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Keywords:

  • cold acclimation;
  • cold shock domain;
  • flowering time;
  • freezing tolerance;
  • RNA chaperone;
  • silique development

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Bacterial cold shock proteins (CSPs) act as RNA chaperones that destabilize mRNA secondary structures at low temperatures. Bacterial CSPs are composed solely of a nucleic acid-binding domain termed the cold shock domain (CSD). Plant CSD proteins contain an auxiliary domain in addition to the CSD but also show RNA chaperone activity. However, their biological functions are poorly understood.
  • We examined Arabidopsis COLD SHOCK DOMAIN PROTEIN 2 (AtCSP2) using overexpressing and mutant lines.
  • A double mutant, with reduced AtCSP2 and no AtCSP4, showed higher freezing tolerance than the wild-type when cold-acclimated. The increase in freezing tolerance was associated with up-regulation of CBF transcription factors and their downstream genes. By contrast, overexpression of AtCSP2 resulted in decreased freezing tolerance when cold-acclimated. In addition, late flowering and shorter siliques were observed in the overexpressing lines.
  • AtCSP2 negatively regulates freezing tolerance and is partially redundant with its closest paralog, AtCSP4.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The cold shock domain (CSD) is a highly conserved nucleic acid-binding domain found in bacteria, animals and higher plants (Yamanaka et al., 1998; Karlson & Imai, 2003; Mihailovich et al., 2010). The CSD structure contains a five-stranded β-barrel sheet with two consensus RNA binding motifs (RNP-1 and RNP-2) that are essential for single-stranded DNA/RNA binding (Schroder et al., 1995; Hillier et al., 1998; Wang et al., 2000). Bacterial cold shock proteins (CSPs) are composed of a single CSD and act as RNA chaperones that destabilize secondary structures in RNAs (Jiang et al., 1997; Bae et al., 2000; Phadtare et al., 2002). In Escherichia coli, nine members of the CSP gene family (cspA to cspI) have been identified and four of them (cspA, cspB, cspG and cspI) are highly induced after cold shock (Yamanaka et al., 1998; Wang et al., 1999). A quadruple deletion mutant that lacks cspA, cspB, cspG and cspE exhibits growth defects at low temperature (Xia et al., 2001). At low temperatures, RNA molecules form secondary structures that may inhibit RNA functions including translation and transcription (Rajkowitsch et al., 2007). Therefore, RNA chaperone activity is likely to be critical for RNA function at low temperatures. In addition, CspA, CspC and CspE also function as transcription antiterminators and regulate the expression of a set of cold-responsive genes (Bae et al., 2000).

Vertebrate Y-box proteins are the most extensively studied eukaryotic CSD proteins and Y-box CSDs show about 40% amino acid sequence identity to bacterial CSDs (Wistow, 1990; Wolffe et al., 1992). All vertebrate Y-box proteins contain a single N-terminal CSD and C-terminal auxiliary domains (Kohno et al., 2003). The vertebrate Y-box binding protein-1 (YB-1) acts in transcriptional and translational regulation, and plays critical roles in many biological processes such as stress responses, cell proliferation and DNA repair (Kohno et al., 2003). YB-1-deficient mice die during late embryonic development, indicating its essential function in animals (Lu et al., 2005). In addition, YB-1-depleted chicken cells cease cell division after a temperature downshift (Matsumoto et al., 2005). YB-1 binds to DNA and RNA, and shows concentration-dependent melting and annealing activities (Matsumoto & Wolffe, 1998; Skabkin et al., 2001). These activities are probably necessary for the pleiotropic functions of YB-1. A Xenopus Y-box binding protein, FRGY2, is involved in translational repression of maternal mRNAs that accumulate in oocytes, preventing premature mRNA translation (Matsumoto et al., 1996). In Caenorhabditis elegans, another CSD protein, LIN-28, was first characterized as a critical regulator of larval development (Moss et al., 1997). LIN-28 is an evolutionarily conserved RNA-binding protein containing an N-terminal CSD and retroviral-like CCHC zinc fingers at the C-terminus. Mammalian LIN-28, together with other factors, can reprogram human somatic cells into pluripotent stem cells (Yu et al., 2007; Liao et al., 2008). LIN-28 post-transcriptionally inhibits the biogenesis of let-7 miRNA, which is a regulator of cell growth and differentiation in embryonic cells (Roush & Slack, 2008; Viswanathan et al., 2008). Taken together, these studies suggest that animal CSD proteins are essential regulators of a variety of biological processes.

Plant CSD proteins typically contain an N-terminal CSD and a glycine-rich region interspersed with various numbers of retroviral-like CCHC zinc fingers at the C-terminus (Sasaki & Imai, 2012). The first studied plant CSD protein was wheat cold shock domain protein 1 (WCSP1; Karlson et al., 2002), which accumulates in crown tissue during prolonged cold acclimation. WCSP1 mRNA is not modulated by other environmental stresses such as salt, drought and heat, or treatment with ABA (Karlson et al., 2002), which suggests that the function of WCSP1 is specific to cold adaptation. WCSP1 has nucleic acid-binding activity (Karlson et al., 2002; Nakaminami et al., 2005) and unwinds nucleic acid duplexes in vitro and in vivo (Nakaminami et al., 2006). These studies indicated that, similar to bacterial CSPs, WCSP1 functions as an RNA chaperone to destabilize RNA secondary structures during cold acclimation.

In Arabidopsis, four CSD protein genes (AtCSP1AtCSP4) have been identified in the genome sequence (Karlson & Imai, 2003; Sasaki & Imai, 2012) and AtCSP3 (At2 g17870) is essential for the acquisition of freezing tolerance in Arabidopsis (Kim et al., 2009). C-repeat binding factors (CBFs) regulate freezing tolerance through activation of cold-regulated (COR) gene expression (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Thomashow, 1998). However, AtCSP3 does not affect expression of CBFs or COR genes (Kim et al., 2009). AtCSP3 regulates expression of stress-related genes but their roles in freezing tolerance are unknown (Kim et al., 2009). Transgenic plants overexpressing AtCSP1 (CSDP1, At4 g36020) show delayed seed germination under dehydration and salt stress conditions, and plants overexpressing AtCSP2 (AtGRP2/CSDP2, At4 g38680) exhibit accelerated seed germination under salt stress conditions (Park et al., 2009). Overexpression of AtCSP1 or AtCSP2 did not enhance freezing tolerance of wild-type plants but did complement the freezing sensitive phenotype of grp7, a mutant of glycine-rich RNA binding protein 7 (Park et al., 2009). Expression of bacterial CSPs in plants confers stress tolerance against drought and cold (Castiglioni et al., 2008). These studies support the hypothesis that plant and bacterial CSD proteins have conserved biochemical functions and are involved in stress adaptation.

Plant CSD proteins also regulate developmental processes similar to animal CSD proteins. AtCSP2 is highly expressed in meristematic tissues and ovules (Fusaro et al., 2007; Sasaki et al., 2007; Nakaminami et al., 2009). AtCSP2 knockdown plants showed early flowering, reduced number of stamens and high rates of abnormal development of seeds/embryos (Fusaro et al., 2007). Recently, Yang & Karlson (2011) demonstrated that overexpression of AtCSP4 (AtGRP2b, At2 g21060) reduces silique length and induces embryo lethality. Overexpression of AtCSP4 affects the expression of several MADS box and endosperm development genes during floral and silique development (Yang & Karlson, 2011).

In this paper, we describe the functional characterization of AtCSP2 in planta. Genetic studies with overexpressor and mutant lines indicate that AtCSP2 is a negative regulator of freezing tolerance. In addition, AtCSP2 is functionally redundant with AtCSP4 in freezing tolerance and flowering time.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and growth condition

Arabidopsis (Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col-0)) was used in this study. T-DNA mutant atcsp2-2 (SALK_048476), atcsp2-3 (SALK_083561) and atcsp4-1 (GK-623B08.01) were obtained from the Arabidopsis Biological Resource Center (http://abrc.osu.edu/) or GABI-kat (http://www.gabi-kat.de/) and genotyped by PCR using primers flanking the insertions (Supporting Information, Table S1). To generate the double mutant, we crossed atcsp2-3 and atcsp4-1, and genotypes of F2 plants were identified by PCR. Seeds of Arabidopsis were surface-sterilized, and planted on MS medium (Murashige & Skoog, 1962) containing 2% sucrose. Plates were maintained in the dark at 4°C for 2 d to synchronize germination and were transferred to a growth chamber (16 h light / 8 h dark) at 22°C. The flowering time was determined by counting the days and rosette leaves once a flower bud was visible.

Generation of AtCSP2 overexpression plants

For overexpression of AtCSP2, the coding region of AtCSP2 was cloned into the XbaI–SacI site of the pBI121 vector (Clontech, Mountain View, CA, USA) under the control of the cauliflower mosaic virus 35S promoter. The resulting 35S:AtCSP2 construct was transformed into Arabidopsis to get 35S:AtCSP2 transgenic plants. Transformation was performed by the floral dip method (Clough & Bent, 1998) using Agrobacterium tumefaciens strain GV3101. For selection, seeds were plated on MS medium containing 2% sucrose and 50 μg ml−1 kanamycin. Accumulation of AtCSP2 protein in independent transgenic lines was examined by western blot analysis. Homozygous T4 plants were used for analysis.

Western blot analysis

Total protein was prepared from freshly harvested 10-d-old plants. Tissue (100 mg FW) was ground in 200 μl of extraction buffer (62.5 mM Tris-HCl, pH 7.0, 20% glycerol, 4% SDS, 1.4 M β-mercaptoethanol) and then centrifuged at 13 000 g for 15 min at 4°C to remove cell debris. After centrifugation, the supernatant was collected and dissolved in sodium dodecyl sulfate (SDS) sample buffer. Extracted total protein was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond-C extra membrane (GE Healthcare, Buckinghamshire, UK). The membrane was hybridized with custom rabbit polyclonal antibodies against the CSD of WCSP1 (1 : 5000 v/v; Sasaki et al., 2007) and antirabbit IgG peroxidase-linked secondary antibodies (1 : 10 000 v/v; GE Healthcare). Chemiluminescent detection of the signal was carried out using the ECL kit (GE Healthcare) according to the manufacturer's instructions.

Reverse transcription PCR (RT-PCR) and quantitative real time RT-PCR (qRT-PCR)

Total RNA was extracted from 10-d-old plants with or without cold acclimation using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. To remove genomic DNA, total RNA was treated with DNase I (Takara, Shiga, Japan) following the manufacturer's instructions. First-strand cDNA synthesis was carried out using High Capacity RNA-to-cDNA Kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions. For RT-PCR, PCR amplifications were performed with Expand High Fidelity PCR System (Roche), using gene-specific primers for AtCSP2 and AtCSP4 (Table S1). Reaction products were visualized after electrophoresis on a TAE (Tris-acetate-EDTA) buffer plus 1% agarose gel using ethidium bromide. Tubulin transcript was used as a loading control. qRT-PCR analysis was performed in 96-well blocks with an ABI7500 Real-Time PCR System (Applied Biosystems) using the SYBR Green PCR master mix -Plus- (Toyobo, Osaka, Japan). PCR was performed as follows: 95°C for 1 min, 40 cycles of 95°C for 15 s, 60°C for 1 min. Transcripts were normalized to the housekeeping gene CITRATE SYNTHASE3 (CSY3; Pracharoenwattana et al., 2005) for Figs 1(b) and (d) and 3, and ACTIN2 for Fig. 5. The specificity of the PCR was determined by melting curve analysis of the amplified products using the standard method installed in the system. The RNA preparation and qRT-PCR procedure were performed using at least two different biological replicates, and very similar results were obtained. The primers used for RT-PCR and qRT-PCR are listed in Table S1.

Freezing tolerance analysis

The freezing tolerance analysis was performed as described previously (Chinnusamy et al., 2003), with some modifications. Non-acclimated and cold-acclimated 10-d-old seedlings were transferred to a programmed freezer LU-112 (ESPEC, Osaka, Japan) set at −1°C. After 1–2 h, the plates were sprinkled with ice chips and maintained at −1°C for at least 16 h. The temperature was lowered at a rate of −1°C h−1 and plates were removed from the freezer at specific temperature points. The plates were thawed overnight at 4°C in the dark and then incubated at 22°C under constant light. The survival rate was scored after 10 d. Experiments were performed in quadruplicate, and the percentage of plants that survived was calculated.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Increased freezing tolerance in atcsp2-3 atcsp4-1 mutant

To analyze the functional roles of AtCSP2 in planta, we isolated two T-DNA insertion lines, atcsp2-2 (SALK_048476) and atcsp2-3 (SALK_083561), from the Arabidopsis Biological Resource Center collection. The atcsp2-2 and atcsp2-3 alleles contain T-DNA insertions at 182 and 126 bp upstream of the AtCSP2 start codon, respectively (Fig. 1a). qRT-PCR analyses confirmed the reduction in AtCSP2 expression in these lines. The AtCSP2 expression levels in 10-d-old seedlings of atcsp2-2 and atcsp2-3 were 61.0 and 41.6% of that in wild-type, respectively (Fig. 1b). Therefore, we used atcsp2-3 for further studies. Since AtCSP2 expression is induced by cold treatment (Karlson & Imai, 2003; Fusaro et al., 2007; Sasaki et al., 2007), we first characterized freezing tolerance of atcsp2-3. We assessed freezing tolerance by survival rate after a freeze–thaw program. Ten-day-old seedlings of atcsp2-3 and the wild-type grown on agar plates were subjected to freezing temperature with or without cold acclimation (4°C, 7 d). Although AtCSP2 mRNA was significantly reduced in atcsp2-3, the freezing tolerance of atcsp2-3 was the same as the wild-type (Fig. 2a). Since the amino acid sequence and domain structure are highly conserved between AtCSP2 and AtCSP4, we speculated that the two proteins might have overlapping functions. To generate a double mutant of AtCSP2 and AtCSP4, we first isolated a knockout mutant of AtCSP4, atcsp4-1, from the GABI-kat collection (GK-623B08.01; Fig. 1a). RT-PCR confirmed that AtCSP4 mRNA is knocked out in atcsp4-1 (Fig. 1c). Freezing tolerance of atcsp4-1 mutants was also the same as wild-type (Fig. 2a). We crossed atcsp2-3 with atcsp4-1 and selected two double homozygous mutant lines (atcsp2-3 atcsp4-1-#1 and #2). The atcsp2-3 atcsp4-1 mutants showed reduced AtCSP2 and no AtCSP4 expression (Fig. 1d,e). Expression of one gene was not affected by the mutation of the other gene (Fig. S1). Interestingly, the atcsp2-3 atcsp4-1 mutant showed substantially increased freezing tolerance after cold acclimation at 4°C for 3 or 7 d (Fig. 2b). By contrast, the difference in freezing tolerance was not observed in non-acclimated plants (Fig. 2b). Survival rates of 7-d cold-acclimated wild-type plants after −16°C treatment was 37.5% and two lines of atcsp2-3 atcsp4-1 showed survival rates of 79.2 and 75.0%. The single and double mutants grew normally and did not show any abnormal morphological phenotypes under normal growth conditions (Fig. S2a). However, the atcsp2-3 atcsp4-1 mutant flowered earlier than wild-type plants (Fig. S2b). These data suggest that AtCSP2 is a negative regulator of freezing tolerance and flowering time, and is functionally redundant with AtCSP4.

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Figure 1. Isolation of Arabidopsis atcsp2-2, atcsp2-3, atcsp4-1 and atcsp2-3 atcsp4-1 T-DNA mutant lines. (a) Schematic representation of the position of T-DNA insertion within the promoter region of AtCSP2 and the cold shock domain of AtCSP4. The solid, open and hatched boxes indicate the cold shock domain, glycine-rich region and zinc finger motif, respectively. (b) Quantitative real time reverse transcription PCR (qRT-PCR) analysis of AtCSP2 expression in wild-type (WT), atcsp2-2 and atcsp2-3. The data represent means of three replicates ± SE. (c) RT-PCR analysis of AtCSP4 expression in wild-type (WT) and atcsp4-1 (4). Tubulin was amplified as an internal control. RT+ and RT− are samples with and without reverse transcriptase treatment, respectively. (d) qRT-PCR analyses of AtCSP2 expression in WT and atcsp2-3 atcsp4-1 with or without cold treatment (4°C). The data represent means of three replicates ± SE. (e) RT-PCR analysis of AtCSP4 expression in WT and atcsp2-3 atcsp4-1.

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Figure 2. Arabidopsis atcsp2 and atcsp4 double mutants show increased freezing tolerance after cold acclimation. (a) Freezing tolerance of 10-d-old wild-type (WT), atcsp2-3 and atcsp4-1 seedlings grown on MS medium containing 2% sucrose with or without cold acclimation. Non-acclimated plants were exposed to a freezing temperature of −11°C, while cold-acclimated plants were exposed to a freezing temperature of −16°C. Experiments were performed in quadruplicate, and the percentage of plants that survived was calculated (= 12 plants used in each treatment). (b) Freezing tolerance of 10-d-old WT and atcsp2-3 atcsp4-1 seedlings grown on MS medium containing 2% sucrose with or without cold acclimation. Experiments were performed in quadruplicate, and the percentage of plants that survived was calculated (= 12 plants used in each treatment). Data represent means ± SE. * and ** indicate statistically significant differences with < 0.05 and < 0.01 (Student's t test), respectively. NA, non-acclimation; CA, cold acclimation; CA3, CA 3 d; CA7, CA 7 d.

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Enhanced accumulation of cold-inducible mRNAs in atcsp2-3 atcsp4-1 mutant

To determine whether increased freezing tolerance in the atcsp2-3 atcsp4-1 mutant is associated with the CBF-dependent pathway, transcript abundances of the CBFs (CBF1CBF3) and COR genes in atcsp2-3 atcsp4-1 mutant were determined by qRT-PCR. The expression of two CBF genes, CBF2 and CBF3, was up-regulated in atcsp2-3 atcsp4-1-#1 compared with that in the wild-type over 24 h of cold exposure (4°C; Fig. 3). However, CBF1 exhibited similar expression patterns in both the wild-type and atcsp2-3 atcsp4-1-#1 seedlings (Fig. 3). Transcript abundances of the COR genes, COR15A, RD29A and KIN1, were higher in atcsp2-3 atcsp4-1-#1 than in wild-type after 3 d cold treatment (Fig. 3). These data indicate that AtCSP2 and AtCSP4 control cold signaling through the negative regulation of CBF2 and CBF3 expression.

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Figure 3. Expression profiles of cold-responsive genes in the Arabidopsis atcsp2-3 atcsp4-1 double mutant. The expression of CBF1, CBF2, CBF3, COR15A, RD29A or KIN1 mRNAs in 10-d-old wild-type (WT) and atcsp2-3 atcsp4-1-#1 seedlings during cold acclimation was analyzed by quantitative real time reverse transcription PCR (qRT-PCR). The data represent means of three replicates ± SE. The values of non-cold-treated WT were set to 1. WT and atcsp2-3 atcsp4-1-#1 are indicated by open and solid bars, respectively.

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AtCSP2 overexpression alters development of freezing tolerance during cold acclimation

To further understand the biological function of AtCSP2, we generated 35S:AtCSP2 transgenic lines and found three (18, 20 and 26) that show higher AtCSP2 accumulation than the wild-type (Fig. 4a). To evaluate the effects of AtCSP2 overexpression on freezing tolerance, freezing survival was measured for 35S:AtCSP2 and wild-type plants. Ten-day-old 35S:AtCSP2 and wild-type seedlings grown on agar plates were subjected to freezing temperatures with or without cold acclimation. Under non-acclimated conditions, freezing tolerance of 35S:AtCSP2 plants was similar to that of the wild-type (Fig. 4b). However, 35S:AtCSP2 and wild-type plants exhibited a drastic difference in freezing tolerance after cold acclimation (Fig. 4b). For example, the survival rates for wild-type plants after 7 d cold acclimation was 55.6%, but overexpression lines showed 27.8% or lower survival rates after −16°C freezing treatment. Transcript abundances of COR15A, KIN1 and CBF3 were lower in 35S:AtCSP2-18 than that in the wild-type after cold treatment (Fig. 5). These data supported the hypothesis that AtCSP2 negatively regulates freezing tolerance under cold-acclimated conditions.

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Figure 4. Overexpression of AtCSP2 decreases the cold acclimation capability in Arabidopsis. (a) Western blot analysis of AtCSP2 protein accumulation in 35S:AtCSP2 plants. Membrane stained with Ponceau S is shown as a loading control. (b) Freezing tolerance of 10-d-old wild-type (WT) and 35S:AtCSP2 seedlings grown on MS medium containing 2% sucrose with or without cold acclimation. Experiments were performed in quadruplicate, and the percentage of plants that survived was calculated (= 9 plants used in each treatment). Data represent the means ± SE. * and ** indicate statistically significant differences with < 0.05 and < 0.01 (Student's t test), respectively. NA, non-acclimation; CA, cold acclimation; CA 3, CA 3 d; CA7, CA 7 d.

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Figure 5. Expression of cold-regulated genes in an AtCSP2-overexpressing Arabidopsis plants. Expression of CBF3, COR15A, RD29A or KIN1 mRNAs in 10-d-old wild-type (WT) and 35S:AtCSP2-18 seedlings during cold acclimation was analyzed by quantitative real time reverse transcription PCR (qRT-PCR). The data represent means of three replicates ± SE. The values of non-cold-treated WT plants were set to 1. WT and 35S:AtCSP2-18 are indicated by open and solid bars, respectively.

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Pleiotropic phenotype of AtCSP2-overexpressing plants

In addition to the phenotype associated with freezing tolerance, 35S:AtCSP2 plants show pleiotropic phenotypes in plant development. Under long-day conditions (16 h light), 35S:AtCSP2 plants exhibited smaller plant size and late flowering (Fig. S3a,b). In addition, siliques of 35S:AtCSP2 plants were shorter and wider than wild-type siliques (Fig. 6a,b). Examination of opened siliques from wild-type and 35S:AtCSP2 plants indicated that seeds of 35S:AtCSP2 were not arranged uniformly compared with the wild-type seeds (Fig. 6c). We speculated that shorter size and larger width of the siliques in 35S:AtCSP2 are the result of the decreased distance between the seeds within the siliques. To examine this hypothesis, we measured several parameters, including the number of seeds per silique, the number of seeds per 1 cm of silique and the distance between the bases of funiculi. The number of seeds per silique in 35S:AtCSP2 is almost same as that in the wild-type, except that the 35S:AtCSP2-20 silique had fewer seeds (Fig. 6d). However, the number of seeds contained in a unit length was larger in 35S:AtCSP2 siliques than in wild-type siliques (Fig. 6e). The distance between the bases of funiculi in 35S:AtCSP2 siliques was shorter than that in wild-type siliques (Fig. 6f). These results suggest that AtCSP2 negatively regulates silique size by altering spaces between seeds in siliques.

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Figure 6. Changes in silique morphology of AtCSP2-overexpressing Arabidopsis plants. (a) Representative mature siliques from wild-type (WT) and 35S:AtCSP2 plants (18, 20 and 26). Bar, 0.5 cm. (b) Silique length (cm). (c) Representative opened siliques from WT and 35S:AtCSP2 plants. Bar, 0.1 cm. (d) The number of seeds per silique. (e) The number of seeds per 1 cm length of silique. (f) The distance between the bases of funiculi in silique (mm). Data represent the means ± SE. * and ** indicate statistically significant differences with < 0.05 and < 0.01 (Student's t test), respectively. For measurement of silique length and number of seeds, 10 siliques were scored for each line. For measurement of the distance between the base of funiculi, 10 funiculi intervals were measured and averaged for each silique. Data are the averages from three different siliques for each line.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Differences in freezing tolerance between the atcsp2-3 atcsp4-1 mutant and wild-type were observed after cold acclimation but not in nonacclimated conditions (Fig. 2b). In addition, 35S:AtCSP2 plants showed reduced freezing tolerance only when cold treatment was applied (Fig. 4b). These data clearly demonstrate that AtCSP2 negatively regulates cold acclimation in Arabidopsis. AtCSP2 may directly regulate the expression of CBFs or control transcription or translation processes upstream of CBFs. It has been demonstrated that several proteins negatively regulate expression of CBFs (Chinnusamy et al., 2007). HOS1, an ubiquitin E3 ligase, represses expression of CBFs and confers increased sensitivity to freezing under cold-acclimated conditions (Dong et al., 2006). MYB15 (an R2R3 type MYB family protein) is another negative regulator of the CBF pathway and suppresses expression of CBFs (Agarwal et al., 2006). Interestingly, AtCSP2 and MYB15 are both induced during cold acclimation, even though they negatively regulate the CBF pathway. In our study, transcripts of CBFs were transiently up-regulated after 6 h cold treatment and the transcript abundances decreased thereafter (Fig. 3), while expression of AtCSP2 increased after 6 h cold treatment and the increased expression level was maintained for up to 48 h of cold treatment (Fusaro et al., 2007). This may suggest that AtCSP2 is involved in the attenuation of the CBF pathway to fine-tune the levels of freezing tolerance at the later stage of cold acclimation.

Although AtCSP2 and AtCSP3 are both induced by cold treatment and have an RNA chaperone activity in common, their physiological functions clearly differ. AtCSP3 positively regulates freezing tolerance through a CBF-independent pathway under both non-acclimated and cold-acclimated conditions (Kim et al., 2009). 35S:AtCSP3 plants show no morphological or developmental phenotypes. By contrast, AtCSP2 negatively regulates freezing tolerance through a CBF-dependent pathway (Figs 3, 5) and overexpression of AtCSP2 affects plant development (Figs 6, S3). These data suggest that there is no functional interaction between AtCSP2 and AtCSP3 in regulating freezing tolerance and plant development.

The atcsp2 and atcsp4 single mutants did not show differences from the wild-type in freezing tolerance and plant development (Figs 2a, S2). However, the atcsp2 atcsp4 double mutants showed increased freezing tolerance after cold acclimation and early flowering (Figs 2b, S2b). AtCSP2 shows the highest sequence similarity with AtCSP4 and the two genes also show very similar tissue expression patterns and protein subcellular localization (Sasaki et al., 2007; Nakaminami et al., 2009; Yang & Karlson, 2011). These data suggest that AtCSP2 and AtCSP4 function redundantly in freezing tolerance and flowering time. By contrast, neither atcsp2 nor atcsp4 single mutants, nor the double mutant, showed an altered silique phenotype. Therefore, their functional relationship in silique development remains unclear.

Fusaro et al. (2007) demonstrated that AtCSP2 RNAi plants in the C24 ecotype showed early flowering, reduced number of stamens and high rates of abnormal seeds/embryos. The atcsp2-3 atcsp4-1 mutants (Col-0 background) also showed an early flowering phenotype but no significant alterations in the number of stamens and seed development (Fig. S4). Since the atcsp2-3 atcsp4-1 mutant has some remaining AtCSP2 expression (Fig. 1d), it may be that this low level of AtCSP2 expression is sufficient to complete stamen and embryo development in Col-0.

In addition to plant growth and flowering time, AtCSP2 negatively regulates silique length without affecting the number of seeds (Fig. 6). In the wild-type, there is a positive correlation between seed number and silique length (Cox & Swain, 2006). In addition, it was reported that increased seed size in Arabidopsis lba1/atupf1-1 mutant coincides with a decrease in seed numbers within a unit length of silique (Yoine et al., 2006). Therefore, it is possible to hypothesize that there is a mechanism that maintains proper packing of seeds within a silique. This mechanism is probably impaired in 35S:AtCSP2 plants. Since AtCSP2 expression is detected in ovules and transmitting tissue (Fusaro et al., 2007; Sasaki et al., 2007), AtCSP2 may play a role in regulating the distance between funiculi. It is interesting to note that the siliques of ga20ox2 mutants, which have reduced GA4 levels, are shorter than that of wild-type (Rieu et al., 2008). However, the number of seeds per silique is not reduced in the ga20ox2 mutant (Rieu et al., 2008), which indicates that the seeds are more closely packed in the ga20ox2 siliques. Since AtCSP2 negatively regulates GA biosynthesis genes in seed germination (K. Sasaki et al., unpublished), the short silique phenotype of 35S:AtCSP2 plants may possibly involve GA signaling.

The mechanism by which AtCSP2 regulates RNA functions is currently unknown. AtCSP2 may enhance translation of mRNAs that encode negative regulators of freezing tolerance and plant development. YB-1, a well studied CSD protein in animals, is a major component of cytoplasmic messenger ribonucleoprotein particles (mRNPs) and binds to mRNA to inhibit the access of cap-binding factor, resulting in translational arrest, which is called RNA masking (Kohno et al., 2003; Mihailovich et al., 2010). YB-1 stimulates translation when the YB-1/mRNA ratio is low, but inhibits translation when the ratio increases (Pisarev et al., 2002; Nekrasov et al., 2003). RNA masking activity was also described for another CSD protein, FRGY2, in Xenopus oocytes (Matsumoto et al., 1996). In Chlamydomonas, a CSD protein NAB1 from Chlamydomonas reinhardtii stabilizes mRNAs of LHCBM (major light-harvesting complex of photosynthesis II) genes and represses translation at the pre-initiation stage (Mussgnug et al., 2005). Based on these studies, we can propose a putative function of AtCSP2 in translational repression of specific mRNAs during cold acclimation and various developmental stages. In addition to a role in translational regulation, YB-1 plays a role in pre-mRNA splicing (Chansky et al., 2001; Stickeler et al., 2001). The Arabidopsis U11/U12-31K protein harboring one CCHC-type zinc finger motif has RNA chaperone activity and is involved in proper U12 intron splicing and plant development (Kim et al., 2010). Since AtCSP2 localizes to the nucleolus and nucleoplasm in addition to cytoplasm (Sasaki et al., 2007), it is also possible to speculate that AtCSP2 plays a role in pre-rRNA processing and pre-mRNA splicing.

Our data revealed that AtCSP2 has regulatory functions in several physiological processes in addition to cold acclimation. Regulatory mechanisms of these processes can be different. Our study with AtCSP3 revealed that AtCSP3 interacts with a variety of RNA-processing proteins within the nucleus and cytoplasm (Kim et al., 2013). Considering the structural conservation, we speculate that AtCSP2 also functions as a versatile modulator of gene expression within a variety of RNA processing bodies. Therefore, it will be important to determine coregulators and downstream factors of AtCSP2 for each phenotype in the future study.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank the Arabidopsis Biological Resource Center and GABI-kat for supplying the T-DNA mutant seeds. This work was supported by grants from the Japan Society for the Promotion of Science (KAKENHI Scientific Research B nos 22380063 and 19380063) and the National Agriculture and Food Research Organization (NARO; Development of Innovative Crops through the Molecular Analysis of Useful Genes, no. 3204; to R.I. and K.S., respectively). We also thank Derek Goto for critical comments on this manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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Fig. S1 AtCSP2 expression levels in atcsp4 mutant background and AtCSP4 expression levels in atcsp2 mutant background.

Fig. S2 Morphology and flowering time of atcsp mutant lines.

Fig. S3 Overexpression of AtCSP2 results in smaller plant size and late flowering.

Fig. S4 Number of stamens and seed morphology in the atcsp2-3 atcsp4-1 mutant.

Table S1 Primers used in this study