Plant Mol Biol DOI 10.1007/s11103-014-0260-3

Overexpression of a cotton annexin gene, GhAnn1, enhances drought and salt stress tolerance in transgenic cotton Feng Zhang · Shufen Li · Shuming Yang · Like Wang · Wangzhen Guo 

Received: 6 July 2014 / Accepted: 13 October 2014 © Springer Science+Business Media Dordrecht 2014

Abstract  Plant annexins are members of a diverse, multigene protein family that has been associated with a variety of cellular processes and responses to abiotic stresses. GhAnn1, which encodes a putative annexin protein, was isolated from a cotton (Gossypium hirsutum L. acc 7235) cDNA library. Tissue-specific expression showed that GhAnn1 is expressed at differential levels in all tissues examined and strongly induced by various phytohormones and abiotic stress. In vivo and in vitro subcellular localization suggested that GhAnn1 is located in the plasma membrane. In response to drought and salt stress, transgenic cotton plants overexpressing GhAnn1 showed significantly higher germination rates, longer roots, and more vigorous growth than wild-type plants. In addition, plants overexpressing GhAnn1 had higher total chlorophyll content, lower lipid peroxidation levels, increased peroxidase activities, and higher levels of proline and soluble sugars, all of which contributed to increased salt and drought stress tolerance. However, transgenic cotton plants in which the expression of GhAnn1 was suppressed showed the opposite results compared to the overexpressing plants. These findings demonstrated that GhAnn1 plays an important role in the abiotic stress response, and that overexpression of GhAnn1 in transgenic cotton improves salt and drought tolerance. Electronic supplementary material  The online version of this article (doi:10.1007/s11103-014-0260-3) contains supplementary material, which is available to authorized users. F. Zhang · S. Li · S. Yang · L. Wang · W. Guo (*)  State Key Laboratory of Crop Genetics and Germplasm Enhancement, Hybrid Cotton R & D Engineering Research Center, MOE, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, People’s Republic of China e-mail: [email protected]

Keywords  Cotton · Gossypium hirsutum · GhAnn1 · Drought stress · Salt stress · Abiotic stress tolerance

Introduction Cotton (Gossypium hirsutum L.) is an important commercial crop that is grown worldwide as a source of fiber and edible oil. Cotton shows higher drought and salt tolerance than do many other major crops such as rice, wheat, and maize. Even so, abiotic stress can still have a significant effect on the growth and yield of cotton. As a result of global climate change and environmental pollution, abiotic stress is becoming a major environmental factor limiting cotton growth and productivity (Ahuja et al. 2010). In addition, a decrease in the amount of arable land worldwide, coupled with an increase in the amount of cotton planted on marginal soils that are unsuitable for food crops due to salinity and water shortage has created an urgent need to develop multistress-tolerant cotton (Ragauskas et al. 2006). Therefore, mining candidate genes for abiotic stress tolerance and further improving drought and salt tolerance of cotton through genetic engineering could be an efficient strategy to develop novel, multi-tolerant varieties of cotton. Annexins comprise an evolutionarily conserved, multigene family of Ca2+-dependent, phospholipid-binding proteins that are widely conserved in various plants, animals, and fungi (Laohavisit and Davies 2011). In terms of overall structure, annexins contain a four-fold repeat of an approximately 70 amino acid domain, a discrete calcium binding site, and have molecular masses between 33 and 36 kD (Konopka-Postupolska 2007). In animals, annexins have been intensively studied since they were first identified (Creutz et al. 1978). The first plant annexin was isolated from tomato (Boustead et al. 1989), and many subsequent

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reports focused on the roles of plant annexins in growth, development, and responses to environmental stress. Plant annexins are abundant proteins that can comprise as much as 0.1 % of the plant cell protein (Delmer and Potikha 1997; Moss and Morgan 2004). Recently, genomic, proteomic, and transcriptomic approaches have provided data on the diversity, cellular localization, and expression patterns of different plant annexins (Clark et al. 2012). Annexin genes are present in the genomes of a wide range of plant species, including Arabidopsis (Huh et al. 2010; Konopka-Postupolska et al. 2009), rice (Shang et al. 2007), wheat (Breton et al. 2000), and tobacco (Vandeputte et al. 2007). The majority of plant annexins are localized in the cytosol, but some are found to be peripheral or integral membrane proteins targeted to the plasma membrane, endomembrane, or the nuclear envelope (Battey and Blackbourn 1993; Clark et al. 1994; de Carvalho-Niebel et al. 2002; Thonat et al. 1997). Annexin genes are expressed in most tissues and organs throughout the plant life cycle (Mortimer et al. 2008), and expression is regulated by developmental processes and induced by diverse kinds of biotic and abiotic stresses (Cantero et al. 2006; Divya et al. 2010; Jami et al. 2009; Konopka-Postupolska et al. 2009). Some annexin genes are also induced by plant hormones such as abscisic acid (Xin et al. 2005), gibberellic acid (Lu et al. 2012), jasmonic acid (Kiba et al. 2005), auxin (Baucher et al. 2011), and salicylic acid (Konopka-Postupolska et al. 2009). Furthermore, genetic and transgenic approaches have indicated that annexins play a significant role in protecting plants from both abiotic and biotic stresses (Divya et al. 2010; Lee et al. 2004; Huh et al. 2010; Sravan et al. 2008). Taken together, plant annexins participate in a variety of physiological processes, playing important roles in plant growth and development and in responses to environmental stresses. In cotton, studies on annexin proteins have drawn considerable attention because of their role in fiber expansion and their ability to bind calcium and lipid membranes. At present, nine annexins have been isolated from G. hirsutum, and six from G. barbadense. Since the first cotton annexin was identified in 1993, the fuctional roles of several annexins have been systematically elucidated in vivo and in vitro. For example, annexin proteins may be responsible for the inhibition of β-glucansynthase activity (Andrawis et al. 1993). Also, a recombinant cotton annexin was found to possess ATPase/GTPase activities (Shin and Brown 1999). The upregulation of GhFAnnx and GhAnx1 during fiber elongation indicates their functional roles in cotton fiber development (Wang et al. 2010; Zhou et al. 2011). However, the down‑regulating of GhAnn2 inhibited cotton fiber elongation through modulating Ca2+ fluxes and signaling (Tang et al. 2014). AnxGb6, a gene that is predominantly expressed in the fiber, influenced cotton fiber

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Plant Mol Biol

elongation rate during fiber polar expansion, and was also found to interact directly with F-actin and regulate the mode of the actin assembly (Huang et al. 2013). Expression of four genes encoding putative annexin proteins (AnnGh3–AnnGh6) is regulated during fiber development and induced by some plant hormones and Ca2+. Overexpression of AnnGh3 in Arabidopsis resulted in a significant increase in leaf trichome density and length (Li et al. 2013). Although several genes encoding annexin proteins have been reported in cotton, functional studies of these genes concentrated mainly on the regulation of cotton fiber development. Divya et al. (2010) showed that ectopic expression of a mustard annexin gene, AnnBj1, in cotton enhanced abiotic stress tolerance and fiber quality under stress. No endogenous annexin gene associated with cotton abiotic stress has been documented previously. In this study, we cloned a novel annexin gene family member designated GhAnn1 from cotton (Gossypium hirsutum). By overexpression or suppression of GhAnn1 expression in transgenic cotton, we evaluated stress-related phenotypic, physiological, and biochemical parameters under water-deficit and conditions of high salinity. Our results demonstrate that overexpression of the cotton annexin gene GhAnn1 enhances tolerance to drought and salinity stress in cotton.

Materials and methods Plant materials, growth conditions, and abiotic stress treatments Cotton plants (Gossypium hirsutum L.) accessions 7235, TM-1, W0, and Jinmian19 were grown in a greenhouse under long-day conditions with a 16 h light/8 h dark cycle (28/25 °C) at Nanjing Agricultural University. TM-1 is the standard genetic accession of G. hirsutum (Kohel et al. 1970), 7235 is an elite fiber strain of G. hirsutum developed in China (Qian et al. 1992), W0 is used as a acceptor for Agrobacterium tumefaciens-mediated transgenic cotton development, and Jinmian 19 exhibits high tolerance to abiotic stress. G. hirsutum L. cv. Jinmian 19 was used to investigate the expression of GhAnn1 in response to abiotic stress and different signaling compounds; seedlings were treated individually with 100 mM abscisic acid (ABA), 10 mM salicylic acid (SA), 100 mM methyl jasmonate (mJA), 10 mM hydrogen peroxide (H2O2), 200 mM sodium chloride (NaCl), and 20 % polyethylene glycol (PEG6000), with water treatment as the control. Following the treatments, leaves were collected at different times, frozen in liquid N2, and stored at −70 °C until further use. Cotton (G. hirsutum acc. W0) seeds were surface-sterilized with 70 % ethanol for 30–60 s and then 10 % H2O2

Plant Mol Biol

for 60–120 min, followed by washing with sterile water. Sterilized seeds were germinated on 1/2× MS medium at 26 °C under long-day conditions (16 h light/8 h dark cycle) with a light intensity of 150 µmol m−2 s−1. Cotyledons and hypocotyls were excised from sterile seedlings and used as explants for transformation. Transgenic seeds were grown in a greenhouse under long-day conditions at (28 °C day/25 °C night). Seedlings containing two simple leaves and one heart-shaped leaf were subjected to various abiotic stress treatments. Dehydration stress and salt stress treatments were performed by submerging the roots of the plants in 15 % (w/v) PEG 6000 or 200 mM NaCl. Cloning and sequence analysis of GhAnn1 More than 5,000 cDNA clones were sequenced randomly from the Gossypium hirsutum L. acc 7235 fiber cDNA library (He et al. 2008). A GhAnn1 cDNA clone encoding an annexin protein with a complete 3′-cDNA fragment was obtained. In order to obtain a full-length cDNA, we designed three gene-specific primers (Table S1) based on the sequence of the cDNA fragment. The 5′ end of the cDNA was obtained by 5′ rapid amplification of cDNA ends (RACE) according to the instructions in the Gene Racer Kit user’s manual (Invitrogen, Carlsbad, Germany). The conceptual full-length cDNA was generated by splicing the overlap of the 5′-RACE sequence with the cDNA fragment. Primers were then designed from the 5′ and 3′ ends of the cDNA sequence to amplify the complete open reading frame (ORF) from line 7235 (Table S1). The ORF was predicted with ORF Finder (htt p://www.ncbi.nlm.nih.gov/gorf/gorf.html). The molecular weight (Mw) and isoelectric point (pI) were predicted with the Compute pI/Mw tool (http://web.expasy.org/compute_pi/), and the sequence was examined for a predicted amino acid signal peptide with SignalP3.0 (http://www. cbs.dtu.dk/services/SignalP/). Similarity analysis and multiple sequence alignments were performed with ClustalW (http://www.ebi.ac.uk/clustalw/). Identification of potential annexin genes in the G. raimondii genome Genes and proteins annotated in G. raimondii were downloaded from http://www.phytozome.net. Annexins were identified using HMMER software version 3.0 (Finn et al. 2011) and from the pfam protein families database using the IAA domain (PF00191) as a query. Expressed sequence tag (EST) sequences for four cotton species, Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii were downloaded from the GenBank EST database (http:// www.ncbi.nlm.nih.gov/dbEST/).

The exon–intron structures of annexin genes were analyzed in the plant lineage by comparing the genomic and coding or cDNA sequence information obtained from the above-mentioned genome databases. Intron phases between exon–intron junctions were obtained by using an online tool, the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). Phylogenetic analysis of GhAnn1 with annexin proteins from other plants We investigated the evolutionary relationships between annexin proteins in several plant species including rice, Arabidopsis and cotton. The search was performed using ‘‘annexin’’ as a keyword in the SUPERFAMILY (http://supfam.cs.bris. ac.uk/SUPERFAMILY/), Plaza (http://bioinformatics.psb. ugent.be/plaza/news/index) and Phytozome (http://www.ph ytozome.org) databases; the sequences were retrieved from the corresponding plant genome annotation resources and analyzed. Partial and redundant sequences were excluded. A phylogenetic tree was constructed using the neighbor-joining (NJ) method (Saitou and Nei 1987) as implemented in MEGA version 5.1 (Tamura et al. 2011). Bootstrap analysis was performed using 1,000 replicates. Subcellular localization of GhAnn1 The coding region of GhAnn1 was cloned into the PJIT166GFP vector to generate the PJIT166-35s::GhAnn1-GFP construct under control of the cauliflower mosaic virus (CaMV) 35S promoter. For transsient expression in onion epidermal cells, particle bombardments were performed using the Biolistic® PDS-1000/He™ Particle Delivery System (Bio-Rad, Hercules, CA) with gold particles (containing DNA 1 µg/µl) and 1,350 psi helium pressure. After transformation, the onion cells were incubated at 25 ± 1 °C for 16 h in dark condition. The pJIT166-GFP plasmid vector was used as the control. In order to further investigate the subcellular localization of GhAnn1, the coding sequence of GhAnn1 was cloned into the pDONR201 vector to generate the pDONRGhAnn1 construct. The GhAnn1 sequence was then recombined into the pBIB vector by the Gateway LR recombination reaction (Invitrogen, CA, USA) to generate the pBIB-35S::GhAnn1:GFP expression vector, which was then transformed into Agrobacterium strain EHA105. The Agrobacterium bacterial cells were grown, harvested, and re-suspended in a buffer composed of 10 mM MgCl2 and 10 mM MES (pH 5.6). The cells were induced for transformation by the addition of 100 mM acetosyringone and incubated for 3–4 h at 28 °C. The bacterial suspensions (OD600nm = 0.6) were infiltrated into fully expanded leaves of N. benthamiana plants (Sparkes et al. 2006).

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Protein subcellular localization was analyzed 2–4 days after infiltration. No matter in onion or tobacco cell, membrane marker FM4-64 was used for plasma membrane staining according to the plasma membrane labeling protocol (FM® 4-64, Invitrogen, USA). All images were observed and photographed using a Confocal Laser Scanning Microscopy (Zeiss LSM 780, Germany) fitted with nomarski differential interference contrast (DIC) optics and an LP560 filter (Ex543/Em560) or LP505 filter (Ex488/Em518) for FM464 and GFP visualization under fluar 40×/0.50 M27 objective lens, respectively. Isolation of total RNA, RT‑PCR, and qRT‑PCR analysis Total RNA was extracted from roots, stems, leaves, ovules, and fibers in different developmental stages by the cetyl trimethylammonium bromide (CTAB)-acid phenol extraction method (Jiang and Zhang 2003). Each RNA sample was treated with DNaseI after the extraction to remove contaminating genomic DNA. Total RNA samples (2 μg per reaction) from different tissues were reverse transcribed into cDNA using AMV reverse transcriptase. The cDNAs were then used as templates to assay the expression of GhAnn1 in cotton tissues by semi-quantitative and real-time RTPCR. A cotton elongation factor (EF1α) gene was used as a standard expression control in the RT-PCR reactions. Real-time RT-PCR assays were performed on the ABI Prism 7500 instrument (Applied Biosystems, USA) using a light cycler fast start DNA Master SYBR Green I kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The relative expression levels of GhAnn1 were calculated using the 2−ΔCt method (Livak and Schmittgen 2001). ΔCt is the difference in the Ct of the target GhAnn1 and the control Ghhis3 signals (i.e., ΔCt = CtGhAnn1 − CtGhhis3) with three biological replicates and three experimental replicates. GhAnn1 sense, antisense, and 3′‑antisense vector construction The full-length ORF and 114 bp of the C-terminal region of GhAnn1 were amplified from cotton cDNA using three pairs of primers that had XbaI and BamHI sites added to the ends of the target gene (Table S1). The PCR products were cloned into PBI121 to generate recombinant plasmids in which the GhAnn1 sequences were under the control of the strong constitutive CaMV35S promoter for cotton transformation. The constructed vectors included a sense expression vector with GhAnn1 fused with GUS (SAnn1), an antisense expression vector containing the full-length GhAnn1 (AsAnn1), and an antisense expression vector containing the 3′-cDNA fragment of GhAnn1 (3AsAnn1). The vector

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Plant Mol Biol

constructs were sequenced to verify the sequences of the GhAnn1 inserts. Plant transformation and generation of GhAnn1 transgenic cotton lines Cotton transformation was performed as previously described (Li et al. 2009). Genomic DNA was isolated from young leaves of non-transgenic and transgenic cotton plantlets following the protocol of Paterson et al. (1993). Homozygosity of transgenic plants was determined by segregation analyses based on the presence or absence of the kanamycin resistance selection marker and PCR analysis for the GhAnn1 transgene using a set of primers for determining the presence of the promoters and the antisense and sense GhAnn1 constructs (Table S1). The obtained homozygous transgenic lines were analyzed by Southern blotting to determine the copy numbers of GhAnn1, and real-time RT-PCR to determine whether GhAnn1 was overexpressed or suppressed in the transgenic lines. For Southern blot analysis, genomic DNA of transgenic plants was digested with EcoRI, fractionated on 0.8 % agarose gels, and transferred to HybondN+ nylon membranes by capillary blotting. A 750-bp fragment of the NPTII coding region, amplified using the NPTII-F/R primers (Table S1), was DIG-labeled as a hybridization probe according to the instructions described in the Dig High Prime DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland). Drought and salt stress tolerance assays in transgenic cotton Seed germination tests and measurement of root elongation For seed germination analysis, sterilized seeds were stratified for 24 h at 26 ± 1 °C and then plated on MS agar medium containing 2 % sucrose supplemented with NaCl (200 mM) or PEG (15 %). Germination was scored daily for 7 days. The germination rate was expressed as a percentage of the total number of seeds plated. A 5-mm radicle emergence from seeds was considered to be germination. The number of germinated seeds was counted and the length of the root was measured after 7 days of exposure to MS-sucrose (2 %) agar medium supplemented with NaCl (200 mM) and PEG (15 %), respectively. Three replicates were used for each treatment to ensure reproducibility of the data. Each replicate contains 30 seeds. Stress tolerance tests in seedlings For drought and salt tolerance tests, 2-week-old plants grown in soil were irrigated separately with 15 % PEG

Plant Mol Biol

or 200 mM NaCl solutions for 20 days. For measurement of physiological indexes, leaves after the treatment were collected at similar developmental stages, wrapped in aluminum foil, frozen in liquid N2 and stored at −70 °C until further use. Prior to collecting the samples, plant height was measured using vernier calipers. Under drought conditions,for water-loss assay, leaves of WT and transgenic cotton plants were detached and the changes in fresh weight were recorded over time by electronic balance. In addition, stomatal change was observed by Microscopy (Zeiss CFM-500, Germany) and the stomatal apertures were recorded. For ABA sensitivity analysis, the stamatal apertures in epidermal peel from the leaves of transgenic and WT plants treated with 1 μM ABA were observed using Microscopy (Zeiss CFM-500, Germany) under fluar 40×/0.50 M27 objective lens. At least 50 stomatal apertures were measured for each line.

All the assays described above were repeated at least three times on three biological replicates. The data were subjected to analysis of variance (ANOVA) to detect statistical differences, and the least significant difference (LSD) of means was determined by using the t test at the level of significance (defined as α = 0.01) using IBM SPSS Statistics (IBM Corporation, New York, USA).

5′-RACE extended the cDNA length to 1,800 bp, which contained a 951-bp-long ORF. The cDNA sequence with the full-length ORF was verified using gene-specific primers (Table S1). Sequence analysis predicted that the cloned gene encoded an annexin protein of 316 amino acids, which we designated GhAnn1 (Genbank No: KM062523). GhAnn1 has a molecular mass of 36.06 kDa, a theoretical pI of 6.19, and no signal peptides. Furthermore, a search of the PROSITE database (http://cn.Expasy.org/prosite/) for motifs revealed that this protein contains 20 potential phosphorylation sites, including seven serines, seven threonines, and six tyrosines, which could provide sites for post-translational modifications. At present, nine annexins have been isolated from G. hirsutum and six from G. barbadense. Multiple alignments of GhAnn1 and the 15 annexin protein sequences reported previously showed that the structures of cotton annexins are highly conserved (Fig. 1). All of them contain a typical annexin domain, which consists of four repeats (I–IV) of 70 amino acids, with the core repeat at the C-terminus. In the first repeat, there is a type II Ca2+ (G-X-GTD-{38}-E/D) binding domain, and another typeII-like Ca2+ binding site is present in the fourth repeat. Except for the Ca2+ binding motif, there is also a W residue (position 27) found in the first repeat which is present in most plant annexins and may be important for phospholipid-binding (Delmer and Potikha 1997; Mortimer et al. 2008). In addition to the Ca2+-binding motif, this protein has several other conserved sites important for annexin function such as a heme-binding site (position 1–40) with a highly conserved H residue (position 39) that is related to the peroxidase activity of annexins (Mortimer et al. 2008), an F-actin-binding site (position 189–190, IRV) in which the I has been substituted with a V residue in the fourth repeat, and a GTP-binding site (position 297–300; DXXG) in the fourth repeat. The cotton proteins also contain S3 clusters that are putatively involved in redox reactions formed by the C residue (Hofmann et al. 2003). These results suggest that GhAnn1 encodes a typical annexin protein, possessing the classical characteristics of annexin family proteins.

Results

Phylogenetic relationships of GhAnn1 with other plant annexins

Measurement of physiological indexes Total chlorophyll was measured spectrophotometrically following the method of Arnon (1949). Free proline content of drought- and salt-treated WT and transgenic cotton plants was measured spectrophotometrically according to the method of Bates et al. (1973). The total soluble sugar content of plants was assayed using the phenol–sulfuric acid assay (Chyzhykova and Palladina 2005). Lipid peroxidation levels were assessed by measuring thiobarbituric acid reactive substances (TBARS) (Heath and Packer 1968). Superoxide dismutase (SOD) was determined according to Paoletti et al. (1986). Statistical analysis

Cloning and characterization analysis of GhAnn1 cDNAs We sequenced a cDNA library from G. hirsutum line 7235, and isolated a 1,200-bp cDNA fragment with a poly (A) tail and an incomplete open reading frame (ORF). Genespecific primers for 5′-RACE (rapid amplification of cDNA ends) analysis were designed from the known sequence.

To investigate the evolutionary relationships between GhAnn1 and other known plant annexins, we first identified 14 G. raimondii annexins based on sequence information downloaded from http://www.phytozome.net, using HMMER software version 3.0 (Finn et al. 2011) and the pfam protein families database with the IAA domain (PF00191) as a query (Table S2 and Fig. S1). The 15

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annexins reported previously from tetraploid cotton, fourteen from G. raimondii, eight from Arabidopsis, ten from rice and GhAnn1 were selected for phylogenetic analysis.

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As shown in Fig. 2, 48 annexin proteins could be clustered distinctly into six groups (Groups A to F). GhAnn1 clustered with group A, the largest group that contained 20

Plant Mol Biol

◂ Fig. 1  Multiple sequence alignment GhAnn1 and other predicted cot-

ton annexin protein sequences. The putative annexin repeats (I–IV) are shown enclosed in black boxes. The red boxes in repeats I and IV delineate type II G-X-GTD-{ca. 38}-E/D calcium binding sites; red triangles indicate putative S3 clusters thought to be involved in redox reactions; the yellow box indicates the IRV: motif for binding actin; the red star marks the conserved His residue; the green box in repeat IV shows the putative GTP-binding motif. Amino acid sequence alignment was performed using ClustalW. GenBank accession numbers (in parentheses) are as follows: AnnGhl (AAB67993), AnnGh2 (AAB67994), AnnGh3 (JX897059), AnnGh4 (JX897060), AnnGh5 (JX897061), AnnGh6 (JX897062), GhAnX1 (AAR13288), AnxGh1(AAC33305), GhFAnnx (ACJ11719), and AnxGb1 to AnxGb6 (KC316004 to KC316009)

annexins apart from GhAnn1; seven from G. hirsutum, four from G. barbadense, three from G. raimondii, four from Arabidopsis, and three from rice. Other than the annexin

Fig. 2  Unrooted phylogenetic tree showing the evolutionary relationships between GhAnn1 and orthologous annexin proteins from different plant species. The evolutionary history was inferred using the neighbor-joining method as implemented in MEGA version 5.1, and the bootstrap consensus tree inferred from 1,000 replicates. G. hirsutum proteins—AnnGhl (AAB67993), AnnGh2 (AAB67994), AnnGh3 (JX897059), AnnGh4 (JX897060), AnnGh5 (JX897061), AnnGh6 (JX897062), GhAnX1 (AAR13288), AnxGh1(AAC33305), and GhFAnnx (ACJ11719); G. barbadense proteins—AnxGb1 to AnxGb6 (KC316004 to KC316009); Arabidopsis thaliana proteins— AnnAt1 (NP_174810), AnnAt2 (NP_201307), AnnAt3 (NP_181410),

proteins from Gossypium, GhAnn1 showed the highest homology with AnnAt1. Plants overexpressing AnnAt1 were previously shown to be more drought-tolerant than wild-type (WT) plants (Konopka-Postupolska et al. 2009), and AnnAt1 and AnnAt4 interact with each other to regulate drought and salt stress responses in Arabidopsis (Huh et al. 2010). This provided evidence that GhAnn1 may play an important role in abiotic stress tolerance. Each of the other five groups also includes annexin proteins from both the monocot plant rice and the dicots Arabidopsis and/or cotton, indicating that even if there is biological and functional divergence between monocots and dicots, the divergence of annexin proteins from the different species probably occurred before the monocot-dicot split.

AnnAt4 (NP_181409), AnnAt5 (NP_564920), AnnAt6 (NP_196584), AnnAt7 (NP_196585), and AnnAt8 (NP_568271); G.raimondii proteins—Gorai.007G239000, Gorai.009G237900, Gorai.007G060900, Gorai.001G068900, Gorai.006G190800, Gorai.011G212900, Gorai. 011G212700, Gorai.009G329000, Gorai.009G077400, Gorai.009 G295800, Gorai.013G194300, Gorai.005G219600, Gorai.009G296 000, and Gorai.009G29590; Oryza sativa proteins—OS01G31270, OS05G31750, OS05G31760, OS06G11800, OS07G46550, OS08 G32970, OS09G20330, OS09G23160, and OS09G27990. GhAnn1 from G. hirsutum is indicated by a red circle

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Plant Mol Biol

Fig. 3  Subcellular localization of GhAnn1. a Subcellular localization of the fused PIJT166-35S::GhAnn1:GFP in onion epidermal cells. The PJIT166-35S::GFP construct was used as the control. b Transient expression of GhAnn1 fused to the pBIB-35S::GFP vector in tobacco leaf cells. The pBIB-35S::GFP construct was used as the control. GFP GFP fluorescence; FM4-64 FM4-64 used for plasma

membrane labeling for PJIT166-35S:GhAnn1::GFP and pBIB35S:GhAnn1::GFP transformed cells in onion epidermal cells or tobacco leaf cells; Merged overlay of bright field and green/red fluorescence images; Bright field bright field images of onion epidermal cells or tobacco leaf cells; Bars 50 μm

GhAnn1 is localized in the plasma membrane

mosaic virus (CaMV) 35S promoter, and we introduced PJIT166-35s::GhAnn1-GFP and PJIT166-GFP constructs into the onion epidermal cells using biolistic bombardment. Following a subculture of 16 h, laser confocal imaging microscopy revealed that fluorescence microscopy visualizations of GhAnn1::GFP and plasma membrane

To examine the subcellular localization of GhAnn1, we cloned the GhAnn1 into the PJIT166-GFP vector to generate the PJIT166-35s::GhAnn1-GFP construct. The gene fusion construct was under control of the cauliflower

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Plant Mol Biol

GhAnn1 is significantly upregulated when induced by different stresses

Fig. 4  Semi-quantitative RT-PCR assay for tissue-specific expression analysis of GhAnn1. Lane 1 Root (R). Lane 2 Stem (S). Lane 3 Leaf (L). Lanes 4–8 mixtures of ovules and fibers from −1, 1, 3, 5, and 8 dpa, respectively. Lanes 9–11 fiber cells from 11, 14, and 17 dpa, respectively. Dpa days post-anthesis. GhEF1a was used as an internal control. The experiments were repeated in triplicate with similar results

stained with FM4-64 showed that the GFP signals and the red fluorescence signals overlapped in the plasma membrane (Fig. 3a), whereas GFP alone distributed throughout the cell. In order to further define the subcellular localization of GhAnn1 in plant cells, we introduced pBIB-35s::GhAnn1GFP and pBIB-GFP constructs into the tobacco leaves mediated by Agrobacterium strain EHA105. Protein subcellular localization was analyzed 3 days after infiltration using a confocal laser fluorescence microscope (Zeiss LSM510 META, Germany). Confocal microscopy showed that GFP signals emitted by GhAnn1::GFP fusion protein and red fluorescence signals stained by FM4-64 colocalized in the plasma membrane of tobacco leaf cells (Fig. 3b). Taken together, in vivo and in vitro results suggest that GhAnn1 is a plasma membrane-localized protein. GhAnn1 is preferentially expressed both in vegetative and reproductive tissues To investigate the tissue-specific expression pattern of GhAnn1, total RNA from various tissues including roots, stems, leaves, ovules, and fibers at different fiber developmental stages, were extracted and used for RT-PCR. As shown in Fig. 4, GhAnn1-specific mRNA was detected in all tissues examined, and the transcripts were abundant in various tissues, but relatively less abundant in roots. These results indicated that GhAnn1 is preferentially expressed both in vegetative and reproductive tissues.

To analyze the role of GhAnn1 in abiotic stress, we examined transcriptional regulation of GhAnn1 after treatment of cotton leaves with several stress-related signaling compounds (ABA, SA, JA), an oxidative stress inducer (H2O2), and osmotic stress generators (NaCl and PEG) for various lengths of time. qRT-PCR showed that expression of GhAnn1 was significantly upregulated after these stress treatments. Following ABA treatment, the expression of GhAnn1 was significantly upregulated from 0.5 to 24 h, with a peak at 4 h post-treatment (Fig. 5a). The other three phytohormone treatments showed similar expression profiles, with a peak at 6 h after SA treatment (Fig. 5b), at 8 h after H2O2 treatment (Fig. 5c), and at 6 h after JA treatment (Fig. 5d). Treatments with both 200 mM NaCl and PEG 6000 (20 %, w/v) caused a significant accumulation of GhAnn1 mRNA after 1 h, which persisted for 24 h, with a peak at 10 h under salt stress (Fig. 5e), and 12 h under drought stress (Fig. 5f). These results indicated that GhAnn1 may be involved in the response to different phytohormones and stresses, and may also play an important role in the crosstalk between multiple stress responses. Characterization of transgenic cotton lines showing overexpression or suppression of GhAnn1 In order to evaluate the functional significance of GhAnn1 in transgenic cotton plants, GhAnn1 sense (SAnn1), antisense (AsAnn1), and 3′ antisense (3AsAnn1) vectors were individually introduced into the cotton line W0 via Agrobacterium-mediated transformation. A total of 20 independent T0 transgenic plants were obtained by screening regenerated kanamycin-resistant cotton plants; this included eight SAnn1 sense clones and six each of the AsAnn1 and 3AsAnn1 antisense clones. The T1 population (progeny of the T0 plants) was then used for molecular analysis. Genomic PCR analysis using specific primers showed that 14 of the T1 plants had the expected 951 bp band, corresponding to the GhAnn1 cDNA, and six plants had the expected 114 bp band, correponding to the 3′-downstream fragment of GhAnn1 (Table S1); no amplification was observed from DNA of WT (Wild Type) cotton plants (Fig. S2A). Southern blot analysis further confirmed that the transgenic lines had 1–6 copies of GhAnn1 in the cotton genome, indicating independent integration events in different transgenic plants. No hybridization signal was detected in WT plants (Fig. S2C). We also examined the expression level of GhAnn1 in various T2 transgenic cotton lines. The results showed that the relative expression levels of GhAnn1 varied in the different transgenic plants. In eight of the overexpressing

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Plant Mol Biol

Fig. 5  Expression timecourse of GhAnn1 in response to different stress treatments by qRT-PCR. qRT-PCR analysis was performed with total RNA extracted from leaves at the indicated times after treatment with 100 μM ABA (a), 10 mM SA (b), 10 mM H2O2 (c), 100  μM JA (d), 15 % PEG (e), or 200 mM NaCl (f). Cotton seed-

lings treated with distilled water under the same conditions served as controls (Mock). The error bars represent the standard deviations of three biological replicates. Asterisk indicates a significant difference at P 

Overexpression of a cotton annexin gene, GhAnn1, enhances drought and salt stress tolerance in transgenic cotton.

Plant annexins are members of a diverse, multigene protein family that has been associated with a variety of cellular processes and responses to abiot...
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