Identification of chilling-responsive transcripts in peanut (Arachis hypogaea L.)

¹ Ocean University of China, College of Marine Life Sciences, Qingdao, PR China

² Shandong Peanut Research Institute, Qingdao, PR China

³ Jilin Academy of Agricultural Sciences, Institute of Economic Plants, Gongzhuling, PR China

⁴ Zhengzhou Tobacco Research Institute of CNTC, China Tobacco Gene Research Center, Zhengzhou, PR China

*Corresponding author: chinapeanut@126.com

^§These authors equally contributed to this work.

Financial support: This research was supported by the earmarked fund for Modern Agro-industry Technology Research System (MATRS) Peanut Program (Grant No. nycytx-19), Ministry of Agriculture of China; Qingdao Science & Technology Support Program (Grant No. 10-3-3-20-nsh, Grant No. 09-1-3-67-jch); Shandong Natural Science Foundation (Grant No. Y2008D11) and Shandong Key Project of Science & Technology (Grant No. 2009GG10009008)

Keywords: chilling response, peanut, real-time quantitative PCR, suppression subtractive hybridization.

To isolate differentially expressed peanut genes responsive to chilling, a suppression subtractive hybridization (SSH) cDNA library was constructed for a chilling tolerant peanut cultivar A4 with mRNAs extracted from the seeds imbibed at 2ºC and 15ºC, respectively, for 24 hrs. A total of 466 cDNA clones were sequenced, from which 193 unique transcripts (73 contigs and 120 singlets) were assembled. Of these unique transcripts, 132 (68.4%) were significantly similar to the sequences in GenBank non-redundant (nr) protein database, which belonged to diverse functional categories including metabolism, signal transduction, stress response, cell defense and transcriptional regulation. The remaining 61 (31.6%) showed no similarity to either hypothetical or known proteins. Six differentially expressed transcripts were further confirmed with real-time quantitative PCR (RT-qPCR).

Low temperature (LT) is a major environmental factor limiting plant growth, distribution, and productivity (Boyer, 1982). Different plant species, even individual genotypes of a single species, may respond differently to LT stress. In the long course of evolution, some plant genotypes have developed resistance mechanisms that protect themselves from being severely affected by LT.

Thus far, a large number of genes related to LT stress have been identified. These mainly include, late embryogenesis abundant (LEA) genes, oleosin genes, C-repeat/dehydration-responsive element binding factor (CBFs/DREB1s), NAC, WRKY-type, basic leucine zipper (b-ZIP) and MYB transcription factors (Thomashow, 1999; Shimada et al. 2008; Survila et al. 2010). Dehydrins are probably the best characterized group of LEA protein and have been demonstrated to be responsive to LT in several plant species (Survila et al. 2010). Simultaneous over-expression of two different Arabidopsis dehydrins resulted in enhanced tolerance of transgenic Arabidopsis plants to freezing (Puhakainen et al. 2004). Similarly, a citrus (Citrus unshiu Marcov.) dehydrin, when expressed in tobacco, led to improved chilling tolerance (Hara et al. 2003). Oleosin genes were associated with chilling tolerance in Arabidopsis (Shimada et al. 2008). Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt and freezing stresses (Dubouzet et al. 2003). Over-expression of NAC-coding genes in rice enhanced cold tolerance (Hu et al. 2008). Of 64 WRKY-type transcription factors identified in soybean, 8 were low temperature responsive, and over-expression of GmWRKY21 in Arabidopsis increased tolerance to freezing (Zhou et al. 2008). Over-expression of GmbZIP44, GmbZIP62, GmbZIP78 and GmMYB76 from soybean in Arabidopsis improved freezing tolerance of the plants (Liao et al. 2008a; Liao et al. 2008b).

In contrast to the abundant information on LT stress in other plant species, in peanut (Arachis spp), there are scanty reports in this regard, even though in many portions of the world, LT is a major constraint to peanut production, especially during seeding period. Studies aimed at screening for peanut genotypes with chilling tolerance either at the period of seeding or emergence were conducted and differential responses were noted (Wang et al. 1985; Feng, 1991; Upadhyaya et al. 2001; Upadhyaya et al. 2009). Dave and Mitra (1998) isolated a peanut cold shock protein (AHCSP33) which was secreted into leaf apoplast during low temperature exposure. They also identified a putative Ahlti (A. hypogaea low temperature induced) gene in a leaf cDNA library (Dave and Mitra, 2000). However, to the best of our knowledge, there is no report on genes related to chilling tolerance at the seeding period in peanut.

The objective of the present study is to isolate and characterize chilling responsive transcripts from peanut with chilling tolerance by exploiting a suppression substractive hybridization (SSH) strategy, which is of relevance to the development of peanut cultivars with stabilized high yields.

Plant materials and chilling treatment

The seeds of A4, a chilling tolerant peanut cultivar of Valencia type, were surface sterilized with 75% (v/v) ethanol, rinsed with distilled water and imbibed in a Petri dish. For chilling treatment, the seeds were kept in a growth chamber set at 2ºC for 1, 6 and 24 hrs, respectively. The control seeds were kept in a growth chamber set at 15ºC. The seeds were frozen in liquid nitrogen at appropriate times and stored at -70ºC for further analysis.

Isolation of total RNA and mRNA

Total RNA was isolated from the frozen seeds using RNAprep pure Plant Kit (Tiangen, Beijing, China) with its quantity and quality determined with spectrophotometery and agarose gel electrophoresis and Gelred (Biotium, USA) staining. The poly (A)⁺ RNA was purified from total RNA using Oligotex mRNA Mini Kit (Qiagen, Germany).

SSH library construction

A subtraction cDNA library was constructed using the PCR Select^TM cDNA subtraction kit (Clontech, Mountain View, CA, USA) basically following the manufacturer’s instructions. Two micrograms of poly(A)⁺ RNA were used to synthesize cDNA. The cDNAs of A4 (2ºC, 24 hrs) and A4 (15ºC, 24 hrs) were used as tester and driver, respectively. PCR amplification was conducted using the Advantage 2 Polymerase Mix (Clontech, Mountain View, CA, USA). The subtracted and enriched DNA fragments were purified by QIAquick PCR Purification Kit (Qiagen, Germany). The differential cDNA fragments derived from SSH forward subtractive library were cloned into pGEM-T easy vector (Promega, USA). The positive colonies were identified with colony-PCR method. PCR products were resolved on a 2% agarose gel to confirm the positive colonies and analyze the size of inserts.

Sequencing and sequence analysis

The clones were sequenced in an ABI3700 DNA Sequencer from two ends with SP6 and T7 primers, respectively. Quality read were trimmed with DNAStar (DNASTAR Inc., London, UK) and assembled using CAP3 program (http://deepc2.psi.iastate.edu/aat/cap/cap.html) with default parameters. Each contig or singlet was assumed to represent a unique transcript.

Transcript annotation and functional assignment were done with BLAST2GO (http://blast2go.org) and GenBank nr database. Of the hits with an e-value of < 1.0E-6, the most similar one was considered as the homologue.

RT-qPCR analysis

Total RNA for RT-qPCR analysis was extracted from chilling-treated (1, 6, 24 hrs) and untreated (control) seeds (1, 6, 24 hrs). First-strand cDNA was synthesized using an oligo (dT)18 primer (TaKaRa, Japan) and reverse transcriptase M-MLV (RNase Hˉ) (TaKaRa, Japan).

Primer pairs were designed with the Beacon Designer 7.91 (Table 1) and checked for specificity by aligning with peanut DNA sequences. RT-qPCR was carried out in a Lightcycler 2.0 (Roche, USA) PCR machine. Thermal cycling profile was 95ºC for 30 sec, followed by 45 cycles of 95ºC for 5 sec, 60ºC for 20 sec, and 72ºC for 15 sec. Melting curves were obtained by slow heating from 65 to 95ºC at 0.1ºC/s and continuously monitoring the fluorescence signal. A negative control without a cDNA template was run with each analysis to evaluate the overall specificity. The reaction mixture (20 μl) contained 2 μl of cDNA solution, 10 μl SYBR Premix Ex Taq ^TM(TaKaRa, Japan), and 5 pmol of each primer. The reactions were performed in triplicate with the resultant data averaged. Fold changes of RNA transcripts were calculated with the 2^-ΔΔCt method (Livak and Schmittgen, 2001) using β-actin gene as an internal control.

Construction of SSH cDNA library

After subtraction and transformation, 500 well isolated clones were randomly selected and checked by colony PCR prior to sequencing. Agarose gel electrophoresis showed that the length of cDNA inserts ranged from 100 to 1500 bp (Figure 1). Plasmid DNAs were then extracted from these clones and inserts sequenced, resulting in 466 high-quality reads, which were assembled into 193 unique cDNAs (73 contigs and 120 singlets).

Annotation and functional assignment

Each unique transcript was aligned with the sequences in GenBank nr protein database. As showed in Table 2, 132 transcripts (68.4% of the total) were significantly similar to the sequences in GenBank nr database, while the remaining 61 (31.6%) were unclassified.

The transcripts with known functions involved in metabolism, signal transduction, stress response, cell defence and transcriptional regulation.

The protective mechanisms induced during cold acclimation include alterations in membrane structure, solute biosynthesis and production of protective proteins (Survila et al. 2010). Gene expression regulation under chilling stress is also crucial for plant survival; as it is involved in signal transduction in cold acclimation, transcriptional/post-transcriptional regulation and abscisic acid-dependent cold signal pathway (Survila et al. 2010). To date, a large number of chilling responsive genes have been identified from plants (Survila et al. 2010), which can be divided into two categories. Those genes in category I encode proteins directly relating to the improvement in chilling resistance, for example, the LEA proteins that enhance the ability of resisting freezing injury and cellular dehydration, the membrane proteins that protect enzymes, heat shock proteins (HSPs), proline synthase, chaperones that prevent degeneration of proteins and membranes, FAD8 that increases the content of unsaturated fatty acids and decreases the temperature of phase transition, and the kinase-regulated proteins that stabilize RNAs. Those in category II are involved in the regulation of signal transduction, expression of chilling-resistant genes, and proteins with chilling-resistant activities. Those in category II also include transcription factors such as MYB, bZIP, WRKY, AP2/EREBP, Zinc finger protein and protein kinases. The differential cDNAs obtained in this study almost covered all the chilling tolerance/resistance relating components described above (Table 2).

Verification of differential transcripts

Six transcripts encoding heat shock related proteins, oleosin, LEA, NAC family transcription factor and MYB family transcription factor, respectively, were verified with RT-qPCR. The results showed that these transcripts existed differentially in stressed and control peanut seeds (with overall relative expression ranging from 0.65 to 5.40) (Figure 2). Specifically, relative expression of the 6 genes ranged from 0.65 to 2.23 at 1 hr, 1.06 to 1.62 at 6 hrs, and 2.26 to 5.40 at 24 hrs, respectively, indicating that there was no remarkable difference in the relative expression level of the 6 genes at the earlier stages (1 hr and 6 hrs).

To investigate changes in gene expression in stressed seeds over time, the expression of the 6 genes at 2ºC (1 hr, 6 hrs) relative to that at 24 hrs was analyzed. Gene expression across 1 hr, 6 hrs and 24 hrs as illustrated in Figure 3 exhibited 3 different tendencies: decrease (TS36 and TS54); increase and then decrease (TS110 and TC36); and decrease and then increase (TC50 and TS98). For TS110, TC36, TC50 and TS98, the relative expression level was lower than 2.0, indicating a narrow range of variation in expression; for TS36 and TS54, however, the relative expression level was higher than 2.4, showing that the expression at 1 hrs and 6 hrs was significantly higher than that at 24 hrs. Though the expression of the 6 genes in stressed seeds exhibited a narrow range of fluctuation or remarkably decreased (Figure 3), its relative expression at 24 hrs relative to that in the control (15ºC) was much higher (Figure 2), due to drastic decline in expression of the genes under unstressed condition, possibly suggesting their protective roles against LT injury.

The 6 transcripts were chosen based on their roles in plant chilling tolerance/resistance as described in previous reports (Thomashow, 1999; Zhu et al. 2005; Hu et al. 2008; Shimada et al. 2008; Jan et al. 2009; Duan et al. 2011). In many plants, a large and ubiquitous group of stress influenced genes encoding LEA proteins have been identified, especially under stress conditions such as cold, drought, or high salinity (Thomashow, 1999). HSPs are a class of ubiquitous and highly conserved proteins which show up-regulated expression in response to various environmental stresses, including heat, cold, heavy metal, water deficit, oxidative stress, and wounding (Duan et al. 2011). NAC (NAM, ATAF, and CUC) family transcription factors have been found to play important roles in plant development and responses to environmental stresses (Ohnishi et al. 2005; Hu et al. 2008). Over expression of cold stress-inducible rice SNAC2 in transgenic rice resulted in high cell membrane stability under cold stress. Microarray analysis showed up-regulation of several stress regulated genes in SNAC2-over expressing plants (Hu et al. 2008). These results suggested that several transcriptional networks function during cold acclimation and cold stress in plants. Although NAC genes were isolated from peanut (Shao et al. 2008), thus far no research has been conducted on their relationship to cold-resistance. MYB is also an important transcription factor under the cold stress. Over expression of the cold regulated rice transcription factors MYB4 and OsMYB3R-2 enhanced freezing tolerance in Arabidopsis (Vannini et al. 2004; Zhu et al. 2005). The molecular function of oleosins contributes healthy germination and freezing tolerance to seeds by maintaining nuclear structure (Takashi and Ikuko, 2010).

In this report, a series of chilling responsive genes were identified from peanut, which may provide new insights into the underlying molecular events involved. Nevertheless, the exact function of these genes is still unknown, although the importance of their orthologues to stress responses in other plant species has been well documented. Our future work will focus on the elucidation of the roles played by these genes in peanut using transgenesis. Notably, this study also resulted in some transcripts with unknown functions. It is anticipated that further study on these transcripts may lead to the identification of novel genes involved in chilling tolerance in peanut.

We are most grateful to Dr. Cai Yun Xin and Dr. Xiao Jing Jiang for their valuable comments and suggestions.

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