.40. The protein includes α-helixes, β-turns, and random coils (Fig. 1b 1b ). The protein is a hydrophilic with the aliphatic index of 81.64. Further analysis of AnAPX protein according to the GenBank of known sequences shows that the protein belongs to a typical peroxidase heme-ligand superfamily. This protein has a peroxidase active site (33–44: APLMLRLAWHSA) and a peroxidase proximal heme-ligand site (155–165: DIVALSGAHTL) (Fig. 1a 1a ). The conserved domains of AnAPX were found with coding sequence (CDS) in NCBI, showing that APX protein belongs to a plant-peroxidase-like superfamily (Fig. 1a 1a ). Sequence analysis indicated that the deduced amino acid residues of AnAPX were highly similar to the known APX homologs from other plants, such as Zantedeschia aethiopica (No. AAC08576, 93%; No. AAK57005, 88%), V. pseudoreticulata (No. AAZ79357, 87%), Gossypium hirsutum (No. ABR18607, 87%; No. ACJ11731, 86%), Elaeis guineensis (No. ACF06591, 87%), and Zea mays (No. ACF87724, 86%; No. NP_001170482, 86%). Cluster analysis revealed that the deduced amino acid residues were conserved in all APX sequences. Highly conserved protein sequences appeared on the C-terminal regions, whereas non-conserved sequences existed on the N-terminal regions (Fig. 2 2 ). A phylogenetic tree was created with the basis of AnAPX amino acids and other 16 typical plants (Fig. 3 3 ). The phylogenetic relationship between AnAPX and APXs of other 16 plants showed that AnAPX had the closest relationship with Z. aethiopica (No. AAC08576), while it had the most distant relationship with A. thaliana (No. NP_172267). Multiple alignments of those 17 sequences revealed that all amino acid sequences were highly conserved, implying their functions are evolutionarily conserved. Expression of AnAPX in plant tissues 2 The expression of AnAPX in different tissues was analyzed to understand the spatial expression pattern of AnAPX . The abundance of the AnAPX gene in different tissues such as root, stem, leaf, spathe, and spadix was measured by quantitative RT-PCR. Our results revealed that AnAPX was expressed in all those tissues. The relative expression level varied insignificantly between root and spathe, as well as among stem, leaf, and spadix. However, the difference was significant in root vs. stem, root vs. leaf, root vs. spadix, leaf vs. spathe, and spathe vs. spadix (Fig. 4 4 ). Nevertheless, all of these showed that the expression of AnAPX could be detected in all those organs, indicating that AnAPX might have different functions in different tissues during the growth and development of A. andraeanum . Data are expressed as mean±SD, n =3. Different letters above the bar show P < 0.05 Expression of AnAPX under cold stress 2 To find out the possible function of AnAPX in response to cold stresses, we analyzed its expression level under low temperature. The results showed that AnAPX ’s expression was remarkably up-regulated under low temperature. After 12-h treatment, the expression level of AnAPX was increased to 3.44 times of the control, whereas the relative expression level varied insignificantly during 12–36 h treatment, reaching the maximum after 48-h treatment (Fig. 5 5 ). These results indicated that AnAPX might be related to plant cold response. Data are expressed as mean±SD, n =3. Different letters above the bar show P < 0.05 Enzyme activity of AnAPX 2 To understand the changes of APX activity under low temperature stress, we measured the APX activity of A. andraeanum treated with 6 °C temperature (Fig. 6 6 ). The results showed that APX activity declined sharply during the first 12-h cold treatment in comparison with the control, then it averted to increase and reach a maximum level at 36 h; afterwards, the activity returned to normal level (Fig. 6 6 ). On the whole, the changes showed the trend of first declining, then rising and finally declining. Data are expressed as mean±SD, n =3. Different letters show P < 0.05 Analysis of AnAPX transgenic plants 2 The function of AnAPX has been investigated by transforming an expression construct of AnAPX cDNA with the CaMV 35S promoter into tobacco. The malondialdehyde (MDA) content which indicated the extent of lipid peroxidation was employed to estimate the degree of cold injury in transgenic plants (Fig. 7 7 ). The content of MDA was increased by the cold treatments, but the increased amount in 35S:: AnAPX transgenic plants was lower than that in WT plants. In addition, less MDA content was detected in high-expression lines than in low-expression strains after cold treatment (Fig. 7a 7a ). Similar results were obtained when EL tests were applied to both transgenic plants and WT plants. When exposed to 4 °C for 12 h, the EL values were 78.4% and 69.3% for WT plants and transgenic plants, respectively, which indicated the increasing degree of WT plants was higher than that of transgenic lines (Fig. 7b 7b ). In contrast, the transgenic tobacco APX activity increased by 14.5%, while decreased by 3.3% in WT plants (Fig. 7c 7c ). In short, overexpression of AnAPX in tobacco plants slowed down the increase of MDA and EL due to the responses to cold stress but stimulated APX activity and consequently enhanced the cold tolerance in the transgenic plants. Data are expressed as mean±SD, n =3. Different letters above the bar show P < 0.05 Discussion 1 Under adverse conditions, a large number of ROS are often produced and accumulated in plant cells, which will bring the plants into an oxidative stress condition resulting into retarded plant growth and development. However, plants have developed its defense system against such oxidative stress both through nonenzymatic and enzymatic ROS scavenging systems. A nonenzymatic system uses antioxidants to quench ROS such as glutathione (GSH), AsA, and flavonoids, while an enzymatic system employs many enzymes including APX, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT). Hydrogen peroxide, the natural product originated mostly from the photosynthetic electron transport chain and some enzymatic reaction in plant chloroplast, is toxic to the plant photosynthetic system. High concentration of H 2 O 2 can also inhibit the enzymes of Calvin cycle. APX is a key enzyme in the enzymatic ROS scavenging system called AsA-GSH redox system that can remove the H 2 O 2 especially in the chloroplast (Shen et al., 1997 1997 ). Cold temperature will bring plants into an oxidative stress and it was shown that APX activity was up-regulated in plants such as rice (Oidaira et al., 2000 2000 ), Coffea (Fortunato et al., 2010 2010 ), haw (Eltelib et al., 2011 2011 ), and pepper (Airaki et al., 2012 2012 ) when subjected to low temperature. This is thought to be a normal defense reaction when plants are subjected to cold stress. Therefore, the increased APX activity acquired during cold acclimation can help plants tolerate or survive under even lower temperature or longer cold stress (Baek and Skinner, 2003 2003 ; Kuk et al., 2003 2003 ). In this work, the APX activity of A. andraeanum declined first, then gradually increased to a maximum level and finally returned to a normal level when subjected to cold stress (Fig. 6 6 ). Accordingly, we speculated that the early 12−36 h low temperature treatment on A. andraeanum was just like the plants acclimatizing to cold temperature conditions. When the plants got adapted to such conditions after 12 h, some unknown mechanisms triggered the adjustment of the ROS scavenging system and consequently the APX activity was reverted from a decrease into an increase trend. The rise of APX activity indicated that the capacity of A. andraeanum against membrane lipid peroxidation under the lower temperature was increased. Obviously, further research is required to illustrate the mechanism how A. andraeanum has such a transition of APX activity during the cold stress. Previous research also indicated that the APX activity was tightly associated with the expression of APX gene, which could be up-regulated after the plants treated with adversities, such as salt, wounding, drought, and heat. For example, the APX activities in leaves of salt-tolerant pea cultivars increased after treated with NaCl (Hernández et al., 1995 1995 ); the APX expression of Betula platyphylla remarkably increased after treated with NaCl (Wang et al., 2009 2009 ). The APX expression of sweetpotato was remarkably up-regulated after wounding for a long time (Park et al., 2004 2004 ). Drought combined with heat could induce the culmination of APX1 mRNA and protein in A. thaliana (Koussevitzky et al., 2008 2008 ). In this study, the expression of A. andraeanum APX mRNA was evidently elevated by cold stress, which is consistent to the other previous reports. Therefore, we believed that APX plays defense roles in plants under oxidative stress and its activity might be transcriptionally regulated. Since the expression of APX was closely related to the degree of resistance to cold stress, it could be considered rational to increase its expression by introducing any exogenous APX gene to plants, then to improve their stress tolerance. As of now, APX genes were cloned from many kinds of higher plants, and some transgenic plants were studied and proven that the overexpression of APX genes in these plants can indeed provide additional protection against oxidative stress under adverse plant growth conditions. For example, overexpression of chloroplast APX gene in cotton has conferred higher photochemical activity of photosystem II (PSII) and stronger antioxidant capacity to transgenic plants (Kornyeyev et al., 2001 2001 ). Overexpression of chloroplast APX gene in A. thaliana could increase its resistance to herbicide (Murgia et al., 2004 2004 ). Not alone, overexpression of tobacco chloroplast APX gene improved the resistance to drought and salt stresses (Badawi et al., 2004 2004 ). The peroxidase activity of the CAPOA1 (a pepper ascorbate peroxidase-like 1) transgenic tobacco increased, resulting in stress resistance and pathogen tolerance (Sarowar et al., 2005 2005 ). The peroxisomal type ascorbate peroxidase ( pAPX ) gene originated from barley was transferred to A. thaliana , and the transgenic plants exhibited higher resistance to salt stress (Xu et al., 2008 2008 ). APX gene originated from Hordeum vulgare ( HvAPX ) could enhance the resistance to high temperature stress in A. thaliana (Shi et al., 2001 2001 ). As well as tAPX (thylakoid-bound ascorbate peroxidase gene from tomato leaf) transgenic tobacco plants, their resistance to extreme temperature has been improved (Sun et al., 2010 2010 ). Therefore, we reckon that APX -overexpressed A. andraeanum plants should also have a higher resistance to oxidative outburst caused by cold stress and cloning this gene is certainly the first requisite for such an effort to make a transgenic plant. In this paper, we described that a novel APX gene has been cloned from A. andraeanum . Transcript levels of APX could be quickly induced by the cold stress, and functional analysis also proved that the anti-cold characteristic of transgenic tobacco was enhanced by overexpression of AnAPX . The results implied that AnAPX may be a functional gene related to cold stress. Thus, it is feasible to apply AnAPX gene to create anti-cold plants by genetic engineering. Further investigation is obviously required to understand how many isoforms of APX in A. andraeanum and their respective subcellular localizations in order to maximize the utilization of the right isoform genes. Previous researches such as on tobacco (Lim et al., 2007 2007 ), sweetpotato (Wang et al., 2005 2005 ), and tomato (Sreenivasulu et al., 2007 2007 ) also has reported. Nevertheless, the experiment in this study has just characterized the expression of AnAPX against one kind of stress. In the field, multiple stresses often occurred simultaneously (Chen et al., 2006 2006 ), which will be undoubtedly more harmful to the growth of plants. Therefore, further studies are required to figure out whether AnAPX is also involved in the plant tolerance to multiple abiotic stresses. Conclusions 1 An APX gene from A. andraeanum ‘Alabama’ was cloned for the first time. The full length of the APX cDNA was 888 bp, which consisted of a 110-bp 5′ noncoding region, a 28-bp 3′ noncoding region, and a 750-bp ORF, named AnAPX (GenBank accession No. JQ838071). The ORF of AnAPX encodes a 250-amino acid protein, which has a peroxidase active site (APLMLRLAWHSA) and a peroxidase proximal heme-ligand site (DIVALSGAHTL). Cluster analysis showed that AnAPX was most related to Z. aethiopica , which belongs to Araceae family. Biochemical analysis of AnAPX showed that there was a high activity in plants at low temperature. Molecular expression and functional analysis suggested that AnAPX may be a cold stress responsive gene. These important data shed light on the molecular mechanisms of cold stresses in Anthurium and provide theoretical basis for the improvement of varieties with higher cold resistance through genetic engineering. References M Airaki , M Leterrier , RM Mateos , R Valderrama , M Chaki , JB Barroso , LAD Rio , JM Palma , FJ Corpas . 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Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves Compliance with ethics guidelines Hui-chun LIU, Dan-qing TIAN, Jian-xin LIU, Guang-ying MA, Qing-cheng ZOU, and Zhu-jun ZHU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300105 Project supported by the Science and Technology Key Project of Zhejiang Province (No. 2009C12095) and the National Natural Science Foundation of China (No. 31200527) 2013-04-08 2013-11-25 2013-08-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 14