Cold-induced Genes Research Articles

Application of data analysis in cold stress: a case study of Nicotiana benthamiana

Cold stress is a major environmental factor in plant life cycles. Nicotiana benthamiana, which belongs to the family Solanaceae, is one of the most commonly used model species in plant-microbe interaction studies. In total, 5205 differentially expressed genes were identified under cold stress in N. Benthamiana. Of these, 5029 were upregulated and 176 were downregulated within four time periods (4 h, 12 h, 24 h, and 48 h). The common up- and downregulated genes were identified as 692 and 6, respectively. The functional annotations of these genes were studied and these common genes involved in protein, RNA, miscellaneous enzyme families, signaling, stress, lipid, and carbohydrate metabolisms were enriched by using MapMan ontology. In addition, a total of 22 cold-inducible transcription factors were enriched, including subsets of the zinc finger family, bHLH, E2F/DP, bZIP, SET domain, GRAS, MYB, ARF, CO-like, Homeobox, and DOF zinc finger family members. Our findings will pave the way for understanding the expression of cold-inducible genes as a response to cold stress in Nicotiana species. This study will also be a valuable resource for crop improvement studies under abiotic stress conditions for Nicotiana plants.

Overexpression of ShDHN, a dehydrin gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses in tomato

Dehydrins (DHNs) play important roles in plant adaptation to abiotic stress. In this study, a cold-induced SK3-type DHN gene (ShDHN) isolated from wild tomato species Solanum habrochaites was characterized for its function in abiotic stress tolerance. ShDHN was constitutively expressed in root, leaf, stem, flower and fruit. ShDHN was continuously up-regulated during cold stress and showed higher expression level in the cold-tolerant S. habrochaites than in the susceptible S. lycopersicum. Moreover, ShDHN expression was also regulated by drought, salt, osmotic stress, and exogenous signaling molecules. Overexpression of ShDHN in cultivated tomato increased tolerance to cold and drought stresses and improved seedling growth under salt and osmotic stresses. Compared with the wild-type, the transgenic plants accumulated more proline, maintained higher enzymatic activities of superoxide dismutase and catalase, and suffered less membrane damage under cold and drought stresses. Moreover, the transgenic plants accumulated lower levels of H2O2 and O2− under cold stress, and had higher relative water contents and lower water loss rates under dehydration conditions. Furthermore, overexpression of ShDHN in tomato led to the up- or down-regulated expression of several genes involved in ROS scavenging and JA signaling pathway, including SOD1, GST, POD, LOX, PR1 and PR2. Taken together, these results indicate that ShDHN has pleiotropic effects on improving plant adaptation to abiotic stresses and that it possesses potential usefulness in genetic improvement of stress tolerance in tomato.

Fine mapping of the qLOP2 and qPSR2-1 loci associated with chilling stress tolerance of wild rice seedlings.

Using leaf osmotic potential and plant survival rate as chilling-tolerant trait indices, we identified two major quantitative trait loci qLOP2 and qPSR2 - 1 (39.3-kb region) and Os02g0677300 as the cold-inducible gene for these loci. Chilling stress tolerance (CST) at the seedling stage is an important trait affecting rice production in temperate climate and high-altitude areas. To identify quantitative trait loci (QTLs) associated with CST, a mapping population consisting of 151 BC(2)F(1) plants was constructed by using chilling-tolerant Dongxiang wild rice (Oryza rufipogon Griff.) as a donor parent and chilling-sensitive indica as a recurrent parent. With leaf osmotic potential (LOP) and plant survival rate (PSR) as chilling-tolerant trait indexes, two major QTLs, qLOP2 (LOD = 3.8) and qPSR2-1 (LOD = 3.3), were detected on the long arm of chromosome 2 by composite interval mapping method in QTL Cartographer software, which explained 10.1 and 12.3% of the phenotypic variation, respectively. In R/QTL analyzed result, their major effects were also confirmed. Using molecular marker RM318 and RM106, qLOP2 and qPSR2-1 have been introgressed into chilling-sensitive varieties (93-11 and Yuefeng) by marker-assisted selection procedure (MAS), which resulted in 16 BC(5)F(3) BILs that chilling tolerance have significantly enhanced compare with wild-type parents (P < 0.01). Therefore, two large segregating populations of 11,326 BC(4)F(2) and 8,642 BC(4)F(3) were developed to fine mapping of qLOP2 and qPSR2-1. Lastly, they were dissected to a 39.3-kb candidate region between marker RM221 and RS8. Expression and sequence analysis results indicated that Os02g0677300 was a cold-inducible gene for these loci. Our study provides novel alleles for improving rice CST by MAS and contributes to the understanding of its molecular mechanisms.

Transcriptional fingerprinting of “browning” white fat identifies NRG4 as a novel adipokine

Brown adipocytes help to maintain body temperature by the expression of a unique set of genes that facilitate cellular metabolic events including uncoupling protein 1-dependent thermogenesis. The dissipation of energy in brown adipose tissue (BAT) is in stark contrast to white adipose tissue (WAT) which is the body's primary site of energy storage. However, adipose tissue is highly dynamic and upon cold exposure profound changes occur in WAT resulting in a BAT-like phenotype due to the presence of brown-in-white (BRITE) adipocytes. In our recent report, transcription profiling was used to identify the gene expression changes that underlie the browning process as well as the intrinsic differences between BAT and WAT. Neuregulin 4 was categorized as a cold-induced BAT gene encoding an adipokine that signals between adipocytes and nerve cells and likely to have a role in increasing adipose tissue innervation in response to cold.

Ultrastructural changes and dynamic expressions of FAD7, Cu/Zn-SOD, and Mn-SOD in Neosinocalamus affinis under cold stress

As one of the fast-growing species, bamboo plays an important role in ecological stability and wood processing industry. However, low temperature limitation is the basic problem for the cultivation and introduction of bamboo. In this study, the symptoms of cold stress influence on the native bamboo (Neosinocalamus affinis (Rendle) Keng f.) and hybrid bamboo (Bambusa pervariabilis × Dendrocalamopsis grandis) were observed under transmission electron microscope, and the dynamic responses of FAD7, Cu/Zn-SOD, and Mn-SOD genes to cold stress were identified in bamboo by real-time quantitative RT-PCR. Observation by electron microscopy indicated that bamboo is one of the most chilling-sensitive species with severe ultrastructural injury induced by chilling, but the native bamboo (N. affinis) is more cold-tolerant compared with the hybrid bamboo. Results obtained by real-time quantitative RT-PCR analysis revealed that FAD7, Cu/Zn-SOD, and Mn-SOD were all cold-inducible genes in N. affinis. In addition, dynamic response patterns of N. affinis Cu/Zn-SOD and Mn-SOD under cold stress were similar. This work is a fundamental research of hardiness physiology of bamboo and may contribute to the breeding program on obtaining transgenic bamboo species.

Ectopic expression of Arabidopsis RCI2A gene contributes to cold tolerance in tomato.

Cold is a major stress that limits the quality and productivity of economically important crops such as tomato (Solanum lycopersicum L.). Generating a cold-stress-tolerant tomato by expressing cold-inducible genes would increase agricultural strategies. Rare cold-inducible 2a (RCI2A) is expressed in Arabidopsis, but its molecular function during cold stress is not fully understood. Here we ectopically expressed Arabidopsis RCI2A in transgenic tomato to evaluate tolerance to cold stress without altering agronomic traits. Biochemical and physiological study demonstrated that expression of RCI2A in transgenic tomato enhanced the activity of peroxidase and ascorbate peroxidase (APX) and reduced the accumulation of H2O2, alleviated lipid peroxidation, increased the accumulation of chlorophyll, reduced chilling-induced membrane damage, retained relative water content and enhanced cold tolerance. A motif search revealed that the motifs of photosystem II (PSII) phosphoproteins PsbJ and PsbH and reaction-center proteins PsbL and PsbK were common to cold-inducible RCI2A and peroxidase proteins RCI3A, tomato peroxidase (TPX1), TPX2, tomato ascorbate peroxidase (APX1), and horseradish peroxidase (HRP-c). In addition to membrane protection, RCI2A may cross talk with PSII-associated proteins or peroxidase family enzymes in response to cold stress. Our findings may strengthen the understanding of the molecular function of RCI2A in cold-stress tolerance. RCI2A could be used to improve abiotic stress tolerance in agronomic crops.

Identification of a cold-inducible gene encoding calmodulin-binding protein from Eucalyptus dunnii through suppression subtractive hybridization

Low temperature is the main limiting factor for cultivation expansion, fast growth, and high yield of Eucalyptus species. To investigate the mechanism of their cold tolerance, a cDNA subtraction library representing the cold-induced genes of Eucalyptus dunnii was constructed using suppression subtractive hybridization (SSH) technique. A gene encoding a calmodulin-binding protein (CaMBP) was identified from the SSH library, and the expression pattern of CaMBP under cold stress was further evaluated through reverse transcription — quantitative polymerase chain reaction (RT-qPCR). The expressions of EguCBF1a and EdCaMBP increased already after 4 h of the cold stress, supporting the idea that the CaMBP gene may have a function in the survival of Eucalyptus during winter. A full-length mRNA sequence of 1 808 bp was obtained via a rapid amplification of cDNA ends method, and the sequence was subsequently deposited to GenBank (accession No. JX401571). The CaMBP cDNA of E. dunnii contained a single open reading frame of 1 362 bp, a 5t’ untranslated region of 175 bp, and a 3t’ untranslated region of 268 bp. Multiple sequence alignment and phylogenetic analysis indicated that CaMBP of E. dunnii shared 56 to 84 % identities with the CaMBPs of other plants and was similar to that of Ricinus communis, Medicago truncatula, and Gossypium hirsutum.

ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling.

Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize.

The Arabidopsis 14-3-3 protein RARE COLD INDUCIBLE 1A links low-temperature response and ethylene biosynthesis to regulate freezing tolerance and cold acclimation.

In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis.

Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco

The ERF1 gene was well characterized in Arabidopsis to be responsive to salt, drought, heat and necrotrophic fungi. In this paper, an ortholog of ERF1, designated as CsERF that was isolated originally as an ethylene induced gene from Citrus sinensis (L.) Osbeck, was characterized. Sequence analysis revealed that CsERF and ERF1 belonged to the same AP2/ERF superfamily, or more specifically, the same IXc subgroup of the ERF subfamily. Transient expression experiment revealed that CsERF protein was localized in nuclei. Quantitative real-time PCR results showed that the expression of CsERF was induced not only by various stresses including salt, dehydration and cold, but also by stress-related hormones, such as ethylene, jasmonic acid and abscissic acid, with ethylene and cold as the two most potent inducers. Its ethylene induction could be blocked by 1-MCP, indicating ethylene signaling pathway was required for its activation. Overexpressing CsERF conferred tobacco plants with much stronger tolerance to chilling stress, and activated constitutively four indicator genes: two cold responsive transcription factor genes, NtCBF1 and NtCBF3, and two cold-induced genes, NtERD10B and NtERD10C. Under cold treatment, proline content and superoxide dismutase activity increased more rapidly and were much higher in overexpression lines than in wild type. These data suggest that CsERF plays a role in cold response in addition to the roles shared with its ortholog ERF1.

Potato plants ectopically expressing Arabidopsis thaliana CBF3 exhibit enhanced tolerance to high-temperature stress.

CBF3, a known cold-inducible gene that encodes a transcription factor, was isolated from Arabidopsis thaliana and introduced into the potato (Solanum tuberosum cv. 'luyin NO.1') under the control of the CaMV35S promoter or the rd29A promoter. Our results revealed that temperature of 40 °C or higher can significantly induce AtCBF3 expression. After heat stress, the net photosynthetic rate (Pn ), the maximal photochemical efficiency of photosystem II (PSII) (Fv /Fm ) and the accumulation of the D1 protein were higher in the transgenic lines than in the wild-type (WT) line. Moreover, compared with the WT line, O2 (●-) and H2 O2 accumulation in the transgenic lines were reduced. A Q-PCR assay of a subset of the genes involved in photosynthesis and antioxidant defence further verified the above results. Interestingly, under heat stress conditions, the accumulation of heat-shock protein 70 (HSP70) increased in the WT line but decreased in the transgenic lines. These results suggest that potato plants ectopically expressing AtCBF3 exhibited enhanced tolerance to high temperature, which is associated with improved photosynthesis and antioxidant defence via induction of the expression of many stress-inducible genes. However, this mechanism may not depend upon the regulatory pathways in which HSP70 is involved.

A DEAD-box RNA helicase produces two forms of transcript that differentially respond to cold stress in a cryophyte (Chorispora bungeana).

This work demonstrated that a cold-induced DEAD-box RNA helicase, CbDRH, is also post-transcriptionally regulated upon cold stress, and it interacts with a cold-responsive, glycine-rich, RNA-binding protein, CbGRP. Chorispora bungeana (C. bungeana) is a representative alpine subnival plant species that shows strong tolerance to multiple abiotic stresses, especially cold stress. DEAD-box RNA helicases are implicated in almost all RNA metabolic processes and participate in multiple abiotic stress responses. Here, we characterized a cold-induced DEAD-box RNA helicase gene from C. bungeana. We cloned the full-length cDNA of the gene by RACE and called it C. bungeana DEAD-box RNA Helicase (CbDRH). Structurally, CbDRH possesses all nine conserved motifs characteristic of DEAD-box protein family members in its central region, and the N- and C- terminal extensions both harbor a glycine-rich region containing several RGG-box motifs. The CbDRH gene produces two forms of transcripts, CbDRH.2 and CbDRH.1, by alternative splicing. CbDRH.2 comes from the complete excision of all the nine introns, while CbDRH.1 results from the use of an alternative 5' splice site in the eighth intron, retaining part of the intron (the first 260 bp) with an early stop codon. Semi-quantitative RT-PCR analysis showed that CbDRH.2, but not CbDRH.1, is up-regulated by cold stress. However, the abundance of CbDRH.1 transcript can be elevated by cycloheximide (an inhibitor of nonsense-mediated decay) treatment, indicating that CbDRH.1 is targeted to nonsense-mediated decay (NMD). A subcellular localization analysis showed that CbDRH.2 protein is located in the nuclei. Further investigation suggested that CbDRH.2 can interact with a cold-responsive, glycine-rich, RNA-binding protein, CbGRP (Chorispora bungeana glycine-rich, RNA-binding protein). These data suggest that the cold-induced CbDRH is also post-transcriptionally regulated under cold stress and that CbDRH.2 may function together with the glycine-rich, RNA-binding protein, CbGRP, in the cold stress response.

Membrane rigidification functions upstream of the MEKK1-MKK2-MPK4 cascade during cold acclimation in Arabidopsis thaliana.

The MEKK1-MKK2-MPK4 cascade is activated during cold acclimation. However, little is known regarding the perception of low temperature. In this study, we demonstrate that treatment of Arabidopsis with a membrane rigidifier, DMSO, caused MPK4 activation concomitantly with MEKK1 and MKK2 phosphorylation, as well as the cold-inducible gene COR15a expression. These processes are similar to the effects of cold treatment, whereas benzyl alcohol (BA), a membrane fluidizer, prevented such cold-induced events. Moreover, the DMSO-treated seedlings acquired freezing tolerance without cold acclimation. In contrast, the BA-pretreated seedlings did not show freezing tolerance. These results suggest that membrane rigidification activates this MAPK cascade and contributes to the acquisition of freezing tolerance.

Transgenic tobacco plants expressing PicW gene from Picea wilsonii exhibit enhanced freezing tolerance

A cold-induced gene of 669 bp in length without introns, PicW, was cloned from Picea. wilsonii, a cold tolerant conifer species. Sequence analysis showed that it was a member of the dehydrin family because of its conserved amino acid constitution and protein secondary structure. The protein was rich in hydrophilic amino acids such as alanine, lysine, glutamic acid, glutamine and threonine, but devoid of two hydrophobic amino acids, cysteine and tryptophan. The PicW gene contained five repeated motifs homologous to the core K-segment in dehydrins. Protein secondary structure prediction showed that PicW comprised 29 % α-helix, mostly in the K-homologous segment, and random coils. The PicW gene was cloned into the expression vector PEZR(K)-LC under the 35S promoter and transformed into tobacco plants. After treatment at −5 °C for 3 h, all of the tobacco plants were wilting. However, the transgenic plants showed better growth performance than wild-type plants. Further tests of physiological indexes including relative electrolyte leakage and malondialdehyde content, proline and soluble sugar content also revealed significant differences between the wild-type and transgenic tobacco plants. It was concluded that the PicW gene could be an important gene resource for freezing-tolerant plant breeding.

Cold-induced gene expression and ω3 fatty acid unsaturation is controlled by red light in Synechocystis

The expression of cold-induced genes, which are controlled by the cold sensor histidine kinase Hik33, and the formation of ω3 polyunsaturated fatty acids are controlled by light in the cyanobacterium Synechocystis sp. PCC 6803. Cold-induced Hik33-dependent gene expression is initiated by red light (∼700nm), but not by blue or green light. Red light also turns on the ω3 fatty acid desaturation. Different combinations of other wavelengths in red spectral region (635 and 726nm) had no effect on the red-light-activated cold-induced transcription or fatty acid desaturation. Therefore, the involvement of phytochrome-like photoreceptor(s), similar to phytochromes of higher plants, in this regulation was not confirmed. The absence of light-dependence of gene expression in the mutant cells deficient in Hik33 suggests the involvement of this histidine kinase in direct or mediated with red light regulation of cold responses in Synechocystis.

Comparative analysis of gene expression under cold acclimation, deacclimation and reacclimation in Arabidopsis

Cold acclimated plants show an elevated tolerance against subsequent cold stress. Such adaptation requires alterations in gene expression as well as physiological changes. We were interested in gene expression changes at the transcriptional level during adaptation processes. The patterns of transcriptional changes associated with cold acclimation, deacclimation and reacclimation in Arabidopsis leaves were characterized using the Coldstresschip. Gene expression profiles were further analyzed by 'coexpressed gene sets' using gene set enrichment analysis (GSEA). Genes involved in signal transduction through calcium, and cascades of kinases and transcription factor genes, were distinctively induced in the early response of cold acclimation. On the other hand, genes involved in antioxidation, cell wall biogenesis and sterol synthesis were upregulated in the late response of cold acclimation. After the removal of cold, the expression patterns of most genes rapidly returned to the original states. However, photosynthetic light-harvesting complex genes and lipid metabolism-related genes stayed upregulated in cold deacclimated plants compared to non-treated plants. It is also notable that many well-known cold-inducible genes are slightly induced in reacclimation and their expression remains at relatively low levels in cold reacclimation compared to the expression during the first cold acclimation. The results in this study show the dynamic nature of gene expression occurring during cold acclimation, deacclimation and reacclimation. Our results suggest that there is a memory of cold stress and that the 'memory of cold stress' is possibly due to elevated photosynthetic efficiency, modified lipid metabolism, increased calcium signaling, pre-existing defense protein made during first cold acclimation and/or modified signal transduction from pre-existing defense protein.

Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a “brite” transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with “browning,” as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

Evaluation of Reference Genes for Accurate Normalization of Gene Expression for Real Time-Quantitative PCR in Pyrus pyrifolia Using Different Tissue Samples and Seasonal Conditions

We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as β-Tubulin, Histone H3, Actin, Elongation factor-1α, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ΔCt method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ΔCt method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ΔCt method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out reliable reference genes.

Tomato Inducer of CBF Expression 1 (SlICE1) is Involved in Cold and Salt Stress Signaling

Aims: Inducer of CBF Expression 1 (ICE1), which is one of basic Helix-Loop-Helix type transcription factors, has important roles in regulation of cold stress-indiced genes of plants. Sample: To investigate functions of tomato ICE1 in cold and salt tolerance, c index (SI), immunochemical assay of endogenous ICE1 protein and RT-PCR of coldinducible genes were conducted with tomato plants. Methodology: Tomato plants grown for 4 weeks were subjected to cold (4oC) and salt (0.2 M NaCl) in the presence or the absence of cell signaling inhibitors. An antibody was raised against ICE1 specific epitoipe. Immunoblot with the anti-ICE1 antibody was carried out with extractions of tomato plants treated by cold and salt stresses. The expression profiles of tomato ICE1 (SlICE1) and other cold-inducible genes including LeCBF1/2/3 and SlTPS1 were analyzed by semi-quantitative RT-PCR. Results: An ICE1-related protein with molecular masses of approximately 55 is induced in tomato plant under chilling and salt stresses. The expression of a tomato ICE1 gene (SlICE1) under chilling stress was maintained at a constant level in contrast to the protein Original Research Article American Journal of Experimental Agriculture, 4(7): 785-796, 2014 786 level. Chilling stress sequentially upregulated tomato CBF homolog, SlCBF1 and trehalose-6-phosphate synthase (SlTPS1). Based on the whole genome database of tomato, cis-elements potentially binding to ICE1 and CBF were located in upstream sequences in promoter regions of SlCBF1 and SlTPS1, respectively. Conclusion: Tomato ICE1 homolog mediates the expression of SlCBF1in a cold-stressinduced transcription factor cascade via binding to ICE1-specific cis-elements, leading to induction of cold tolerance by trehalose synthesis.

De novo characterization of the Anthuriumtranscriptome and analysis of its digital gene expression under cold stress

BackgroundAnthurium andraeanum is one of the most popular tropical flowers. In temperate and cold zones, a much greater risk of cold stress occurs in the supply of Anthurium plants. Unlike the freeze-tolerant model plants, Anthurium plants are particularly sensitive to low temperatures. Improvement of chilling tolerance in Anthurium may significantly increase its production and extend its shelf-life. To date, no previous genomic information has been reported in Anthurium plants.ResultsUsing Illumina sequencing technology, we generated over two billion base of high-quality sequence in Anthurium, and demonstrated de novo assembly and annotation of genes without prior genome information. These reads were assembled into 44,382 unigenes (mean length = 560 bp). Based on similarity search with known protein in the non-redundant (nr) protein database, 27396 unigenes (62%) were functionally annotated with a cut-off E-value of 10-5. Further, DGE tags were mapped to the assembled transcriptome for gene expression analysis under cold stress. In total, 4363 differentially expressed genes were identified. Among these genes, 292, 805 and 708 genes were up-regulated after 1-h, 5-h and 24-h cold treatment, respectively. Then we mapped these cold-induced genes to the KEGG database. Specific enrichment was observed in photosynthesis pathway, metabolic pathways and oxidative phosphorylation pathway in 1-h cold-treated plants. After a 5-h cold treatment, the metabolic pathways and oxidative phosphorylation pathway were significantly identified as the top two pathways. After 24-h cold treatment, mRNA surveillance pathway, RNA transport pathway and plant-pathogen interaction pathway were significantly enriched. Together, a total of 39 cold-inducible transcription factors were identified, including subsets of AP2/ERF, Zinc figure, NAC, MYB and bZIP family members.ConclusionOur study is the first to provide the transcriptome sequence resource for Anthurium plants, and demonstrate its digital gene expression profiling under cold conditions using the assembled transcriptome data for reference. These data provides a valuable resource for genetic and genomic studies under abiotic conditions for Anthurium plants.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-827) contains supplementary material, which is available to authorized users.