Characterization of an ethylene-inducible, calcium-dependent nuclease that is differentially expressed in cucumber flower development

Authors

  • Hai-Tao Gu,

    1. PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, Beijing, 100871 China
    2. The National Center of Plant Gene Research (Beijing), Beijing, 100871 China
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  • Dong-Hui Wang,

    1. PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, Beijing, 100871 China
    2. The National Center of Plant Gene Research (Beijing), Beijing, 100871 China
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  • Xia Li,

    1. PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, Beijing, 100871 China
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  • Chao-Xing He,

    1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081 China
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  • Zhi-Hong Xu,

    1. PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, Beijing, 100871 China
    2. The National Center of Plant Gene Research (Beijing), Beijing, 100871 China
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  • Shu-Nong Bai

    1. PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology, National Key Laboratory of Protein Engineering and Plant Gene Engineering, College of Life Sciences, Peking University, Beijing, 100871 China
    2. The National Center of Plant Gene Research (Beijing), Beijing, 100871 China
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Author for correspondence:
Shu-Nong Bai
Tel: +86 10 6276 1444
Email: shunongb@pku.edu.cn

Summary

  • Production of unisexual flowers is an important mechanism that promotes cross-pollination in angiosperms. We previously identified primordial anther-specific DNA damage and organ-specific ethylene perception responsible for the arrest of stamen development in female flowers, but little is known about how the two processes are linked.
  • To identify potential links between the two processes, we performed suppression subtractive hybridization (SSH) on cucumber (Cucumis sativus L.) stamens of male and female flowers at stage 6, with stamens at stage 5 of bisexual flowers as a control.
  • Among the differentially expressed genes, we identified an expressed sequence tag (EST) encoding a cucumber homolog to an Arabidopsis calcium-dependent nuclease (CAN), designated CsCaN. Full-length CsCaN cDNA and the respective genomic DNA sequence were cloned and characterized. The CsCaN protein exhibited calcium-dependent nuclease activity. CsCaN showed ubiquitous expression; however, increased gene expression was detected in the stamens of stage 6 female flowers compared with male flowers. As expected, CsCaN expression was ethylene inducible. It was of great interest that CsCaN was post-translationally modified.
  • This study demonstrated that CsCaN is a novel cucumber nuclease gene, whose DNase activity is regulated at multiple levels, and which could be involved in the primordial anther-specific DNA damage of developing female cucumber flowers.

Abbreviations:  ACC, 1-aminocyclopropane-1-carboxylate; AP3, APETALA3; CAD, caspase-activated DNase; CAGL2, cucumber AGAMOUS like gene 2; CaN, Calcium dependent Nuclease; CYCD1, cyclin D1; EREBP, ethylene responding element binding protein; ETR1, ethylene receptor 1; FM, floral meristem; GAAD, granzyme A-activated DNase; IPTG, Isopropyl β-D-1-thiogalactopyranoside; MADS1, MADS BOX gene 1; MBP, maltose binding protein; RACE, Rapid amplification of cDNA end; rad50, a protein involved in DNA double-strand break repair; TAIL-PCR, thermal asymmetric interlaced PCR.

Introduction

Unisexual flower development is considered a successful mechanism to promote cross-pollination (Barrett, 2002). Although mature unisexual flowers vary greatly in phenotype, in most cases they are indistinguishable from each other at early developmental stages. Stamen and carpel primordia are initiated normally from the floral meristem, but, during development, one of the two structures is arrested (Dellaporta & Calderon-Urrea, 1993; Ainsworth, 1999). A major focus of studies on the regulatory mechanisms of plant sex determination is to address how development of the inappropriate organ is impeded in unisexual flowers (Tanurdzic & Banks, 2004).

Cucumber (Cucumis sativus) is a model plant used to elucidate the mechanisms underlying unisexual flower development. In the 1960s, Galun (1961) and Kubicki (1969a,b,c,d)identified three major genes (F, M and A) that determine the ratio of unisexual flowers. Later, ethylene was proposed as a major hormone facilitating female flower development (reviewed by Perl-Treves, 1999), and the prevention of either male or female organ development is dependent on where the organ is positioned in the flower (Kater et al., 2001). Recently, the F and M genes were cloned; each encodes different members of the ACC synthase (ACS) gene family (Trebitsh et al., 1997; Mibus & Tatlioglu, 2004; Knopf & Trebitsh, 2006; Boualem et al., 2009; Li et al., 2009). Cloning of the F and M genes, particularly the detection of carpel preferential expression of the M gene, CsACS2 (Yamasaki et al., 2001, 2003a,b; Saito et al., 2007), successfully linked the previous genetic and physiological observations. However, the mechanism by which the extra ethylene selectively promotes female flower development remains to be elucidated.

In previous studies, we determined that unisexual cucumber flowers resulted from developmental arrest of the anthers (for female flowers) or ovaries (for male flowers) at floral bud stage 6 (Bai et al., 2004). We also found a correlation between the arrest of stamen development in female flowers and primordial anther-specific DNA damage (Hao et al., 2003). Furthermore, we observed organ-specific down-regulation of a cucumber ethylene receptor gene, CsETR1, which may play a key role in stamen arrest (Wang et al., 2010), and organ-specific up-regulation of ethylene production induced ‘female flowers’ in Arabidopsis (Duan et al., 2008). In both cases, altered ethylene signaling components caused DNA damage, as indicated by chromosome condensation (Duan et al., 2008; Wang et al., 2010). These data clearly suggest the presence of a link between the stamen-specific altered ethylene signaling and primordial anther-specific DNA damage. However, the link remains unidentified. As DNase activity has been detected in the sector containing the inappropriate stamen in female flowers (Hao et al., 2003), the most likely link would be a DNase, whose activity is stamen preferential and ethylene inducible.

Many well-characterized DNases are responsible for DNA damage involved in animal programmed cell death (PCD), including CAD in the caspase pathway (Liu et al., 1997; Enari et al., 1998); mitochondrial endonuclease G (Li et al., 2001; Burhans & Weinberger, 2007), GAAD (Fan et al., 2003; Lieberman & Fan, 2003) and cation-dependent and -independent DNases (Counis & Torriglia, 2006). In plants, enzyme assays have shown that various DNase activities are involved in PCD (Stein & Hansen, 1999; Sugiyama et al., 2000; Marchetti et al., 2001; Balk et al., 2003; Ballut et al., 2003; Dominguez et al., 2004; Dominguez & Cejudo, 2006; Fedoreyeva et al., 2007; Jiang et al., 2008; Lesniewicz et al., 2010). DNases are primarily divided into two categories according to activity, namely calcium-dependent and zinc-dependent nucleases. Sugiyama et al. (2000) suggested that calcium-dependent nucleases are activated during the early stages of cell death, and zinc-dependent nucleases during the later stages. To date, only a few of these DNases have been identified at the molecular level (Aoyagi et al., 1998; Ito & Fukuda, 2002; Sundstrom et al., 2009; Taga et al., 2009; Lesniewicz et al., 2010). An Arabidopsis calcium-dependent nuclease was identified as a result of its homology with Staphylococcus aureus nuclease (EC 3.1.31.1) (Isono et al., 2000). In animal PCD, DNase activity is primarily regulated at the protein activity level (Li & Yuan, 2008). However, activities of the Arabidopsis bifunctional nuclease (BFN1) and barley nuclease (Bnuc2) were regulated at the transcriptional level (Perez-Amador et al., 2000; Zaina et al., 2003; Farage-Barhom et al., 2008). Further investigation is required to identify the corresponding DNase, which are involved in plant PCD, and its activity regulation, particularly primordial anther-specific DNA damage, because little is known regarding each of these processes.

In the present study, we conducted a suppression subtractive hybridization (SSH) screen to identify the putative link between ethylene signaling and primordial anther-specific DNA damage from genes differentially expressed in the stamens of stage 5 bisexual floral buds, stage 6 female flowers and stage 6 male flowers (Fig. 1a). Among the 424 ESTs that were differentially expressed and could be annotated in the cucumber genome, we identified one that was preferentially expressed in stamens of stage 6 female flowers, which was annotated as a calcium-dependent nuclease. We cloned its full-length cDNA, designated it CsCaN, and characterized its enzyme activity, expression pattern, and ethylene inducibility. Based on the results, we propose that CsCaN may be involved in the induction of primordial anther-specific DNA damage. The information about regulation of CsCaN activity expand our understanding of the developmental mechanisms of female cucumber flowers.

Figure 1.

Diagram of the suppression subtractive hybridization (SSH) design and photographs of the organ primordia of cucumber (Cucumis sativus) floral buds. (a) The SSH design. S5 represents stamen samples from a stage 5 floral bud; M6 represents stamen samples from stage 6 male flowers; F6 represents stamen samples from stage 6 female flowers. (b–d) Organ primordia of stage 5 floral buds (b), and stage 6 male (c) and female (d) flowers. (e) Needle dissection of floral buds. (f) Stamen primordia following dissection. Bar, 100 μm. St, stamen; C, carpel.

Materials and Methods

Plant material and growth conditions

Seeds of the monoecious cucumber (Cucumis sativus L.) line Zhongnong No.5 were purchased from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The seeds were sown in soil and plants were grown in a growth room under a day : night light regime of 16 h : 8 h and a temperature of 25 : 18°C for sample collection.

Floral buds were cut longitudinally under a dissecting microscope to collect stamen primordia at the appropriate stages according to Bai et al. (2004) from male and female flowers.

RNA isolation and cDNA amplification

Total RNA was extracted from c. 10 stamens per stage with Trizol (Invitrogen, USA) and amplified with a SMART™ PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). The optimal number of PCR cycles for amplification was determined to be 24 on the basis of pilot experiments (Supporting Information Fig. S1).

Suppression subtractive hybridization (SSH) screen and library construction

The amount of RNA obtained from stamen primodia was not adequate for a direct SSH experiment, and so we chose to use SMART-PCR (Clontech) cDNA amplification. SSH was subsequently performed according to the Clontech PCR-Select cDNA Subtraction Kit (PT1117-1) user manual. The quality and efficiency of the key steps in the SSH, including amplified cDNA digestion, adaptor ligation, and subtractive hybridization, among others, were examined and optimized (Fig. S1). Following SSH, the PCR products were cloned into the pGEM-T vector (Promega, USA) and transformed into Escherichia coli (DH5α). Six libraries were constructed, each representing one subtraction with c. 5000 clones. The ratio of positive clones exceeded 70% using X-gal assay and PCR verification. Clones with inserts of > 200 bp were randomly selected from five of the six libraries (Table S1) and sequenced at the Beijing Genomics Institute (BGI), Beijing, China (Table S2).

Cloning the DNA sequence related to CsCaN

The full-length cDNA was cloned by RACE with the SMART™ RACE cDNA Amplification Kit (Clontech). The primers used in RACE are listed in Table S3. According to the full-length cDNA sequence, genomic DNA was cloned and sequenced using the primers listed in Table S3. TAIL-PCR was then conducted and the 1.5-kb upstream sequence was cloned with the primers sequences listed in Table S3.

Southern blot analysis

Genomic DNA was extracted from cucumber leaves using the DNeasy Plant Maxi Kit (Qiagen, Germany), digested with EcoRV and HindIII, and separated on a 1% agarose gel. The probe fragment was amplified with the primer sequences listed in Table S3 from genomic DNA using the PCR DIG Probe Synthesis Kit (Roche, USA). The Southern blot was conducted according to Liu et al., (1995).

Phylogenetic analysis

The calcium-dependent nuclease (CAN) protein sequence was the reference used to search for CAN homologs in published genome sequences of all organisms in the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST/), and the plant transcript database of Arabidopsis, rice (Oryza sativa) and poplar (Populus trichocarpa) (http://sgdb.cbi.pku.edu.cn) using blastp programs with a cut-off of 1e-50. ClustalX (Plate-Forme de Bio-Informatique, Illkirch Cedex, France) was used to perform multiple protein sequence alignments. The most conservative regions were aligned and used to perform neighbor joining analyses; the whole deletion option, Poisson correction model, and 1000 bootstrap replicates were selected in mega version 4.0 (Kumar et al., 2008; http://www.megasoftware.net). The single sequence of PpCaN (Physcomitrella patens) was treated as an outgroup sequence.

Recombinant protein construction

The CsCaN gene was amplified from the cDNA by PCR, and inserted into pET01-T (http://www.t-vector.com, China) by TA cloning and the pMAL c2x (NEB N8076, Ipswich, MA, USA) vector at the XmnI-SalI restriction sites, respectively. The clones containing the correct inserts were verified by sequencing and transformed into E. coli BL21. The BL21 cells were cultured to an optical density (OD600) of 0.7 and treated with 0.5 mM IPTG for 4 h. The bacterial cells were collected and sonicated to release the expressed protein. The MBP fused recombinant protein generated from the pMAL c2x vector was purified using a MBP binding column as described in the manual (NEB, E8000S) (Fig. S2). The Factor Xa protease (NEB P8010S) was used to cut the soluble MBP-CsCaN fusion protein and to release the full-length CsCaN. The soluble CsCaN protein was subjected to DNase activity in-tube assays. The CsCaN recombinant protein without a tag was insoluble, but suitable for the DNase activity in-gel assay.

DNase activity assays

The DNase in-gel assay was conducted according to Hao et al. (2003). The DNase activity in-tube assay was performed with an enzyme buffer containing 10 mM Tris, pH 8.0, 10 mM CaCl2, 50 ng μl−1 salmon sperm DNA and 1 ng per 20 μl of CsCaN protein. The mixture was incubated at 37°C for 30 min, and then run on agarose gels to observe the effect of the enzyme on the DNA.

Protein modification assay

The recombinant modification mixture contained 1 μl of MBP-CsCaN, 8 μl of 0.5 M NaAc, pH 5, 1 μl of 0.2 M EDTA, 1 μl of proteinase inhibitor cocktail (Calbiochem, Merck KGaA, Darmstadt, Germany), 6 μl of senescent leaf protein extract, and 27 μl of H2O. The mixture was incubated at 25°C for 1 h, and subsequently subjected to the DNase activity in-gel assay. The senescent leaf protein was extracted from yellowish leaves with 10 mM Tris buffer (pH 8.0).

Western blot

The antibody was raised against a synthetic short peptide from the CsCaN C-terminus (PEQPWEWRKGKREGK) by the Tianjin Saierbio Company (China). After a schedule of four injections, serum was drawn from the rabbit. The thiosepharose cross-linked antigen was used to purify the serum before use. Total protein extraction from leaves and flowers was accomplished with extraction buffer (10 mM Tris, pH 8.0). The primary antibody was used at 450 ng ml−1, and the secondary antibody was diluted 10 000 times. Signal detection was conducted with the Supersignal West Femto Kit (Thermo Scientific, Waltham, MA, USA).

Detection of CsCaN expression

Seven samples were examined for CsCaN expression with real-time RT-PCR: fully expanded cotyledons, fully expanded first true leaves, fully expanded 8th leaves, root tips of 5-d-old seedlings, stamens of stage 6 male and female flowers, and carpels of stage 6 female flowers. RNA was isolated from each sample using the RNeasy Plant Mini Kit (Qiagen). Reverse transcription was conducted with SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Real-time RT-PCR was performed in a total volume of 30 μl with 1.5 μl of the RT reactions, 0.25 μM of each gene-specific primer (Table S3), and 15 μl of SYBR Green Master mix (Applied Biosystems, Carlsbad, CA, USA) on a real-time PCR detector (Chromo 4; Bio-Rad, USA) according to the manufacturer’s instructions. The cucumber Actin gene was used as the internal control. All primers were annealed at 58°C and PCR was run for 45 cycles. The relative expression level of each gene was normalized using the Actin cDNA level according to the user manual, and averaged over three replicates. In situ hybridization was performed according to Bai et al. (2004).

Results

Identification of preferentially expressed genes in stamen primordia of male and female flowers

We previously found that stage 6 is a critical stage for cucumber flower development, when male and female floral buds could be distinguished in morphology (Bai et al., 2004). We also found stamen-specific down-regulation of CsETR1 expression at this stage, and primordial anther-specific DNA damage after this stage (at stage 7) in female flowers (Hao et al., 2003; Wang et al., 2010). These findings suggested that it is possible to identify genes responsible for the DNA damage and/or for the developmental arrest of the stamens of female flowers by analysis of differentially expressed genes in stage 6 stamens of female flowers.

Before the invention of RNA-seq and equivalent genomic tools, SSH was widely used as an effective method to identify differentially expressed genes in various biological processes. Here, we used stamens of stage 5 bisexual flowers (labeled S5 in Fig. 1a) as one control and stamens of stage 6 male flowers as another (M6) for the screening of differentially expressed genes in the stamens of stage 6 female flowers (F6) (Fig. 1b–f). Each of these three stamen samples was used as both the tester and the driver for comparisons, and we therefore obtained six libraries containing genes that were putatively differentially expressed between sample pairs (Figs 1a, S1; Table S1).

Following library construction, clones with inserts > 200 bp from different SSH libraries were randomly collected and sent for sequencing. Of a total of 933 positive clones sequenced (Table S2), > 424 matched annotated genes in the NCBI database (Table S1). Genes related to flower development, hormones, cell cycle and cell death were identified, including an Arabidopsis AP3 homolog (designated CsMADS1; GenBank accession number AY944060), CAGL2 (AF135962), EREBP (GO897450), CYCD1 (GO897469), and a DNA double-strand break repair rad50 ATPase (GO897425). Notably, an EST homologous to Arabidopsis CAN was obtained (Isono et al., 2000). This EST was identified in the library that contained genes exhibiting increased expression in stage 6 stamens of female flowers compared with stage 5 stamens of bisexual flowers (Table S1). We previously detected DNA damage in stage 7 but not stage 6 stamens of female flowers. Higher expression of the CAN homolog from stage 5 to 6 suggested that this gene might be involved in DNA damage induction later in stamen development. We therefore chose this gene for further investigation.

Full-length cDNA cloning of CsCaN

The full-length cDNA of the EST homologous to CAN was cloned with RACE, and designated CsCaN (GenBank accession number EU144224). The CsCaN coding sequence consisted of 1002 bp and encoded a protein of 334 amino acids. A NCBI protein BLAST search revealed that the CsCaN protein was comprised of a conserved nuclease domain (the staphylococcal nuclease (SNc) domain) at its C-terminus (from the 220th to the 320th amino acid); therefore, CsCaN belongs to the SNc domain-containing gene family (Fig. 2a). The CsCaN genomic sequence, which was subsequently cloned, consists of eight introns and nine exons (Fig. 2b). Southern blot analysis demonstrated that CsCaN is a single-copy gene in the cucumber genome (Fig. 2c), which was also confirmed by BLAST analysis (http://www.icugi.org/). To study the regulatory mechanism responsible for CsCaN transcription, the 1.5-kb sequence upstream of the CsCaN gene (GQ149069) was cloned using TAIL-PCR (Fig. 2b; Table S4).

Figure 2.

Scheme for Cucumis sativus calcium-dependent nuclease (CsCaN) predicted protein and genomic DNA structure, and genomic Southern blot analysis. (a) Diagram of predicted protein structure, showing the staphylococcal nuclease (SNc) domain. (b) Diagram of the genomic DNA structure, showing nine exons, eight introns and the 1.5-kb upstream promoter region. (c) CsCaN genomic DNA fragment detected by Southern blot analysis. A single band was detected when genomic DNA was digested with EcoRV and HindIII (lane 1), or with HindIII alone (lane 2). (d) Phylogenetic analysis of available CAN sequences from the following sequenced plant genomes: CaN and AtPCaN, proteins from Arabidopsis (CAN, NP_567036; AtPCaN, NP_973649); Pt, Populus trichocarpa (PtCaN1, XP_002311254; PtCaN2, AAX21543; PtCaN3, XP_002316184). Vv, Vitis vinifera (VvCan, CAN70624); Rc, Ricinus communis (RcCaN, XP_002520814); Nt, Nicotiana tabacum (NtCaN, AAK52082); Sb, Sorghum bicolor (SbCaN1, XP_002457283; SbCaN2, XP_002441695); Zm, Zea mays (ZmCaN1, ACF87995; ZmCaN2, NP_001147648); Os, Oryza sativa (OsCaN1, NP_001042112; OsCaN2, ABA91143; OsCaN3, ABA96961; OsCaN4, EEE52625); Ps, Picea sitchensis (PsCaN, ABK23214); Eu, Eucommia ulmoidies (EuCaN1, ACD40019; EuCaN2, ABA41005); Sm, Selaginellae moellendorfii (SmCaN, e_gw1.0.670.1; genome data have not been released, so this is the sequence number); Pp, Physcomitrella patens (PpCaN, XP_001757699); Cse, Corydalis sempervirens (CseCaN, Q39635); and Csa, Cucumis sativus (CsaCaN, e.g. CsCaN, ABX56707). Y. Pang et al. (pers comm.) previously named EuCaN ‘EuDNase’.

To date, 91 protein sequences representing 24 plant species contain the SNc domain and are available in NCBI. Arabidopsis CAN is the only SNc domain characterized (Isono et al., 2000). Phylogenetic analysis of similar CAN sequences from plants with an entire sequenced genome revealed that the CsCaN gene was clustered close to other eudicot SNc sequences, further from monocot SNc sequences (Fig. 2d) and even further from the CaN homologs found in the gymnosperm Picea sitchensis, the fern Selaginellae moellendorfii and the moss Physcomitrella patens.

CaCaN is a calcium-dependent nuclease with two active forms

The protein encoded by CsCaN was assessed for DNase activity by expressing and purifying recombinant proteins in E. coli (Fig. S2). DNase activity was investigated using in-tube assays. Results demonstrated that CsCaN cuts single-stranded, double-stranded, and circular plasmid DNA (Fig. 3a). When EDTA (a calcium chelate agent) was added to the reaction buffer, CsCaN only digested cucumber genomic DNA at a higher calcium concentration, indicating that the nuclease activity of CsCaN was calcium dependent (Fig. 3b). The optimal pH for CsCaN activity was alkaline. Cucumber genomic DNA was digested at pH 8.0 and 9.0, but not pH 4.0–6.0 (Fig. 3c). This result was consistent with the EXPASY (http://www.expasy.ch/tools/pi_tool.html) predicted pI 9.55. In addition, the CsCaN protein is thermally stable with continued activity up to 60°C (Fig. 3d)

Figure 3.

Characterization of DNase activity of Cucumis sativus calcium-dependent nuclease (CsCaN). (a) Recombinant protein CsCaN can digest plasmid, single- and double-stranded DNA. Lanes 1, 3, 5 and 7 are, respectively, the DL15000 marker, the pET28a plasmid, double-stranded salmon DNA, and single-stranded salmon DNA. Lanes 2, 4, 6 and 8 are the same set of samples after a 30-min digestion with CsCaN at 37°C. (b) CsCaN activity is calcium-dependent: 2 mM EDTA was added to the reaction buffer to chelate the cation. Lane 1, the DL15000 marker. Lane 2, reaction buffer with cucumber genomic DNA without CsCaN. Lanes 3–6, reaction buffer with cucumber genomic DNA and CsCaN plus 0, 0.001, 0.01 and 0.1 M CaCl2, respectively. (c) The optimal pH for CsCaN DNase activity is alkaline. Lane 1, the DL15000 marker. Lanes 2–7, cucumber genomic DNA was incubated in reaction buffer at pH 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively. DNase activity was detected clearly at pH 8.0 and 9.0. The enzyme concentration in the experiments was 1 ng per 20 μl. (d) CsCaN activity is thermally stable. Lane 1, the DL15000 marker. Lane 2, reaction buffer with cucumber genomic DNA without CsCaN. Lanes 3–6, standard enzyme reaction with cucumber genomic DNA and CsCaN for 30 min at 50, 60, 70 and 80°C, respectively. The enzyme concentration in the experiments was 1 ng per 20 μl.

Previously, we found DNase activity at 35 kDa in the sectors containing the inappropriate stamens of stage 8 female flowers (Hao et al., 2003). This suggested that the DNase responsible for the primordial anther-specific DNA damage should have a molecular weight of 35 kDa. However, EXPASY predicted a molecular weight for the CsCaN protein of 37.4 kDa, not 35 kDa. This prediction was verified by the DNase activity in-gel assay with the recombinant protein (Fig. 4a, lane 8, upper band) and mass spectrometry (MS) analysis (Fig. S3; Table S5). Is CsCaN a candidate for the targeted DNase? When we analyzed DNase activity with a protein extract of E. coli expressing untagged recombinant CsCaN, an additional DNA digestion band at 35 kDa was identified (Fig. 4a, lane 8; Fig. 4b, lane 9). This suggested that CsCaN might be modified at the protein level, resulting in an additional active form at 35 kDa, and that the 35-kDa DNase activity detected in the female flower sector might be the modified form of CsCaN. Therefore, we re-examined DNase activity in male and female floral buds at developmental stages 6–11 and detected protein extracts with antibodies against CsCaN. We found that, consistent with our previous results, no detectable DNase activity in male flowers occurred before stage 9, but substantial DNase activity at 35 kDa in sectors containing the inappropriate stamens of female flowers was present from stage 6 (Fig. 4a). Western blotting revealed a band at 35 kDa in the protein extract from stamens, but not from the corresponding ovaries of female flowers at stages 6–9 (Fig. 4c). To explore whether CsCaN was modified in vivo, we incubated full-length recombinant CsCaN with a protein extract from senescent cucumber leaves and detected DNase activity. We found that, corresponding to increased leaf extract concentration, the DNase activity of the tagged recombinant decreased, while that of the 35-kDa band increased (Fig. 4b lanes 5–7). This result suggested that CsCaN could be modified by unknown components in senescent cucumber leaves, which resulted in a 35-kDa form. The assessment of these results cumulatively indicated the possibility that the modified 35-kDa CsCaN form might be the targeted DNase identified in the sector of female flowers containing the inappropriate stamens.

Figure 4.

Cucumis sativus calcium-dependent nuclease (CsCaN) detection in stamen and its post-translational modification. (a) DNase activity in-gel assay of samples from cucumber (Cucumis sativus) male flowers and female flower sectors containing inappropriate stamens. Lane 1, protein marker. Lanes 2–4, male flowers at stages 6–7, 8–9, and 10–11, respectively. Lanes 5–7, the sectors from female flowers at stages 6–7, 8–9, and 10–11, respectively. Lane 8, lysate of E. coli BL21 with the recombinant CsCaN-pET01 construct, showing two CsCaN sizes with DNase activity. Lane 9, BL21 lysate with no recombinant CsCaN construct as the negative control. (b) Possible modification of the MBP-tagged recombinant CsCaN by senescent leaf extract. Lane 1, protein marker. Lanes 2–4, in-gel assay reaction with 0.6, 2 and 6 μl of senescent leaf extracts added, respectively, showing concentration-dependent endogenous DNase activities at 35 kDa. Lanes 5–7, MBP-CsCaN was added to the same reaction systems, respectively. The recombinant protein (MBP-CsCaN) band intensities were decreased while the 35-kDa band intensities were increased, concurrent with an increase in leaf extract. These results suggested that increased intensities of DNase activity at 35 kDa in lanes 5–7 compared with lanes 2–4 were derived from the recombinant protein. Therefore, the recombinant protein could be modified to generate a 35-kDa form with DNase activity. Lane 8, MBP-CsCaN was incubated without senescent leaf extracts. Lane 9, E. coli lysate expressing no-tagged recombinant CsCaN. (c) CsCaN protein detection at 35 kDa. A clear signal was detected at the 35-kDa position with the CsCaN antibody in the protein, extracted from stamens of stages 6–9 female flowers (Fs). This signal was not detected in the protein extracted from carpels of the same flower (Fc). The 43-kDa bands were probably nonspecific background signals.

CsCaN expressed differentially during flower development

The spatial specificity of DNA damage in the inappropriate anthers of stage 7 female flowers (Hao et al., 2003) implies that the DNase responsible is probably expressed or activated in an organ-specific manner. Although CsCaN was identified because of its preferential expression in the stamens of stage 6 female floral buds, the expression patterns were further clarified by examining CsCaN expression in various organs with real-time RT-PCR, these organs including fully expanded cotyledons, fully expanded first true leaves, fully expanded 8th leaves, 5-d-old seedling root tips, stamens of stage 6 male and female flowers, and carpels of stage 6 female flowers. Fig. 5(a) shows that, although CsCaN was expressed in all organs examined, the CsCaN expression level was approx. twofold higher in the stamens of stage 6 female flowers compared with those of male flowers. This is consistent with SSH screen results.

Figure 5.

Detection of Cucumis sativus calcium-dependent nuclease (CsCaN) expression by real-time RT-PCR in different organs at indicated developmental stages and by in situ hybridization to cucumber (Cucumis sativus) flowers. (a) CsCaN was expressed universally in all detected organs, but was higher in the stamens of stage 6 female flowers than in those of male flowers. Samples labeled as cotyledon, 1st leaves, 8th leaves, root tip, M6St, F6St and F6c represent, respectively, samples of fully expanded cotyledons, fully expanded first true leaves, fully expanded 8th leaves, root tips of 5-d-old seedlings, stage 6 stamens of male and female flowers, and stage 6 carpels of female flowers. (b–k) Detection of CsCaN expression during flower development by in situ hybridization. CsCaN RNA was detected in dividing cells at floral meristems (b; Fm). Very low signals were detected in stage 5 floral buds (c) and stage 6 male flowers (d). The signals were again detectable at stamen tips of stage 7 male flowers (e), and were greater in stamens of stage 9 male flowers (f). By contrast, notable signals were detected in the floral buds committed to being female at late stage 5 or early stage 6 (h), and continued to substantially increase in female floral buds at stage 6 (i), not only in stamens, but also in the carpel primordial region. CsCaN expression was then restricted to dividing cells of carpel primordia of stage 7 (j) and 8 (k) female flowers. (g) Negative control, hybridization with a sense probe. Bars: (b–k) 100 μm. St, stamen; C, carpel. [Correction added after online publication 31 July 2011: the information relating to the scale bars and abbreviations has been added.]

The expression in floral organs revealed by real-time RT-PCR was further confirmed by in situ hybridization. Fig. 5(b) shows that CsCaN RNA was detectable in the meristematic region (FM) of floral buds before stage 4. RNA signals were low in stage 5 floral buds (Fig. 5c) and stage 6–7 male floral buds (Fig. 5d,e), and higher at the stamen tips of stage 9 male flowers (Fig. 5f). By contrast, CsCaN expression was substantially increased in early and late stage 6 female floral buds, not only in the stamens, but also in the carpel primordia (Fig. 5h,i). However, following stage 7, the signals were only detectable in the carpels, and not in the inappropriate stamens (Fig. 5j,k). Differential CsCaN expression in female flower stamens was consistent with differentially detected DNase activity in female flowers (Fig. 5a).

CsCaN expression is ethylene inducible

We hypothesized that, as female flower development is regulated by ethylene, the targeted DNase responsible for primordial anther-specific DNA damage would be ethylene inducible. Although we found multiple ethylene response binding sites in the CsCaN promoter sequence (Table S4), suggesting that CsCaN expression might be ethylene inducible, it was necessary to experimentally determine whether its expression was ethylene inducible. In view of the difficulty of collecting stamen samples and the fact that CsCaN is also expressed in other organs (Fig. 5a), we used cotyledons from 8-d-old seedlings to detect the response of CsCaN expression to ethylene treatment. The ethylene precursor ACC was applied to seedlings at concentrations of 50, 100, 200 and 400 μM. Cotyledons of the seedlings were collected at 4, 8, 12, 16, 20 and 24 h after ACC application. Fig. 6 indicates that CsCaN expression was up-regulated from 2- to 4-fold following ACC treatments in a time- and concentration-dependent manner.

Figure 6.

Induction of Cucumis sativus calcium-dependent nuclease (CsCaN) expression by ACC treatments. The ethylene treatments were conducted by applying ACC at concentrations of 50, 100, 200 and 400 μM to 8-d-old cucumber seedlings. The CsCaN expression levels changed roughly according to ACC concentration. The y-axis is the ratio of CsCaN to Csactin. Error bars are ± SD from three biological replicates.

Wang et al. (2010) observed ethylene induction of DNase activity in cucumber protoplasts. However, in the present study, we did not find ethylene induction of DNase activity and CsCaN protein content in cotyledons and leaves (data not shown). Our results suggest that the relationship between ethylene and CsCaN activity is not simple.

Discussion

The aim of this study was to identify a putative missing link between the stamen-specific altered ethylene signal and primordial anther-specific DNA damage. To achieve this, we performed a SSH screen and identified the CsCaN transcript. The CsCaN-encoded protein was found to belong to an SNc domain-containing gene family, and to have calcium-dependent nuclease activity. This protein can be modified into two forms with different molecular weights, 37.4 and 35 kDa. The latter was detected by the antibody against the CsCaN protein at the inappropriate stamens, but not at the ovaries, of female flowers. This short form of CsCaN was co-localized with the DNase activity correlated to the primordial anther-specific DNA damage in female cucumber flowers. CsCaN expression analysis at the RNA level revealed that, although CsCaN RNA was detected in all organs sampled, the RNA level was higher in the stamens of stage 6 female flowers than in the stamens of male flowers. Furthermore, as predicted, the CsCaN transcript was ethylene inducible.

Taking these results together, particularly the finding that the CsCaN 35-kDa form was only detected in the stamens of female flowers, we concluded that the CsCaN 35-kDa form should contribute, at least in part, to stamen-specific 35-kDa DNase activity. Therefore, it is likely that CsCaN participates in inducing primordial anther-specific DNA damage in female cucumber flowers.

Given the above data, we propose that the regulation of CsCaN as a DNase responsible for primordial anther-specific DNA damage occurs at multiple levels, including the transcriptional, post-translational, and enzyme activity levels. We demonstrated that CsCaN RNA was ethylene regulated at the transcriptional level (Fig. 6), and coordinated by unknown organs and tissue-specific factors (Fig. 5). Carpel preferential expression (Fig. 5) could also be associated with ethylene induction, because the M gene CsACS2 was preferentially expressed in the carpels of female flowers (Saito et al., 2007; Boualem et al., 2009). These results suggest that ethylene is required for organ-specific CsCaN transcript up-regulation for gene function. However, the presence of CsCaN RNA per se does not necessarily lead to DNase activity, because we did not detect DNase activity or DNA damage in the carpels of female flowers. These observations suggest that CsCaN activity must be regulated at levels additional to transcription.

Although we were unable to examine translational regulation, we did identify post-translational regulation. The predicted molecular weight of CsCaN was 37.4 kDa, and we detected DNase activity in the recombinant protein. However, DNase activity in female flowers at this molecular weight was not detectable. By contrast, we established a clear correlation between CsCaN and DNase activity at 35 kDa (Fig. 4a,c). Furthermore, we identified DNase activity at 35 kDa from the recombinant protein raised in untagged E. coli (Fig. 4a, lane 8; Fig. 4b, lane 9). In addition, the recombinant protein was processed into a 35-kDa form by treatment with protein extracts from senescent cucumber leaves (Fig. 4b). Although the details are still unknown, the post-translational regulation of CsCaN activity introduced a new facet to elucidate the regulatory mechanism involved in the induction of primordial anther-specific DNA damage.

Enzyme activity regulation is another level of CsCaN activity, as we demonstrated that CsCaN activity was calcium dependent. Cellular calcium concentrations may therefore be another component involved in female flower development.

To date, in this study and others, many components have been identified that are involved in cucumber unisexual flower development, including F and M genes, CsETR1 organ-specific regulation, and possibly microRNA (Sun et al., 2010). We suggest that CsCaN is responsible (possibly together with other proteins) for primordial anther-specific DNA damage. Its identification provides a new avenue for future investigations of the mechanisms of DNA damage induction in primordial anthers of female cucumber flowers.

Acknowledgements

We thank Su-Lan Bai of Capital Normal University for her contribution to the initiation of SSH during her postdoctoral work in this laboratory; Song-Gang Li of BGI for providing help in large-scale sequencing of the SSH clones; San-Wen Huang and Zhong-Hua Zhang of the Institute of Vegetable and Flowers of CAAS for sharing with us the cucumber sequence information; Rui Cai of Shanghai Communication University for sharing his unpublished results on cucumber M gene cloning; Xiao-Dong Su, Sodmergen and their students for their help with protein purification; Jian-Guo Ji for his help with MS and 2D gels; Ke-Ming Cui, Xing-Qiang He and Yu Pang for discussions about Eucommia CaN (EuCaN or EuDNase); Hong-Wei Guo for his help in providing facilities for ethylene treatments; and Rafael Perl-Terves (Bar-Ilan University, Israel) for his critical reading of the manuscript, comments and suggestions. This work was supported by grants to S.N.B. from the Ministry of Science and Technology of China (MST) (J00-A-005, G19990116), the National Natural Science Foundation of China (NSFC) (30070361) and the International Center of Genetic Engineering and Biotechnology (CRP/CHN03-02); a grant to D.H.W. from NSFC (30470842); and a grant to Z.H.X. from MST.

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