Isolation and characterization of drought-responsive genes from peanut roots by suppression subtractive hybridization of Biotechnology

Background: Peanut ( Arachis hypogaea L.) is an important economic and oilseed crop. Long-term rainless conditions and seasonal droughts can limit peanut yields and were conducive to preharvest a ﬂ atoxin contamination. To elucidate the molecular mechanisms by which peanut responds and adapts to water limited conditions, we isolated and characterized several drought-induced genes from peanut roots using a suppression subtractive hybridization (SSH) technique. Results: RNAwasextractedfrompeanutrootssubjectedtoawaterstresstreatment(45% ﬁ eldcapacity)andfrom control plants (75% ﬁ eld capacity), and used to generate an SSH cDNA library. A total of 111 non-redundant sequences were obtained, with 80 unique transcripts showing homology to known genes and 31 clones with no similarity to either hypothetical or known proteins. GO and KEGG analyses of these differentially expressed ESTs indicated that drought-related responses in peanut could mainly be attributed to genes involved in cellular structure and metabolism. In addition, we examined the expression patterns of seven differentially expressed candidate genes using real-time reverse transcription-PCR (qRT-PCR) and con ﬁ rmed that all were up-regulated in roots in response to drought stress, but to differing extents. Conclusions: We successfully constructed an SSH cDNA library in peanut roots and identi ﬁ ed several drought-related genes. Our results serve as a foundation for future studies into the elucidation of the drought stress response mechanisms of peanut.


Introduction
Peanut (Arachis hypogaea L.) is an important economic and oilseed crop, which is mainly grown under rain-fed conditions in arid and semi-arid regions. Consequently, drought is a major production constraint since rainfall is generally both erratic and inadequate [1,2]. Hence, improving the drought tolerance of peanut is a key objective. Genetic engineering is one approach that could be used, but requires prior information about drought stress-related genes in peanut. However, the molecular mechanisms by which peanut adapts to water stress are not well described. The peanut genome is very large in comparison to other plant species, making it difficult to study. Thus, a detailed understanding of peanut water stress tolerance would be highly informative and, moreover, the altered expression of key genes may enhance peanut drought tolerance.
Studies into the mechanisms of peanut drought resistance have previously focused on aboveground plant tissues. For instance, nearly 700 genes were identified as being enriched in a subtractive cDNA library generated from peanut leaves exposed to a gradual drought stress treatment [3]; and a proteomic analysis of the water-deficit stress response in three contrasting peanut genotypes implicated a variety of stress response mechanisms as being active in peanut [4]. Dang et al. [5] analyzed the gene expression of twelve transcription factors from two drought tolerant peanut genotypes under drought conditions and identified the expression patterns of drought-inducible transcripts.
As the major interface between the plant and the various biotic and abiotic factors in the soil environment, root tissues may produce root-to-shoot chemical signals that regulate stomatal closure and thus reduce transpiration [6,7]. However, there is currently limited information on the root responses of peanut under water deficit conditions, particularly at the molecular level. Suppression subtractive hybridization (SSH) is a powerful technique for the identification of differentially expressed genes and for the enrichment of genes with low expression levels [8]. There are several examples in the literature where the SSH approach has been successfully employed to screen for candidate genes, including the identification of chilling-responsive transcripts in peanut [9], and the isolation of a submergence-induced gene, OsGGT (glycogenin glucosyltransferase) in rice [10]. Hence, we utilized an SSH strategy to isolate and characterize drought-induced transcripts from peanut roots. A better understanding of the key genes involved in peanut stress response is vital for the development of plants that can maintain high yields under drought conditions, and the cultivation of drought-resistant peanut varieties.

Plant growth and drought stress treatment
A. hypogaea cv Huayu 25 were used in this study. Plants were grown in a growth chamber at 28°C/18°C (day/night), and 300 μmoL m -2 s -1 light intensity provided by reflector sunlight dysprosium lamps (DDF 400, Nanjing, China). The water stress treatment was as described by Govind et al. [3]. The amount of water held by the soil is expressed as a mass percentage, and it is considered as 100% field capacity (FC) of soil. Three different water treatments were considered in this study: 75%, 45% and 20% FC with 75% FC serving as the control treatment. Plants were held at one of the three different water treatments (75%, 45% and 20% FC) for the plants planted at 75% FC for 25 d after sowing. The water stress treatment was maintained for a total of 5 d and was monitored gravimetrically by weighing the pots twice daily. The fresh roots, first nodal leaves and the first main stem were harvested at the end of the stress period from three treated plants for RNA isolation. The second fully expanded leaves were harvested for the measurement of leaf relative water content (RWC). The RWC was calculated as described by Barrs   Leaf relative water content (%) Fig. 1. (a) Phenotype of peanut plants exposed to different levels of water deficit. Leaf rolling and leaf thinning were observed in drought stressed plants but not in control plants; (b) changes in RWC of peanut leaves subjected to different water deficit treatments for 5 d. RWC was measured in the upper fully expanded leaves. Bars represent mean ± SD of three samples.

Construction of an SSH cDNA library
A subtractive cDNA library was constructed using the PCR Select™ cDNA subtraction kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. The 45% FC root cDNA was used as the tester and the 75% FC root cDNA as the driver for SSH. The digested cDNA were ligated to adapters 1 and 2R supplied with the PCR-Select cDNA Subtraction Kit. After two rounds of hybridization and PCR amplification, the differentially expressed cDNAs were normalized and enriched. The subtracted and enriched DNA fragments were purified by QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The PCR products were ligated to pGEM-T Easy vector (Promega Co., USA) and transformed into DH5α cells using heat shock. Transformants were isolated from white colonies on X-gal/isopropyl-beta-D-thio-galatopyranoside agar plates. Positive colonies were identified by colony PCR. PCR products were separated on a 2% agarose gel to detect the amplification quality and quantity.

Sequencing and sequence analysis
The clones were sequenced by Sangon (Shanghai, China). The vector and adaptor sequences were removed using the DNAman software, and masked repeats, rRNA and low complicity sequences were eliminated using RepeatMasker. The sequences were searched against the NCBI database using BLASTN and BLASTX. Transcript annotation and functional assignment were performed using Blast2GO (http://blast2go.org).

Quantitative real time PCR analysis (qRT-PCR)
Total RNA for qRT-PCR analysis was treated with recombinant RNase-free DNaseI (Takara, Toyoto, Japan) to remove any contaminating genomic DNA. First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, USA). Primer pairs were designed using the Primer 5.0 software (Table 1). ACT11 was used as a reference gene for the normalization of all data [12]. qRT-PCR was carried out in a Lightcycler 2.0 PCR machine (Roche, USA) based on SYBR Premix Ex Taq polymerase (Takara, Toyoto, Japan). The thermal protocol consisted of 95°C for 30 s, then 40 cycles of amplification at 95°C for 5 s, 60°C for 20 s, and 72°C for 15 s. Melting curves were obtained by slow heating from 65°C to 95°C at 0.1°C/s and continuous monitoring of the fluorescence signal. The reactions were performed in 20 μL volumes containing 2 μL of cDNA solution, 10 μL 2 × SYBR Premix and 0.4 μL (10 μM) of each primer. Each experiment was replicated three times. The comparative Ct method was applied.

Performance of peanut under drought stress
Huayu 25 has been identified as a peanut variety with strong drought tolerance. An obvious difference in phenotype was observed between plants subjected to drought stress and well-watered plants (Fig. 1a). Visible symptoms such as leaf rolling and leaf thinning were seen in the plants subjected to drought stress, and the leaves of the control plants were greener than those of the stressed plants. The  (Fig. 1b).

Construction of an SSH cDNA library
A differential expression cDNA library of peanut roots was constructed utilizing Clontech PCR Select Subtraction Kit. After subtraction and transformation, the blue-white spot screening showed that approximately 95% of transformants contained an insert. A total of 576 clones were randomly selected prior to sequencing and were shown to have an insert size of approximately 200-1000 bp. Sequencing of positive clones yielded a total of 360 EST sequences.
Thus, we successfully constructed a putative drought-stress specific subtracted cDNA library from peanut roots.

Analysis of differentially expressed ESTs
After the removal of vector and adaptor sequences and elimination of masked repeats, rRNA and low complicity sequences, 111 non-redundant sequences were obtained. Based on homology searches to the NCBI database, 80 clones (72.07%) were homologous to known genes and 31 clones were homologous to genes with unknown function or had no matches in the NCBI database (Table 2). For functional annotation, Blast2GO was used to classify the ESTs into three principal GO categories: cellular location, molecular function and biological process.   (24) was obtained for 'cell', followed by 'membrane' (20) (Fig.  2a). Within the category of biological process, 36 ESTs (80%) were assigned to 'metabolic process' and 34 (75.6%) to 'cellular process', which accounted for the majority of the annotated sequences (Fig.  2b). Within the molecular function category, the GO terms with the highest number of ESTs were 'catalytic activity' and 'binding', with 31 and 24 ESTs, respectively (Fig. 2c). Hence, the GO analysis suggested that drought responses in peanut were mainly related to genes involved in cellular structure and metabolism.
The expression patterns of the selected SSH clones in peanut roots, leaves and stems under water stress conditions (45% and 20% FC) were analyzed by qRT-PCR. Amongst the seven ESTs, GolS showed the greatest degree of up-regulation, with the largest increase in expression levels relative to the control observed in the stems under 20% FC conditions (1290 fold-change). The expression pattern of STPK differed in the roots, leaves and stems. In roots subjected to drought stress, the STPK transcript level increased approximately five-fold under 45% FC conditions and 11-fold under 20% FC conditions (Table 3). However, in leaves, STPK levels decreased significantly in the 45% FC treatment but showed no obvious change in the 20% FC conditions. In stems, STPK levels increased approximately two-fold following drought stress. The MnSOD gene showed no obvious expression changes in peanut roots and leaves under 45% FC water treatment, but increased between four-and nine-fold in the 20% FC water treatment ( Table 3). The expression of P5CS in peanut roots and leaves increased with the degree of drought stress, with the highest expression level observed in stems at 45% FC treatment. The remaining three clones (Gsi-83, ANN and ADH) showed a similar pattern of expression in all tissues, with a small increase in the 45% FC treatment and the greatest expression level at 20% FC treatment (Table 3).

Discussion
Drought stress cDNA libraries have previously been constructed for peanut, but these correspond to genes expressed in drought stressed leaves [3] or in immature pods [13,14]. Hence, there is limited molecular information on the root responses of peanut subjected to drought stress conditions. In this study, a total of 111 differentially expressed, non-redundant ESTs were identified in the subtractive cDNA library. Of these 111 ESTs, 80 had significant homology to known genes, many of which are associated with drought stress responses previously reported in soybean and chickpea. Some genes, such as those encoding lea3, lea4 and metallothionein-like protein had confirmed involvement in drought stress in peanut [15,16]. This suggests that we have successfully constructed an SSH cDNA library and have identified drought-stress responsive genes in peanut roots.
We selected seven ESTs for qRT-PCR analysis in drought-stressed and control peanut roots, leaves and stems. The expressions of ANN, ADH and MnSOD were increased in response to drought stress, especially under the 20% FC condition. These three genes are reported to be involved in water stress responses in other plant species [17,18,19,20]. Our study confirms that these genes are also involved in the drought tolerance mechanism of peanut. Protein kinases are widely detected in living organisms and play important roles in signal perception and transduction in cells. Under environment stress conditions, protein kinases perceive and transmit various signals, and activate transcription factors to regulate the expression of downstream genes [21,22]. The expression patterns of STPK differed in the roots, leaves and stems, exhibiting rapid induction in roots under drought stress, but down-regulation in leaves at 45% FC conditions. The expression pattern of this particular protein kinase indicates that its role in the regulation of drought stress response is complex and requires further study.
Some studies have shown that under drought stress conditions, plants can improve their drought tolerance by adjusting the levels of osmoprotectants such as proline [23], galactinol [24] and glycinebetaine [25]. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS is a key regulatory enzyme that plays a crucial role in proline biosynthesis [26]. Raffinose and galactinol are involved in tolerance to drought, high salinity and cold stress. Stress-inducible GolS plays a key role in the accumulation of galactinol and raffinose under abiotic stress conditions [24]. In this study, the mRNA levels of P5CS and GolS in the control leaves and stems were significantly reduced in comparison to roots (data not shown). Furthermore, the expression of P5CS was significantly increased in all three tissues under drought stress, suggesting that proline accumulation in peanut may form a key defense mechanism against drought stress. The up-regulation of GolS under 20% FC conditions was 9-fold, 4.5-fold and 53.8-fold greater than that of P5CS in roots, leaves and stems, respectively. This indicates that, in peanut, the osmotic adjustment ability of soluble sucrose is greater than that of proline under drought stress conditions, which is consistent with our previous report [27].
In addition, some of the genes induced under drought stress were found to be associated with other environmental stresses, such as salt, cold and high temperature stress [28,29]. We identified an EST homologous to nitrate transporter 1.1, and a cold stress responsive protein whose expression was marginally increased in peanut under drought stress conditions. This suggests that some genes respond to both drought stress and other abiotic stresses, and thus implies that similar stress tolerance mechanisms and pathways may exist. The gene expression levels analyzed in this study indicate that the response to drought is a very complex physiological and biochemical process involving multiple metabolism pathways.

Conclusions
We successfully constructed an SSH cDNA library from peanut roots and identified several transcripts encoding proteins with drought-related functions. These proteins were located in different cellular compartments and were involved in various molecular functions and biological processes during normal and water stress conditions in peanut. Our study contributes to a better understanding of the molecular mechanisms of water-stress tolerance in peanut and would facilitate the genetic manipulation of drought-stress resistance in this species.
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