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Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

Isolation and functional characterization of cold-regulated promoters, by digitally identifying... Background: Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset. Results: Digital expression analyses of EST datasets, revealed 164 cold-induced peach genes, several of which show similarities to genes associated with cold acclimation and cold stress responses. The promoters of three of these cold-inducible genes (Ppbec1, Ppxero2 and Pptha1) were fused to the GUS reporter gene and characterized for cold-inducibility using both transient transformation assays in peach fruits (in fruta) and stable transformation in Arabidopsis thaliana. These assays demonstrate that the promoter Pptha1 is not cold-inducible, whereas the Ppbec1 and Ppxero2 promoter constructs are cold-inducible. Conclusion: This work demonstrates that during cold storage, peach fruits differentially express genes that are associated with cold acclimation. Functional characterization of these promoters in transient transformation assays in fruta as well as stable transformation in Arabidopsis, demonstrate that the isolated Ppbec1 and Ppxero2 promoters are cold-inducible promoters, whereas the isolated Pptha1 promoter is not cold-inducible. Additionally, the cold-inducible activity of the Ppbec1 and Ppxero2 promoters suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. These results reveal that digital expression analyses may be used in non-model species to identify candidate genes whose promoters are differentially expressed in response to exogenous stimuli. Page 1 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 demonstrate that Arabidopsis may be used as a heterolo- Background Cold temperature is an environmental factor that plays an gous system to test the functionality of promoters. How- important role in plant growth and development. Tem- ever, this type of heterologous regulation may not exist for perate plants have developed mechanisms to adapt to all promoters and may not be conserved among all plant periods of low non-freezing temperatures, enabling these species. An alternative to functional analyses in heterolo- plants to survive subsequent freezing temperatures. This gous systems is transient transformation of fruits using process is called cold acclimation [1]. Cold acclimation is agro-infiltration. Agro-infiltration of fruits have been per- a complex process that involves physiological, biochemi- formed to test the activity of the 35S CaMV promoter cal and molecular modifications [2-4]. Hundreds of genes fused to reporter genes such as GUS or luciferase in toma- have been shown to have altered expression levels during toes, apples, pears, peaches, strawberries and oranges cold acclimation [5]. These alterations enable the plant to [15,16]. However, to our knowledge, it has not been used withstand freezing by creating a chronic response that to determine the activity of cold-inducible promoters protects the integrity of the cellular membranes, enhances within the fruit (in fruta). anti-oxidative mechanisms and accumulates molecular cryoprotectants [6]. To identify cold-responsive genes expressed in peach fruits, digital expression analyses of ESTs from fruits Under normal conditions, cold acclimation is initiated by exposed to four different postharvest conditions were ana- the cold temperatures of late fall and early winter, when lyzed [17]. Isolation of the promoter regions of three fruit trees lack fruits. Similar cold temperatures have been genes highly expressed in fruits that have undergone long- used in the fruit industry to store fruits for prolonged peri- term cold storage, allowed us to identify common regula- ods of time. These temperatures inhibit fruit ripening, tory elements present in these promoters. Functional thereby extending fruit postharvest life. Despite the bene- characterization of these promoters (stably in A. thaliana fits, peaches that are subjected to long periods of cold stor- and transiently in peach fruits) demonstrates that these age can develop chilling injury symptoms (i.e. woolliness are peach cold-inducible promoters and that there is a and internal breakdown) which reduce the postharvest conserved heterologous regulation of these promoters in quality of these fruits and results in significant economical peach and Arabidopsis. losses [7-9]. Methods Most of the efforts directed towards understanding the Digital expression analyses molecular basis of cold acclimation have been performed We have previously described the contigs used in this in the model plant A. thaliana [1-4]. Little is known about work [17]. The ESTs that make up these contigs represent what occurs under low, non-freezing temperatures in transcripts from peach fruit mesocarp at four different fruits or fruit trees. Since chilling injury occurs in fruits postharvest conditions. The post-harvest conditions that have undergone long-term cold storage, perhaps cold include: fruits processed in a packing plant (E1: non-ripe; acclimation processes are associated with this injury. A no long term cold storage); packing followed by a shelf- better understanding of cold acclimation and cold- life at 20°C for 2-6 days (E2: Ripe; no long term cold stor- responsive genes in peach trees and fruits may provide age; juicy fruits); packing followed by cold storage at 4°C clues about the association of cold acclimation and chill- for 21 days (E3: non-ripe; long term cold storage) and ing injury. packing followed by cold storage at 4°C for 21 days and shelf-life at 20°C for 2-6 days (E4: Ripe; long term cold Several transcription factors associated with cold acclima- storage; woolly fruits). tion have been shown to regulate the expression of cold- inducible genes containing conserved ABRE (abscisic acid As we described in Vizoso et al [17], the contigs that rep- response elements) and/or DRE (dehydration-responsive) resent differentially expressed genes were identified using elements in their promoters [10-13]. The regulation of the Winflat program that submits the sequence data to a cold-inducible promoters in peaches may be mediated by rigorous statistical analysis described by Audic and Clav- the interaction between promoters containing these types erie [18]http://igs-server.cnrs-mrs.fr. This analysis calcu- of cis-elements and orthologous transcription factors. lates the probability that a gene is equally expressed in However, the identification and functional characteriza- two different conditions by observing the distribution of tion of these types of promoters in fruit trees is lacking. tag counts (number of ESTs). Therefore, small probability values (p-values) are associated with non-symmetrical dis- We have demonstrated previously that there is a con- tributions, characteristic of differentially expressed genes served heterologous regulation of the wheat putative [18,19]. high-affinity Pi transporter, TaPT2 in both monocots (wheat) and dicots (Arabidopsis) [14]. These findings Page 2 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 To analyze the co-expression of differentially expressed synthesized from 5 ng of the mRNA in a 20 l final vol- genes, contigs were clustered using the Pearson linear cor- ume. The reaction mix was prepared using the ImProm- relation coefficient [19,20]. Briefly, contigs with at least II™ reverse Transcription System (Promega, Madison, five ESTs were selected to make the expression profile USA) and anchored oligo (dT) of 18-mers, according to matrix, which consisted of 1,402 rows (the contigs) and 4 the manufacturer's instructions. As an internal control for columns (four cDNA libraries). The similarity between normalization, heterologous mRNA (1.2 kb mRNA cod- clusters and libraries was estimated using an un-centered ing for Kanamycin) was added to each mRNA sample. To Pearson's correlation coefficient in the Cluster 3.0 pro- control for genomic DNA contamination, PCR amplifica- gram [20]http://rana.lbl.gov/EisenSoftware.htm. Pearson tion was performed on template RNA that was not reverse correlation coefficients > 0.85 (zero values indicate no transcribed. To confirm that the amplified fragments cor- association and a coefficient equal to 1 indicate a fully respond to the cDNAs of interest, these fragments were correlated pattern) are indicated by an asterisk in Addi- cloned in pBluescript and sequenced (Macrogen, Korea). tional File 1. Dendrograms were constructed from the pair The primer sequences used to amplify the internal regions wise distances using the UPGMA algorithm. The results of the basic endochitinase Ppbec1 (BEC226F and were visualized and analyzed using the Java TreeView pro- BEC576R), dehydrin Ppxero2 (DX-82F and DX176R), gram http://jtreeview.sourceforge.net. thaumatin Pptha1 (THA30F and THA382R), lipoxygenase Pplox1 (LOX982F and LOX1267R) and the actin Ppact7 Gene Ontology molecular function and biological process (ACT-F and ACT-R) genes are shown in Table 1. Primers annotations of the contigs are described in Vizoso et al used to amplify a 323 bp fragment of the cDNA from the [17]. Each annotation and contig assembly was manually Kanamycin mRNA control are: "Upstream Control corrected, when necessary. Primer" (5'-gCCATTCTCACCggATTCAgTCgTC-3') and "Downstream Control Primer" (5'-AgCCgCCgTCCCgT- mRNA isolation and reverse transcriptase (RT)-PCR CAAgTCAg-3'). PCR reactions were performed by diluting The kit Oligotex™ mRNA Spin-Column (Qiagen, New the cDNAs a 100 fold and using 1 l of each dilution as a York, USA) was used to purify mRNA. The mRNA was template in a final reaction volume of 20 l, containing purified from pools of total RNA obtained from peach 0.5 M primers; 0.2 mM dNTPs; 1.5 mM MgCl ; 5U Taq polymerase and 1× buffer. The PCR conditions were: fruit mesocarp (O'Henry var.) representing the stages E1, E2, E3 and E4 as described previously [17,21]. The mRNA 93°C for 5 min and then a variable number of cycles (26 was quantified using the Poly (A) mRNA Detection Sys- to 34) at 93°C for 30 sec, 1 min at 55°C, and 1 min at tem™ (Promega, Madison, USA). First strand cDNA was Table 1: Primers used in this study Primer Sequence (5'3') Method BEC226F gTCAgCAgCgTCgTTAgCTC RT-PCR BEC576R gAgTTggATgggTCCTCTgC DX-82F CCAAACCAAAgCCAgTTTCATTCA DX176R CCAggTTTTgTATgAgTgCCgTA THA30F ACCTTggCCATCCTCTTCTT THA382R AgAAATCTTgACCCCCgTTC LOX982F AAggAgCTCTTgACgTTggA LOX1267R TgCTAACAggTgggAAAACC ACT-F CCTTCCAgCAgATgTggATT ACT-R AgATTAggCAAggCgAggAT BEC87-GSP1 TgCATTTCCAgCTTgCCTCCCACATTg Genome Walker BEC55-GSP2 CTgAgATCCCTAACAgCAAAgCTAgggATA DX85-GSP1 ACCggTTCCggTggTggTgTgATgAACC DX46-GSP2 ACTCATCAgTCTTAgTAggCTCgggTgTT THA82-GSP1 TgATTTTAgCTgCATgTgCACCTgAgAA THA-1-GSP2 CgTCATggAAATgTCTTAATTggCTTgCTg LOX101-GSP1 gAAgAAAACAAATTgggAggAggAgAA LOX63-GSP2 gCgTgTTCCAAAgAACACAATTCAgTgCCTT BEC-32BamHI ggATCCTgATCTgTggATTgggTTTCgTgg Subcloning promoters DX24BamHI ggATCCgggTgTTgAACCAAAATgCgCCATT Page 3 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 72°C. The PCR reaction was with a final step at 72°C for program [25]http://wingless.cs.washington.edu/YMF/ 10 min. YMFWeb/YMFInput.pl. Only the statistically significant motifs (Z score value > 6.5) were selected [26]. Cloning of the promoters Genomic DNA was isolated from peach leaves (Prunus per- Growth, transformation and cold treatments of A. thaliana sica var. persica (L.) Batch cv. O'Henry) as described in Wild-type and transgenic A. thaliana (ecotype Columbia) Manubens et al [22]. The Universal Genome Walker™ Kit were grown in a mixture of soil-vermiculite (3:1) in a -2 - (Clontech Laboratories, Inc., Palo Alto, CA, USA) was growth chamber with a 16-h light cycle (140 mol m s used to isolate the promoters regions of Ppbec1, Ppxero2, ) at 22°C. Alternatively, seeds were surface sterilized as Pptha1 and Pplox1. The isolated genomic DNA was described in Gonzalez et al [27], plated on Murashige- digested with four restriction enzymes (EcoRV, PvuII, SspI, Skoog (1 × MS) media containing 0.8% agar, 0.1% and MlsI). DNA fragments containing adaptors at both sucrose and 50 mg/l Kanamycin for transgenic lines and ends were used as a template for amplifying the promoter grown under the same conditions as the soil-grown regions. GSP1 and GSP2 gene specific primers were plants. designed to isolate the promoters (Table 1). For the first group of PCR reactions, a specific adaptor primer (AP1, 5'- Transgenic Arabidopsis was obtained by using the ggATCCTAATACgACTCACTATAgggC-3') and the GSP1 GV3101 A. tumefaciens-mediated floral dip method [28]. primers specific for each gene were used. The final primer A. tumefaciens previously transformed with the binary vec- concentration in the PCR reaction was 0.2 M in a final tor pBI101.3 harboring the promoter::uidA fusions: volume of 50 L. Manual Hot Start was performed using Ppbec1::uidA (PBIPpbec1); Ppxero2::uidA (pBIPpxero2); 5 U of the Synergy DNA polymerase (Genecraft, Münster, Pptha1::uidA (pBIPptha1), or the control vectors pBI121 Germany). The conditions for this first round of amplifi- (containing the 35S CaMV promoter) and pBI101.3 (pro- cations was: 1 cycle at 93°C for 10 min, 7 cycles of 93°C moterless), were used. In cold treatments, T homozygous for 30 sec, 72°C for 15 min, followed by 37 cycles of 93°C transgenic Arabidopsis seedlings were grown on plates for 30 sec, 67°C for 15 min. For the nested PCR, the spe- containing 1× MS media, 0.8% agar, and 0.1% sucrose in -2 - cific adaptor primer 2 (AP2, 5'-ACTATAgggCACgCgTggT- a growth chamber with a 16-h light cycle (140 mol m s ) at 24°C for two weeks, and then transferred to 4°C for 3') and the gene specific GSP2 primers were used. As a DNA template in these reactions, 1 L of a 50 fold dilu- 7 days. A minimum of three independent transgenic lines tion of end-product of the first round of amplifications were used for each construct. was used. The conditions for the second round of ampli- Peach fruit transient transformation and cold treatments fication were: 1 cycle at 93°C for 10 min, 5 cycles (7 cycles in the case of Ppxero2) of 93°C for 30 sec, 72°C for 15 A. tumefaciens transformed with the vectors pBIPpbec1, min, followed by 20 cycles (30 cycles in the case of pBIPpxero2, pBIPptha1, pBI121 or pBI101.3 were grown Ppxero2) of 93°C for 30 sec, 67°C for 15 min. The ampli- in LB medium supplemented with Kanamycin (100 g/ fied products were cloned in pGEM-T vector and ml), Rifampicin (10 g/ml) and Gentamycin (100 g/ sequenced (Macrogen, Korea). The Ppbec1 and Ppxero2 ml). The cultures were grown for two days at 28°C until promoters were subsequently amplified from the pGEM- they reached an OD between 0.6 and 0.8. The culture T clones using the AP2 and BEC-32BamHI or was then centrifuged and the pellet re-suspended in MMA DX24BamHI primers, respectively (Table 1). The products medium (1× MS, MES 10 mM (pH 5.6), 20 g/l sucrose, of this amplification were also cloned in the pGEM-T vec- and 200 M acetosyringone) to reach an OD of 2.4. tor and re-sequenced (Macrogen, Korea). The promoter Approximately 0.7 mL of this bacterial suspension was fragments were extracted from the pGEM-T vector (includ- used to infiltrate mature fruits from O'Henry, Elegant Lady ing the Pptha1 promoter), with a BamHI-SalI sequential and Florida King varieties of peach as described by Spo- digestion, and transcriptionally fused to the uidA reporter laore et al [15]. gene in the promoterless binary vector pBI101.1 [23]. The binary vector was introduced into A. tumefaciens To analyze the promoter activity at 20°C, the fruits infil- (GV3101) for subsequent Arabidopsis and peach fruit trated with the different constructs, were stored in a dark transformations. growth chamber for five days. To analyze the cold-respon- sive promoter activity, the infiltrated fruits were stored 2 Promoter sequences analysis days post-infiltration (dpi) in a dark growth chamber at Analysis of putative transcription factor binding sites was 4°C for 10 days. After the growth chamber incubation carried out using the database PLACE http:// time, the infiltrated region of the fruit was extracted with www.dna.affrc.go.jp/htdocs/PLACE/[24] coupled with a cork bore and stained for GUS activity as described by visual analyses. To identify predicted conserved motifs, Tittarelli et al [14]. the promoter sequences were analyzed using the YMF 3.0 Page 4 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 GUS activity measurement suggesting that these may be putative orthologs. The puta- Histochemical staining of Arabidopsis seedlings for -glu- tive Arabidopsis orthologs that are induced or repressed curonidase (GUS) activity was performed as described by by cold, based on ColdArrayDB analyses http://cold.stan Jefferson et al [23], with the following modifications: ford.edu/cold/cgi-bin/data.cgi are shown in Table 2. Only transgenic Arabidopsis seedlings used in the cold-treat- 29 contigs (18% of the 164 cold-induced genes) share sig- ments described earlier were vacuum infiltrated in 50 mM nificant sequence identity with genes of unknown func- NaH PO , pH 7.0; 0.1 mM X-Gluc; 10 mM EDTA and tion. Approximately 38% of these contigs (11 contigs) 2 4 0.1% Triton X-100. These samples were incubated in the share significant sequence identity with plant gene dark at 37°C for 24-72 h. Samples that did not develop sequences annotated as expressed proteins. Six of the con- color after 72 h were considered negative for GUS activity. tigs with unknown function do not share sequence iden- Plant material was subsequently fixed in 0.04% formalde- tity with any sequences in the public databases, suggesting hyde, 0.04% acetic acid and 0.285% ethanol for 30 min, that these are novel genes. followed by an ethanol dilution series to remove chloro- phyll from the plant tissue (70% ethanol for 1 h, 100% Annotation frequency comparative analyses of cold- ethanol for 1 h, 70% ethanol for 1 h and distilled water). induced (164 contigs), cold-repressed (138 contigs) or contigs unrelated to cold (1,238 contigs), revealed an Slices (2 mm) of transiently transformed peaches were overrepresentation of stress response genes and an under- imbibed in the GUS staining solution (0.72 M K HPO ; representation of genes related to energy metabolism in 2 4 0.17 M KH PO ; 0.5 mM K Fe(CN) ; 0.5 mM K Fe(CN) ; fruits that were stored in the cold (Figure 1). Among the 2 4 3 6 4 6 1× Triton X-100; 12.7 mM EDTA; 20% (v/v) methanol genes related to stress response we identified four contigs and 0.5 mM X-Gluc) [15]. Samples were vacuum-infil- that are similar to thaumatin-like proteins: C1708, trated for 30 min at room-temperature and then incu- C2177, C2317 and C2147 (98%, 99%, 98% and 93% bated overnight at 37°C. Fluorometric GUS assays were amino acid identity with P. persica thaumatin-like protein performed as described by Jefferson et al [23]. The Arabi- 1 precursor, respectively, GenBank accession number: dopsis seedlings were ground in a mortar using liquid P83332). Three of the stress response genes are similar to nitrogen, and the tissue powder was transferred to a chitinases: C910 (76% amino acid identity with Malus microtube. One ml of the extraction buffer (50 mM domestica class III acidic endochitinase, GenBank acces- NaH PO , pH 7.0; 1 mM EDTA; 0.1% Triton X-100; 0.1% sion number: ABC47924); C2131 (74% amino acid iden- 2 4 (w/v) sodium laurylsarcosine and 5 mM dithiothreitol) tity with Galega orientalis class Ib basic endochitinase, was added. Samples were centrifuged for 10 min at 12,000 GenBank accession number: AAP03087) and C2441 g at 4°C and the supernatant was transferred to a new (72% amino acid identity with A. thaliana class IV chiti- microtube. The fluorogenic reaction was carried out in 2 nase, GenBank accession number: NP_191010). Two of ml volume containing 1 mM 4-methyl umbelliferyl glu- the stress response genes are similar to dehydrins: C254 curonide (MUG) in an extraction buffer supplemented (97% amino acid identity with P. persica Ppdhn1, Gen- with a 50 L aliquot of the protein extract supernatants. Bank accession number: AAC49658) and C304, 100% The protein quantity of the sample extracts was deter- amino acid identity with P. persica type II SK2 dehydrin mined as described previously [29], using bovine serum Ppdhn3 (Genbank accession number: AAZ83586). albumin (BSA) as a standard. Cold-induced expression of Ppbec1, Ppxero2 and Pptha1 We evaluated the expression levels of three cold-induced Results Identification of peach cold-regulated genes by digital candidate genes by RT-PCR: a basic endochitinase expression analyses of EST datasets (C2131, Ppbec1), a dehydrin (C254, Ppxero2) and a thau- Coordinated gene expression analyses of peach fruit ESTs matin-like protein (C2317, Pptha1). These genes were datasets revealed 10 major hierarchical clusters (Addi- chosen due to the high number of ESTs in cold-stored tional File 1), containing unique contigs. We identified fruits (E3), as revealed by the digital expression analyses 164 contigs with preferential expression in fruits stored at (Figure 2). The expression level of a contig similar to 4°C (E3: non-ripe; long term cold storage). Table 2 con- lipoxygenase (C3336, Pplox1) that does not express pref- tains a complete list of these contigs together with their erentially in cold stored fruits (E3) as well as the expres- annotations, GO biological process annotations and the sion level of a contig (C407, Ppact7) that does not origin of the ESTs in each contig. Contigs with statistically significantly change expression under the different post- differential expression, in E3 compared to the other stages harvest conditions, were analyzed (Figure 2). Interest- are also indicated. ingly, all five genes analyzed showed an expression pat- tern significantly similar to the ones predicted by the Approximately 95% of the 164 cold-induced peach genes digital expression analyses (Figure 2). The genes Ppbec1, share significant identity with sequences in Arabidopsis, Ppxero2 and Pptha1 have an increased expression in cold- Page 5 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Table 2: Putative function of 164 genes preferentially expressed in cold stored peach fruits. 1 2 Contig E3 E1+E2+E4 AC test Putative Function;Arabidopsis ortholog Biological process unknown (GO:0000004) C517 10 6 E4 NC domain-containing protein (located in mitochondrion); At5g06370 C675 12 4 E2; E4 Expressed protein; At3g03870 C774* 11 4 E2; E4 Novel gene C2089 20 0 E1; E2; E4 Expressed protein (located in endomembrane system); At5g64820 C2112 31 2 E1; E2; E4 Cupin family protein (nutrient reservoir activity); At1g07750 C2139 12 0 E1; E2; E4 Novel gene C4065 13 8 E2 Expressed protein; At5g52870 C273 5 2 Expressed protein; At5g24660 C477 7 6 Expressed protein (located in endomembrane system); At5g64510 C1207* 8 7 Novel gene C2134 3 2 Expressed protein; At1g71080 C2148 4 1 Novel gene C2155 4 1 Expressed protein; At5g11730 C2167 3 2 RWD domain-containing protein; At1g51730 C2173 7 1 Expressed protein (located in mitochondrion);At5g60680 C2193 3 2 Novel gene C2211 8 1 Ankyrin repeat family protein (protein binding); At2g28840 C2241 6 2 Expressed protein (located in mitochondrion); At5g51040 C2267 7 0 Integral membrane family protein; At4g15610 C2315 5 3 Expressed protein; At1g70780 C2318 3 2 Ribosome associated membrane protein RAMP4; At1g27350 C2343 9 9 Novel gene C2560 6 1 Expressed protein; At3g27880 C2591 6 1 Expressed protein (located in mitochondrion); At5g24600 C2682* 4 2 N-methyl-D-aspartate receptor-associated protein; At4g15470 C2713 4 1 Glycine-rich protein; At4g22740 C2778 12 7 Zinc finger (AN1-like) family (DNA and zinc ion binding); At3g52800 C2806 8 2 C2 domain-containing protein; At1g22610 C3094 3 2 Reticulon family protein (located in ER and mitochondrion); At3g10260 Cell homeostasis (GO:0019725) C2265 91 38 E1; E2; E4 Metallothionein-like protein; At5g02380 C2202* 5 1 Metallothionein-like protein; NSM Cell organization and biogenesis (GO:0016043) C734 17 9 E2; E4 Proline-rich/extensin family; At2g27380 C1240 62 20 E1; E2; E4 Proline-rich/extensin family; At1g54215 C2494* 10 3 E2 Actin-depolymerizing factor 4; At5g59890 C2831 20 6 E1; E2; E4 Leucine-rich repeat/extensin family; At4g13340 C3041 12 5 E2; E4 Leucine-rich repeat/extensin family; At4g13340 C831 4 2 BON1-associated protein (BAP2); At2g45760 C1062 4 1 Invertase/pectin methylesterase inhibitor family; At5g62360 C2060 7 3 Expansin family; At4g38400 C2086* 6 1 Arabinogalactan-protein; At5g64310 C2073 6 2 Zinc finger protein (CYO1); At3g19220 C2574 7 3 Invertase/pectin methylesterase inhibitor family; At2g01610 C2762* 4 1 Profilin 4; At2g19770 C2815 4 1 Phytochelatin synthetase; At4g16120 Cellular protein metabolism (GO:0044267) C228* 112 51 E1; E2; E4 DJ-1 family protein/protease-related; At3g02720 C379* 50 21 E1; E2; E4 DJ-1 family protein/protease-related; At3g02720 C1027* 47 46 E1; E2; E4 Heat shock cognate 70 kDa protein 1; At5g02500 C1660 51 25 E1; E2; E4 Cysteine proteinase inhibitor-related; At2g31980 C2099* 13 1 E1; E2; E4 DJ-1 family protein/protease-related; At3g02720 C2436 17 3 E1; E2; E4 Rhomboid family protein; At1g63120 C2715 41 21 E1; E2; E4 Luminal binding protein 1 (BiP-1); At5g28540 C2066* 3 2 60S ribosomal protein L23A; At3g55280 Cellular protein metabolism (GO:0044267) C2072* 6 2 DNAJ heat shock protein; At3g44110 C2217* 7 3 20S proteasome beta subunit A; At4g31300 C2308* 9 0 Heat shock protein 70; At3g12580 C2345* 4 2 Ubiquitin carrier protein E2; At2g02760 C2364 5 2 Phosphatase-related (SGT1B); At4g11260 C2388 5 3 F-box family protein (AtSKP2;2); At1g77000 Page 6 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Table 2: Putative function of 164 genes preferentially expressed in cold stored peach fruits. (Continued) C2593 4 1 C3HC4-type RING finger family protein; At1g26800 C2597 6 2 26S proteasome regulatory subunit S3; At1g20200 C2691 7 6 C3HC4-type RING finger family protein; At5g47610 C2360 10 7 Structural constituent of ribosome; At5g15260 C2735 9 4 40S ribosomal protein S9; At5g39850 C3022 6 2 Translation initiation factor IF5; At1g36730 C3051* 5 2 DJ-1 family protein/protease-related; At3g02720 C3520 4 1 60S ribosomal protein L36; At3g53740 C3551* 11 4 Cysteine proteinase inhibitor; At3g12490 C3656 6 4 40S ribosomal protein S26; At3g56340 C4131 3 2 C3HC4-type RING finger family protein; At5g48655 Development (GO:0007275) C2802 10 2 E1 Senescence-associated protein; At1g78020 C2919 10 1 E1; E2 Senescence-associated protein; At5g20700 C1113 6 3 Auxin-responsive protein; At3g25290 C3887* 4 1 Maternal effect embryo arrest 60; At5g05950 C3942 6 4 SIAMESE, cyclin binding protein; At5g04470 C2457 6 0 Nodulin MtN3 family protein; At5g13170 Generation of precursor metabolites and energy (GO:0006091) C2304 7 1 NADH dehydrogenase; At4g05020 C2541 8 1 Uclacyanin I; At2g32300 C2552 5 0 Flavin-containing monooxygenase family protein; At1g48910 Metabolism (GO:0008152) C1017 15 9 E2 Xyloglucan endotransglycosylase; At4g25810 (carbohydrate) C1258* 19 2 E1; E2; E4 Phosphoesterase family protein; At3g03520 (phospholipid) C2373 15 8 E2; E4 -alanine-pyruvate aminotransferase; At2g38400 (amino acid) C2397* 27 9 E1; E2; E4 S-adenosylmethionine decarboxylase; At3g02470 (polyamine) C2554* 17 3 E1; E2; E4 UDP-glucoronosyl/UDP-glucosyl transferase; At5g65550 (anthocyanin) C2957 11 0 E1; E2; E4 Glycosyl hydrolase family 3; At5g49360 (carbohydrate) C2669 61 28 E1; E2; E4 Phosphoserine aminotransferase; At4g35630 (amino acid) C656 4 3 Nucleoside diphosphate kinase 3; At4g11010 (nucleotide) C821* 4 1 UDP-glucoronosyl/UDP-glucosyl transferase; At5g49690 (anthocyanin) C926* 7 6 (1-4)--mannan endohydrolase; At5g66460 (carbohydrate) C1000* 8 2 Alkaline alpha galactosidase; At1g55740 (carbohydrate) C1693 9 3 Haloacid dehalogenase-like hydrolase; At5g02230 C1943 4 3 2-oxoglutarate-dependent dioxygenase; At1g06620 (ethylene) C2424 5 0 -amylase; At4g17090 (starch) C2495 8 1 Cinnamoyl-CoA reductase; At4g30470 (lignin) C2522 11 8 Glycosyl hydrolase family 5; At1g13130 (carbohydrate) C2569 7 1 Short-chain dehydrogenase/reductase family; At3g61220 C2602 5 0 Short-chain dehydrogenase/reductase family; At4g13250 C2610 5 0 Galactinol synthase; At3g28340 (carbohydrate) C2222 6 0 Carboxyesterase 5; At1g49660 C2635 6 4 GNS1/SUR4 membrane family protein; At4g36830 (fatty acid) C2705 7 4 DSBA oxidoreductase family protein; At5g38900 (organic acid) C669 4 2 Dehydrogenase; At5g10730 C2936 4 1 Pyruvate decarboxylase; At5g17380 (glycolisis) C2940 4 1 Farnesyl pyrophosphate synthetase 1; At5g47770 (lipid) C2976 6 1 Aminoalcoholphosphotransferase; At1g13560 (phospholipid) C3047* 7 4 Dienelactone hydrolase; At3g23600 (alkene) Metabolism (GO:0008152) C3058* 5 1 Cellulose synthase; At4g39350 (cellulose) C3152 8 3 Purple acid phosphatase; At3g52820 (phosphate) C3225 4 1 Acyl-activating enzyme 12; At1g65890 (phospholipid) C4127 6 2 -3fatty acid desaturase; At5g05580 (fatty acid) C86 6 3 Embryo-abundant protein; At2g41380 C677 4 2 Cyclic phosphodiesterase; At4g18930 (RNA) C802 4 3 RNA recognition motif-containing protein; At5g04600 (RNA) C2798 3 2 Small nuclear ribonucleoprotein G; At2g23930 (RNA) Response to stress (GO:0006950) C30 57 27 E1; E2; E4 Cold acclimation WCOR413-like protein; At3g50830 C254 71 10 E1; E2; E4 Dehydrin Xero2; At3g50970 C304* 189 124 E1; E2; E4 Type II dehydrin SKII; (ERD14) At1g76180 C910 126 38 E1; E2; E4 Class III acidic endochitinase; At5g24090 C1479 96 25 E1; E2; E4 Harpin inducing protein; At5g06320 C1708 30 12 E1; E2; E4 Thaumatin-like protein; At1g20030 C2131 65 2 E1; E2; E4 Class Ib basic endochitinase; At3g12500 Page 7 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Table 2: Putative function of 164 genes preferentially expressed in cold stored peach fruits. (Continued) C2177 15 4 E1; E4 Thaumatin-like protein; At1g20030 C2317 67 6 E1; E2; E4 Thaumatin-like protein; At1g20030 C2514* 20 15 E2 Glutathione peroxidase; At4g11600 C2528 22 7 E1; E2; E4 Hevein-like protein; At3g04720 C2655* 10 6 E4 DREPP plasma membrane polypeptide; At4g20260 C2988* 37 6 E1; E2; E4 Polygalacturonase inhibiting protein; At5g06860 C2473* 10 0 E1; E2; E4 Major allergen Pru p 1; At1g24020 C2147 8 0 Thaumatin-like protein; At1g20030 C2441 8 1 Class IV chitinase; At3g54420 C2507 5 2 Pyridoxine biosynthesis protein; At5g01410 C2556 5 0 4-aminobutyrate aminotransferase; At3g22200 C2578 3 2 Aldehyde dehydrogenase; At1g44170 C2926 7 2 Wounding stress inducimg protein; At4g24220 C3613* 3 2 Harpin inducing protein; At3g11660 C1889* 5 4 Major allergen Pru p 1; At1g24020 C3858* 4 2 Late embryogenesis abundant protein 3; At4g02380 Signal transduction (GO:0007165) C815 9 1 Leucine-rich repeat family protein; At3g49750 C1192* 6 5 CBL-interacting protein kinase 12; At4g18700 C2205 5 4 Ser/Thr kinase; At2g47060 C2312* 8 3 Touch-responsive/calmodulin-related protein 3; At2g41100 C2430* 6 6 Remorin family protein; At5g23750 C2548 10 6 Fringe-related protein; At4g00300 C2829* 3 2 Protein kinase, 41K; At5g66880 C2853 5 3 GTP-binding protein Rab2; At4g17170 C3690* 10 8 Ser/Thr kinase; At4g40010 Transcription (GO:0006350) C452 4 2 Myb family; At5g45420 C2742* 5 1 DREB subfamily A-6; At1g78080 C3420* 8 4 MADS-box protein (AGL9); At1g24260 C3812 3 2 WRKY family; At4g31550 Transport (GO:0006810) C716 13 5 E2; E4 Proton-dependent oligopeptide transport family; At5g62680 C1846 15 10 E4 Auxin efflux carrier family protein; At2g17500 C2091 18 0 E1; E2; E4 Protease inhibitor/seed storage/lipid transfer family; At1g62790 C163 4 1 Vesicle-associated membrane protein; At1g08820 C208 9 2 GTP-binding secretory factor SAR1A; At4g02080 C235 5 4 Sugar transporter; At1g54730 C484 11 6 Porin; At5g67500 C1526 5 4 emp24/gp25L/p24 protein; At3g22845 Transport (GO:0006810) C2062 3 2 Ripening-responsive protein; At1g47530 C2236 3 2 Ras-related GTP-binding protein; At4g35860 C2476 9 1 Bet1 gene family; At4g14450 C2679 5 0 Sulfate transporter ST1; At3g51895 C3063 4 2 Amino acid carrier; At1g77380 C3066 4 1 Sulfate transporter; At3g15990 C3099 3 2 Ras-related GTP-binding protein; At1g52280 Statistically significant cold-induced contigs detected with the Audic and Claverie test (p < 0.01) vs. E1, E2 or E4 cDNA libraries. The column shows the cDNA library with differences to E3. The column described the locus identifier (id) of the Arabidopsis most similar protein. The locus ids with  [37] are the Arabidopsis cold response genes similarly up-regulated; the locus ids with  [31] are the genes with opposite response, down-regulated in Arabidopsis (ColdArrayDB; http:// cold.stanford.edu/cgi-bin/data.cgi). Between parentheses: the principal subcategory of the biological process "metabolism" associated to the annotation. 4 -10 NSM: Not significant match (E value < 10 ) with A. thaliana sequences. -10 * Contigs that shown significant sequence homology (e value > 10 ) with contigs from others hierarchical clusters. Page 8 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 A Figure 1 nnotation frequency comparison of cold-induced, cold-repressed or unrelated to cold-induction contigs Annotation frequency comparison of cold-induced, cold-repressed or unrelated to cold-induction contigs. The frequency of contigs that are associated with a specific Gene Ontology are expressed as the percentage of the total annota- tions for each analyzed group (164 for the cold-induced, 138 for the cold-repressed and 1,238 for unrelated to cold-induction). The numbers of contigs in each group, belonging to each biological process classification, are show at the top of each bar. The category "others process" are: cell adhesion (GO: 0007155, 1 contig); cell communication (GO: 0007154, 1 contig); cell cycle (GO: 0007049, 5 contigs); cell death (GO: 0008219, 1 contig); cell homeostasis (GO: 0019725, 4 contigs); organism physiolog- ical process (GO: 0050874; 1 contig); regulation of GTPase activity (GO: 0043087; 1 contig); response to stimulus (GO: 0050896; 10 contigs) and viral life cycle (GO: 0016032; 1 contig). stored fruits, whereas the Pplox1 gene increased expression Cis-element regulatory motifs related to cold gene expres- in woolly fruits rather than cold-stored fruits. sion regulation such as ABRE [13], MYCR [31,32], MYBR [31,33] and DRE/CRT [34] were identified in all three pro- Identification of conserved motifs in the promoters of cold- moters of these cold-inducible genes (Figure 3). In addi- inducible genes Ppbec1, Ppxero2 and Pptha1 tion, three statistically significant predicted motifs were We cloned 826 bp, 1,348 bp and 1,559 bp fragments cor- present in the promoters of these cold-inducible genes responding to the regions upstream of the translation start (TACGTSGS, TGTGTGYS and CTAGAASY (Figure 3). codons of Ppbec1, Ppxero2 and Pptha1, respectively. The These motifs were not found in the Pplox1 promoter iden- sequences of these promoter regions as well as the cDNA tified in this work (Additional File 5). of their corresponding genes are shown in the Additional Files 2, 3 and 4. Cold-induced Ppbec1 and Ppxero2 promoters in transiently transformed peach fruits and stably transformed Arabidopsis The high sequence identity between the Ppxero2 contig with the coding region of Ppdhn1[30] was also observed Transient transformation assays of peach fruits revealed within the promoter sequences of these two genes. Only that all three cloned promoters (pBIPpbec1, pBIPxero2 one nucleotide difference at position -469 was found, sug- and pBIPptha1) were able to activate GUS (uidA) expres- gesting that Ppxero2 and Ppdhn1 may be the same gene sion (Figure 4). However, only the pBIPpbec1 and (Additional File 3). However, the promoter isolated in pBIPxero2 promoter constructs showed cold-inducible this work is about 230 bp longer (at the 5' end) than the increases in GUS activity (Figure 4). The pBIPtha1 con- previously published promoter [30]. struct was expressed at both 20°C and 4°C. Comparable Page 9 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 All three constructs were able to activate GUS expression, but only the Ppbec1 and Ppxero2 promoters (pBIPpbec1 and pBIPxero2, respectively) induced expression in response to cold (Figure 5). As observed with the fruit transient transformation assays, the Pptha1 promoter (pBIPtha1) expressed GUS under all conditions analyzed. Discussion and Conclusion Digital expression analyses of EST datasets have permitted us to identify a large diversity of cold-inducible genes in peach fruits, three of which were chosen for further anal- yses (Ppbec1, Ppxero2 y Pptha1). Both digital expression analyses and RT-PCR suggest that the Ppbec1, Ppxero2 and Pptha1 are cold-inducible genes. The promoters of these cold-inducible genes were isolated and characterized using both transient transformation assays in peach fruits and stable transformation in Arabidopsis. These analyses have revealed that the isolated Ppbec1 and Ppxero2 pro- moters are cold-inducible promoters, whereas the isolated Pptha1 promoter was not cold-inducible. These results, therefore, demonstrate that the isolated Ppbec1 and Ppxero2 promoters are sufficient for cold-induced gene expression. Furthermore, these results suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. Plants respond to cold temperatures by modifying the E tern Figure 2 valu s of selected ation of the accuracy of genes by RT-PCR the predicted expression pat- transcription and translation levels of hundreds of genes Evaluation of the accuracy of the predicted expres- [35,36]. These acute molecular changes are related to sion patterns of selected genes by RT-PCR. (A) RT- plant cell physiological and biochemical modifications PCR analysis of RNA expression of three cold-induced genes: (cold acclimation) that lead to stress tolerance and cold Ppbec1, Ppxero2, and Pptha1 under different post-harvest adaptation (a chronic response). In peach fruits, cold tem- conditions. These post-harvest conditions include: fruits peratures induce chilling injury, possibly due to global processed in a packing plant (E1: non-ripe; no long term cold transcriptome changes [37]. With the exception of studies storage); packing followed by a shelf-life at 20°C for 2-6 days in the model organism A. thaliana [4] and work published (E2: Ripe; no long term cold storage; juicy fruits); packing fol- recently [17,38], little is known about the peach global lowed by cold storage at 4°C for 21 days (E3: non-ripe; long transcriptional response to cold. Using the Pearson corre- term cold storage) and packing followed by cold storage at 4°C for 21 days and shelf-life at 20°C for 2-6 days (E4: Ripe; lation coefficient, we analyze the coordinated gene expres- long term cold storage; woolly fruits). The expression level sion of 1,402 contigs. This analysis revealed 164 genes of Pplox1 was analyzed as a control for genes that do not preferentially expressed in peach fruits, of which digital express preferentially in cold stored fruits (E3). Ppact7 was expression analyses [18] revealed 45 of these genes (27%) analyzed as a control for genes that do not significantly with statistically significant cold-induction. A large pro- change expression levels between the four post-harvest con- portion of the contigs preferentially expressed at 4°C ditions analyzed. The two arrows associated with each gel (around 74% of the total) do not exhibited significant represent 500 bp (upper) and 300 bp (lower). The number of -10 sequence homology (e-value < e ) with the rest of the ESTs associated with each contig and library source is indi- analyzed contigs (Table 2). This result could suggest that cated. (B) Densitometry quantification of the expression these contigs represent genes with non-redundant func- level obtained by RT-PCR, the figure shows the bands inten- tions that will have a special importance during the expo- sities for each gene relative to Ppact7 intensity. sure of the fruits to low temperatures. results were seen in fruits from three different peach vari- Among the highly expressed genes in cold stored fruits, we eties (data not shown). found genes related to stress response in plants, including three dehydrins (C30, C254 and C304), three chitinases Similar results were seen when these promoter-GUS con- (C910, C2131 and C2441), four thaumatin-like proteins structs were analyzed in stably transformed Arabidopsis. (C1708, C2177, C2317 and C2147), and polygalacturo- Page 10 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Putative Figure 3 cis-regulatory elements identified in Ppbec1, Ppxero2 and Pptha1 promoter sequences Putative cis-regulatory elements identified in Ppbec1, Ppxero2 and Pptha1 promoter sequences. Topologies of the Ppbec1 (A), Ppxero2 (B) and Pptha1 (C) promoters are shown. The promoters are draw proportionally (the bar correspond to 100 bp). Boxed regions: predicted 5' UTR region. Black arrow shows the position of different cis-regulatory elements related to low temperature responses: ABRE, DRE/CRT, MYBR and MYCR. The putative cis-regulatory elements identified by the motif prediction program YMF3.0 are shown as grey triangle, black circle and asterisk. The sequences, the symbol and the sig- nificance score (Zscore) of the motifs, are shown in the upper left corner. The degenerate bases allowed in the motifs are S (C or G) and Y (C or T). Note: in order to ensure at the legibility of the figure, not all cis-elements are marked in (B) and (C). However, the complete sequences of these promoters are available in Additional Files 3 and 4. Cold-inducible peach Figure 4 Ppbec1 and Ppxero2 promoters in transiently transformed peach fruits Cold-inducible peach Ppbec1 and Ppxero2 promoters in transiently transformed peach fruits. (A) Structure of the binary vector constructs used for functional analysis of the Ppbec1, Ppxero2 and Pptha1 promoter-uidA fusions. LB and RB: left and right T-DNA border. (B) Histochemical GUS staining of fruit slices from agro-infiltrated peaches stored at 20°C for 5 days post-inoculation or 4°C for 10 days. These images correspond to the transient transformation of O'Henry variety fruits. How- ever, similar results were seen in all varieties assayed (data not shown). Page 11 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Figure 5 Conserved heterologous regulation of the cold-inducible peach Ppbec1 and Ppxero2 promoters in transgenic Arabidopsis plants Conserved heterologous regulation of the cold-inducible peach Ppbec1 and Ppxero2 promoters in transgenic Arabidopsis plants. The upper panel shows histochemical GUS staining of representative transgenic Arabidopsis lines carry- ing the Ppbec1 promoter-uidA fusion, Ppxero2 promoter-uidA fusion and Pptha1 promoter-uidA fusion. The lower panel shows the results of fluorometric GUS-assays of three independent Arabidopsis transgenic lines (L1, L2 and L3) containing the Ppbec1 promoter-uidA fusion, Ppxero2 promoter-uidA fusion or Pptha1 promoter-uidA fusion. Homozygous T3 plants were grown for 14 days in MS plates with 0.8% agar at 24°C (white bars) and then transfer to 4°C for 7 days (blacks bars). The aster- isk above each bar represents those samples that have a statistically significant increase in GUS activity in the cold treated plants when compared to the untreated plants. Bars represent the mean ± standard deviation, n = 5. t-student * p < 0.01. nase inhibiting protein (C2988), similar to what was We also found some genes related to protein folding and reported by Ogundiwin et al [38]. Dehydrins are degradation, such as heat shock proteins, BiP-1 and DJ-1 hydrophilic proteins that belong to the subgroup D-11 of family proteins (Table 2). These processes are very active the LEA ("late-embryogenesis-abundant") proteins [39]. when plants face low temperatures, chemical and oxida- There is some evidence that suggests that dehydrins pro- tive stress. These proteins participate in the prevention tect macromolecules such as membranes and proteins and repair of damage produced by cold, through the sta- against the damages associated with water deficiency [40- bilization of protein structure and the degradation of pro- 42]. In peach, these genes are induced during cold accli- teins that are not folded correctly [50,51]. mation and in cold-stored fruits [30,38]. It has been observed that pathogenesis-related (PR) proteins such as In this work we were interested in isolating and function- chitinases and thaumatins are accumulated in the apo- ally characterizing promoters of cold-inducible peach plastic space in winter rye during cold acclimation. These genes. To date, only a few inducible promoters have been proteins also may have antifreeze properties that will pro- identified in crop plants. The Pptha1, Ppbec1 and Ppxero2 tect the integrity of the plant cell avoiding the formation genes were chosen for promoter cloning and characteriza- of ice [43,44]. It has also been observed that these types of tion based on the up-regulation that these genes showed proteins retain their enzymatic activity under low temper- in the in silico analysis and RT-PCR. The promoter atures, and may form part of a general response mecha- sequences of these genes contain several cis-regulatory ele- nism associated with unfavorable conditions, by ments such as DRE/CRT, ABRE, MYCR (MYC recognition providing protection from opportunist pathogen attack site) and MYBR (MYB recognition site) [13,31-34] that are whilst the plant is in a weakened state [45-47]. A similar related to stress response, specifically to cold/dehydra- role is shared by polygalacturonase inhibiting proteins in tion. These cis-regulatory elements are conserved in sev- different plants models [48,49]. eral plant species [52]. The presence of these conserved Page 12 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 motifs suggests that these promoters may respond to the Additional file 2 cold. Using transient transformation in peach fruit we Sequence of the Ppbec1 promoter and open reading frame. The data confirmed that the promoters isolated from Ppbec1 and provided represents the sequences of the Ppbec1 promoter and open read- Ppxero2 are induced during low temperature storage, but ing frame. not at room temperature. On the other hand, the Pptha1 Click here for file promoter is active under all the temperatures analyzed. [http://www.biomedcentral.com/content/supplementary/1471- This could indicate that the Pptha1 promoter sequence 2229-9-121-S2.DOC] might not contain all the elements needed to regulate Additional file 3 expression in a cold-inducible manner. Alternatively, the Sequence of the Ppxero2 promoter and open reading frame. The data agro-infiltration technique may induce stress signals that provided represents the sequences of the Ppxero2 promoter and open read- will activate this promoter. However, this last possibility ing frame. is not likely because the activation of the Pptha1 promoter Click here for file at all analyzed temperatures is also seen in the stably [http://www.biomedcentral.com/content/supplementary/1471- transformed transgenic Arabidopsis plants. The promot- 2229-9-121-S3.DOC] ers Ppbec1 and Ppxero2, however, are cold-induced both in Arabidopsis transgenic plants as well as transient express- Additional file 4 Sequence of the Pptha1 promoter and open reading frame. The data ing fruits, suggesting that the Ppbec1 and Ppxero2 promot- provided represents the sequences of the Pptha1 promoter and open read- ers are cold-inducible peach promoters. The cold- ing frame. inducibility of these promoters in A. thaliana also suggests Click here for file that this model plant may be used to functionally analyze [http://www.biomedcentral.com/content/supplementary/1471- peach cold-induced genes as well as their corresponding 2229-9-121-S4.DOC] cis-elements and trans-acting factors. Additional file 5 The identification of these fruit tree cold-inducible pro- Sequence of the Pplox1 promoter and open reading frame. The data provided represents the sequences of the Pplox1 promoter and open read- moters as well as the conserved heterologous regulation of ing frame. these promoters in peach and Arabidopsis, demonstrates Click here for file that these two transformation assays may be used to [http://www.biomedcentral.com/content/supplementary/1471- molecularly define the cis-elements and trans-acting regu- 2229-9-121-S5.DOC] latory factors that are associated with cold-responsive genes. By better understanding the regulatory mecha- nisms associated with cold-responsive genes, we may bet- ter understand the molecular differences and similarities Acknowledgements between cold acclimation and chilling injury as well as the This work was supported by ICM P06-065-F; FDI G02P1001 (Chilean Genome Initiative) with funding from the Chilean government as well as role these processes play in fruit tree growth and fruit ASOEX (Asociación de Exportadores de Chile A.G.), FDF (Fundación para quality. el Desarrollo Frutícola) and Fundación Chile; Proyecto Consorcio BIOF- RUTALES S.A.; PBCT R11 and CONICYT Fellowship D-21080654 to AM. Authors' contributions AT: identified and cloned the promoters. AT, MS, LM and References HS drafted the manuscript. AT and MS: performed the dig- 1. Thomashow MF: So what's new in the field of plant cold accli- ital expression analysis. AM and AT: performed the con- mation? Lots! Plant Physiol 2001, 125(1):89-93. 2. Sharma P, Sharma N, Deswal R: The molecular biology of the struction of Arabidopsis transgenic plants as well as the low-temperature response in plants. Bioessays 2005, transient assay. HS: conceived, supervised and partici- 27(10):1048-1059. 3. Thomashow MF: PLANT COLD ACCLIMATION: Freezing pated in all the analysis. All authors read and approved Tolerance Genes and Regulatory Mechanisms. Annu Rev Plant the manuscript. Physiol Plant Mol Biol 1999, 50:571-599. 4. Zhu J, Dong CH, Zhu JK: Interplay between cold-responsive gene regulation, metabolism and RNA processing during Additional material plant cold acclimation. Curr Opin Plant Biol 2007, 10(3):290-295. 5. Fowler S, Thomashow MF: Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated Additional file 1 during cold acclimation in addition to the CBF cold response pathway. Plant Cell 2002, 14(8):1675-1690. Identification of fruit cold-induced contigs using correlated expression 6. 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Trends Plant Sci 2004, 9(12):591-596. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 15 of 15 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Plant Biology Springer Journals

Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

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Springer Journals
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Copyright © 2009 by Tittarelli et al; licensee BioMed Central Ltd.
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Life Sciences; Plant Sciences; Agriculture; Tree Biology
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1471-2229
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10.1186/1471-2229-9-121
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19772651
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Abstract

Background: Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset. Results: Digital expression analyses of EST datasets, revealed 164 cold-induced peach genes, several of which show similarities to genes associated with cold acclimation and cold stress responses. The promoters of three of these cold-inducible genes (Ppbec1, Ppxero2 and Pptha1) were fused to the GUS reporter gene and characterized for cold-inducibility using both transient transformation assays in peach fruits (in fruta) and stable transformation in Arabidopsis thaliana. These assays demonstrate that the promoter Pptha1 is not cold-inducible, whereas the Ppbec1 and Ppxero2 promoter constructs are cold-inducible. Conclusion: This work demonstrates that during cold storage, peach fruits differentially express genes that are associated with cold acclimation. Functional characterization of these promoters in transient transformation assays in fruta as well as stable transformation in Arabidopsis, demonstrate that the isolated Ppbec1 and Ppxero2 promoters are cold-inducible promoters, whereas the isolated Pptha1 promoter is not cold-inducible. Additionally, the cold-inducible activity of the Ppbec1 and Ppxero2 promoters suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. These results reveal that digital expression analyses may be used in non-model species to identify candidate genes whose promoters are differentially expressed in response to exogenous stimuli. Page 1 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 demonstrate that Arabidopsis may be used as a heterolo- Background Cold temperature is an environmental factor that plays an gous system to test the functionality of promoters. How- important role in plant growth and development. Tem- ever, this type of heterologous regulation may not exist for perate plants have developed mechanisms to adapt to all promoters and may not be conserved among all plant periods of low non-freezing temperatures, enabling these species. An alternative to functional analyses in heterolo- plants to survive subsequent freezing temperatures. This gous systems is transient transformation of fruits using process is called cold acclimation [1]. Cold acclimation is agro-infiltration. Agro-infiltration of fruits have been per- a complex process that involves physiological, biochemi- formed to test the activity of the 35S CaMV promoter cal and molecular modifications [2-4]. Hundreds of genes fused to reporter genes such as GUS or luciferase in toma- have been shown to have altered expression levels during toes, apples, pears, peaches, strawberries and oranges cold acclimation [5]. These alterations enable the plant to [15,16]. However, to our knowledge, it has not been used withstand freezing by creating a chronic response that to determine the activity of cold-inducible promoters protects the integrity of the cellular membranes, enhances within the fruit (in fruta). anti-oxidative mechanisms and accumulates molecular cryoprotectants [6]. To identify cold-responsive genes expressed in peach fruits, digital expression analyses of ESTs from fruits Under normal conditions, cold acclimation is initiated by exposed to four different postharvest conditions were ana- the cold temperatures of late fall and early winter, when lyzed [17]. Isolation of the promoter regions of three fruit trees lack fruits. Similar cold temperatures have been genes highly expressed in fruits that have undergone long- used in the fruit industry to store fruits for prolonged peri- term cold storage, allowed us to identify common regula- ods of time. These temperatures inhibit fruit ripening, tory elements present in these promoters. Functional thereby extending fruit postharvest life. Despite the bene- characterization of these promoters (stably in A. thaliana fits, peaches that are subjected to long periods of cold stor- and transiently in peach fruits) demonstrates that these age can develop chilling injury symptoms (i.e. woolliness are peach cold-inducible promoters and that there is a and internal breakdown) which reduce the postharvest conserved heterologous regulation of these promoters in quality of these fruits and results in significant economical peach and Arabidopsis. losses [7-9]. Methods Most of the efforts directed towards understanding the Digital expression analyses molecular basis of cold acclimation have been performed We have previously described the contigs used in this in the model plant A. thaliana [1-4]. Little is known about work [17]. The ESTs that make up these contigs represent what occurs under low, non-freezing temperatures in transcripts from peach fruit mesocarp at four different fruits or fruit trees. Since chilling injury occurs in fruits postharvest conditions. The post-harvest conditions that have undergone long-term cold storage, perhaps cold include: fruits processed in a packing plant (E1: non-ripe; acclimation processes are associated with this injury. A no long term cold storage); packing followed by a shelf- better understanding of cold acclimation and cold- life at 20°C for 2-6 days (E2: Ripe; no long term cold stor- responsive genes in peach trees and fruits may provide age; juicy fruits); packing followed by cold storage at 4°C clues about the association of cold acclimation and chill- for 21 days (E3: non-ripe; long term cold storage) and ing injury. packing followed by cold storage at 4°C for 21 days and shelf-life at 20°C for 2-6 days (E4: Ripe; long term cold Several transcription factors associated with cold acclima- storage; woolly fruits). tion have been shown to regulate the expression of cold- inducible genes containing conserved ABRE (abscisic acid As we described in Vizoso et al [17], the contigs that rep- response elements) and/or DRE (dehydration-responsive) resent differentially expressed genes were identified using elements in their promoters [10-13]. The regulation of the Winflat program that submits the sequence data to a cold-inducible promoters in peaches may be mediated by rigorous statistical analysis described by Audic and Clav- the interaction between promoters containing these types erie [18]http://igs-server.cnrs-mrs.fr. This analysis calcu- of cis-elements and orthologous transcription factors. lates the probability that a gene is equally expressed in However, the identification and functional characteriza- two different conditions by observing the distribution of tion of these types of promoters in fruit trees is lacking. tag counts (number of ESTs). Therefore, small probability values (p-values) are associated with non-symmetrical dis- We have demonstrated previously that there is a con- tributions, characteristic of differentially expressed genes served heterologous regulation of the wheat putative [18,19]. high-affinity Pi transporter, TaPT2 in both monocots (wheat) and dicots (Arabidopsis) [14]. These findings Page 2 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 To analyze the co-expression of differentially expressed synthesized from 5 ng of the mRNA in a 20 l final vol- genes, contigs were clustered using the Pearson linear cor- ume. The reaction mix was prepared using the ImProm- relation coefficient [19,20]. Briefly, contigs with at least II™ reverse Transcription System (Promega, Madison, five ESTs were selected to make the expression profile USA) and anchored oligo (dT) of 18-mers, according to matrix, which consisted of 1,402 rows (the contigs) and 4 the manufacturer's instructions. As an internal control for columns (four cDNA libraries). The similarity between normalization, heterologous mRNA (1.2 kb mRNA cod- clusters and libraries was estimated using an un-centered ing for Kanamycin) was added to each mRNA sample. To Pearson's correlation coefficient in the Cluster 3.0 pro- control for genomic DNA contamination, PCR amplifica- gram [20]http://rana.lbl.gov/EisenSoftware.htm. Pearson tion was performed on template RNA that was not reverse correlation coefficients > 0.85 (zero values indicate no transcribed. To confirm that the amplified fragments cor- association and a coefficient equal to 1 indicate a fully respond to the cDNAs of interest, these fragments were correlated pattern) are indicated by an asterisk in Addi- cloned in pBluescript and sequenced (Macrogen, Korea). tional File 1. Dendrograms were constructed from the pair The primer sequences used to amplify the internal regions wise distances using the UPGMA algorithm. The results of the basic endochitinase Ppbec1 (BEC226F and were visualized and analyzed using the Java TreeView pro- BEC576R), dehydrin Ppxero2 (DX-82F and DX176R), gram http://jtreeview.sourceforge.net. thaumatin Pptha1 (THA30F and THA382R), lipoxygenase Pplox1 (LOX982F and LOX1267R) and the actin Ppact7 Gene Ontology molecular function and biological process (ACT-F and ACT-R) genes are shown in Table 1. Primers annotations of the contigs are described in Vizoso et al used to amplify a 323 bp fragment of the cDNA from the [17]. Each annotation and contig assembly was manually Kanamycin mRNA control are: "Upstream Control corrected, when necessary. Primer" (5'-gCCATTCTCACCggATTCAgTCgTC-3') and "Downstream Control Primer" (5'-AgCCgCCgTCCCgT- mRNA isolation and reverse transcriptase (RT)-PCR CAAgTCAg-3'). PCR reactions were performed by diluting The kit Oligotex™ mRNA Spin-Column (Qiagen, New the cDNAs a 100 fold and using 1 l of each dilution as a York, USA) was used to purify mRNA. The mRNA was template in a final reaction volume of 20 l, containing purified from pools of total RNA obtained from peach 0.5 M primers; 0.2 mM dNTPs; 1.5 mM MgCl ; 5U Taq polymerase and 1× buffer. The PCR conditions were: fruit mesocarp (O'Henry var.) representing the stages E1, E2, E3 and E4 as described previously [17,21]. The mRNA 93°C for 5 min and then a variable number of cycles (26 was quantified using the Poly (A) mRNA Detection Sys- to 34) at 93°C for 30 sec, 1 min at 55°C, and 1 min at tem™ (Promega, Madison, USA). First strand cDNA was Table 1: Primers used in this study Primer Sequence (5'3') Method BEC226F gTCAgCAgCgTCgTTAgCTC RT-PCR BEC576R gAgTTggATgggTCCTCTgC DX-82F CCAAACCAAAgCCAgTTTCATTCA DX176R CCAggTTTTgTATgAgTgCCgTA THA30F ACCTTggCCATCCTCTTCTT THA382R AgAAATCTTgACCCCCgTTC LOX982F AAggAgCTCTTgACgTTggA LOX1267R TgCTAACAggTgggAAAACC ACT-F CCTTCCAgCAgATgTggATT ACT-R AgATTAggCAAggCgAggAT BEC87-GSP1 TgCATTTCCAgCTTgCCTCCCACATTg Genome Walker BEC55-GSP2 CTgAgATCCCTAACAgCAAAgCTAgggATA DX85-GSP1 ACCggTTCCggTggTggTgTgATgAACC DX46-GSP2 ACTCATCAgTCTTAgTAggCTCgggTgTT THA82-GSP1 TgATTTTAgCTgCATgTgCACCTgAgAA THA-1-GSP2 CgTCATggAAATgTCTTAATTggCTTgCTg LOX101-GSP1 gAAgAAAACAAATTgggAggAggAgAA LOX63-GSP2 gCgTgTTCCAAAgAACACAATTCAgTgCCTT BEC-32BamHI ggATCCTgATCTgTggATTgggTTTCgTgg Subcloning promoters DX24BamHI ggATCCgggTgTTgAACCAAAATgCgCCATT Page 3 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 72°C. The PCR reaction was with a final step at 72°C for program [25]http://wingless.cs.washington.edu/YMF/ 10 min. YMFWeb/YMFInput.pl. Only the statistically significant motifs (Z score value > 6.5) were selected [26]. Cloning of the promoters Genomic DNA was isolated from peach leaves (Prunus per- Growth, transformation and cold treatments of A. thaliana sica var. persica (L.) Batch cv. O'Henry) as described in Wild-type and transgenic A. thaliana (ecotype Columbia) Manubens et al [22]. The Universal Genome Walker™ Kit were grown in a mixture of soil-vermiculite (3:1) in a -2 - (Clontech Laboratories, Inc., Palo Alto, CA, USA) was growth chamber with a 16-h light cycle (140 mol m s used to isolate the promoters regions of Ppbec1, Ppxero2, ) at 22°C. Alternatively, seeds were surface sterilized as Pptha1 and Pplox1. The isolated genomic DNA was described in Gonzalez et al [27], plated on Murashige- digested with four restriction enzymes (EcoRV, PvuII, SspI, Skoog (1 × MS) media containing 0.8% agar, 0.1% and MlsI). DNA fragments containing adaptors at both sucrose and 50 mg/l Kanamycin for transgenic lines and ends were used as a template for amplifying the promoter grown under the same conditions as the soil-grown regions. GSP1 and GSP2 gene specific primers were plants. designed to isolate the promoters (Table 1). For the first group of PCR reactions, a specific adaptor primer (AP1, 5'- Transgenic Arabidopsis was obtained by using the ggATCCTAATACgACTCACTATAgggC-3') and the GSP1 GV3101 A. tumefaciens-mediated floral dip method [28]. primers specific for each gene were used. The final primer A. tumefaciens previously transformed with the binary vec- concentration in the PCR reaction was 0.2 M in a final tor pBI101.3 harboring the promoter::uidA fusions: volume of 50 L. Manual Hot Start was performed using Ppbec1::uidA (PBIPpbec1); Ppxero2::uidA (pBIPpxero2); 5 U of the Synergy DNA polymerase (Genecraft, Münster, Pptha1::uidA (pBIPptha1), or the control vectors pBI121 Germany). The conditions for this first round of amplifi- (containing the 35S CaMV promoter) and pBI101.3 (pro- cations was: 1 cycle at 93°C for 10 min, 7 cycles of 93°C moterless), were used. In cold treatments, T homozygous for 30 sec, 72°C for 15 min, followed by 37 cycles of 93°C transgenic Arabidopsis seedlings were grown on plates for 30 sec, 67°C for 15 min. For the nested PCR, the spe- containing 1× MS media, 0.8% agar, and 0.1% sucrose in -2 - cific adaptor primer 2 (AP2, 5'-ACTATAgggCACgCgTggT- a growth chamber with a 16-h light cycle (140 mol m s ) at 24°C for two weeks, and then transferred to 4°C for 3') and the gene specific GSP2 primers were used. As a DNA template in these reactions, 1 L of a 50 fold dilu- 7 days. A minimum of three independent transgenic lines tion of end-product of the first round of amplifications were used for each construct. was used. The conditions for the second round of ampli- Peach fruit transient transformation and cold treatments fication were: 1 cycle at 93°C for 10 min, 5 cycles (7 cycles in the case of Ppxero2) of 93°C for 30 sec, 72°C for 15 A. tumefaciens transformed with the vectors pBIPpbec1, min, followed by 20 cycles (30 cycles in the case of pBIPpxero2, pBIPptha1, pBI121 or pBI101.3 were grown Ppxero2) of 93°C for 30 sec, 67°C for 15 min. The ampli- in LB medium supplemented with Kanamycin (100 g/ fied products were cloned in pGEM-T vector and ml), Rifampicin (10 g/ml) and Gentamycin (100 g/ sequenced (Macrogen, Korea). The Ppbec1 and Ppxero2 ml). The cultures were grown for two days at 28°C until promoters were subsequently amplified from the pGEM- they reached an OD between 0.6 and 0.8. The culture T clones using the AP2 and BEC-32BamHI or was then centrifuged and the pellet re-suspended in MMA DX24BamHI primers, respectively (Table 1). The products medium (1× MS, MES 10 mM (pH 5.6), 20 g/l sucrose, of this amplification were also cloned in the pGEM-T vec- and 200 M acetosyringone) to reach an OD of 2.4. tor and re-sequenced (Macrogen, Korea). The promoter Approximately 0.7 mL of this bacterial suspension was fragments were extracted from the pGEM-T vector (includ- used to infiltrate mature fruits from O'Henry, Elegant Lady ing the Pptha1 promoter), with a BamHI-SalI sequential and Florida King varieties of peach as described by Spo- digestion, and transcriptionally fused to the uidA reporter laore et al [15]. gene in the promoterless binary vector pBI101.1 [23]. The binary vector was introduced into A. tumefaciens To analyze the promoter activity at 20°C, the fruits infil- (GV3101) for subsequent Arabidopsis and peach fruit trated with the different constructs, were stored in a dark transformations. growth chamber for five days. To analyze the cold-respon- sive promoter activity, the infiltrated fruits were stored 2 Promoter sequences analysis days post-infiltration (dpi) in a dark growth chamber at Analysis of putative transcription factor binding sites was 4°C for 10 days. After the growth chamber incubation carried out using the database PLACE http:// time, the infiltrated region of the fruit was extracted with www.dna.affrc.go.jp/htdocs/PLACE/[24] coupled with a cork bore and stained for GUS activity as described by visual analyses. To identify predicted conserved motifs, Tittarelli et al [14]. the promoter sequences were analyzed using the YMF 3.0 Page 4 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 GUS activity measurement suggesting that these may be putative orthologs. The puta- Histochemical staining of Arabidopsis seedlings for -glu- tive Arabidopsis orthologs that are induced or repressed curonidase (GUS) activity was performed as described by by cold, based on ColdArrayDB analyses http://cold.stan Jefferson et al [23], with the following modifications: ford.edu/cold/cgi-bin/data.cgi are shown in Table 2. Only transgenic Arabidopsis seedlings used in the cold-treat- 29 contigs (18% of the 164 cold-induced genes) share sig- ments described earlier were vacuum infiltrated in 50 mM nificant sequence identity with genes of unknown func- NaH PO , pH 7.0; 0.1 mM X-Gluc; 10 mM EDTA and tion. Approximately 38% of these contigs (11 contigs) 2 4 0.1% Triton X-100. These samples were incubated in the share significant sequence identity with plant gene dark at 37°C for 24-72 h. Samples that did not develop sequences annotated as expressed proteins. Six of the con- color after 72 h were considered negative for GUS activity. tigs with unknown function do not share sequence iden- Plant material was subsequently fixed in 0.04% formalde- tity with any sequences in the public databases, suggesting hyde, 0.04% acetic acid and 0.285% ethanol for 30 min, that these are novel genes. followed by an ethanol dilution series to remove chloro- phyll from the plant tissue (70% ethanol for 1 h, 100% Annotation frequency comparative analyses of cold- ethanol for 1 h, 70% ethanol for 1 h and distilled water). induced (164 contigs), cold-repressed (138 contigs) or contigs unrelated to cold (1,238 contigs), revealed an Slices (2 mm) of transiently transformed peaches were overrepresentation of stress response genes and an under- imbibed in the GUS staining solution (0.72 M K HPO ; representation of genes related to energy metabolism in 2 4 0.17 M KH PO ; 0.5 mM K Fe(CN) ; 0.5 mM K Fe(CN) ; fruits that were stored in the cold (Figure 1). Among the 2 4 3 6 4 6 1× Triton X-100; 12.7 mM EDTA; 20% (v/v) methanol genes related to stress response we identified four contigs and 0.5 mM X-Gluc) [15]. Samples were vacuum-infil- that are similar to thaumatin-like proteins: C1708, trated for 30 min at room-temperature and then incu- C2177, C2317 and C2147 (98%, 99%, 98% and 93% bated overnight at 37°C. Fluorometric GUS assays were amino acid identity with P. persica thaumatin-like protein performed as described by Jefferson et al [23]. The Arabi- 1 precursor, respectively, GenBank accession number: dopsis seedlings were ground in a mortar using liquid P83332). Three of the stress response genes are similar to nitrogen, and the tissue powder was transferred to a chitinases: C910 (76% amino acid identity with Malus microtube. One ml of the extraction buffer (50 mM domestica class III acidic endochitinase, GenBank acces- NaH PO , pH 7.0; 1 mM EDTA; 0.1% Triton X-100; 0.1% sion number: ABC47924); C2131 (74% amino acid iden- 2 4 (w/v) sodium laurylsarcosine and 5 mM dithiothreitol) tity with Galega orientalis class Ib basic endochitinase, was added. Samples were centrifuged for 10 min at 12,000 GenBank accession number: AAP03087) and C2441 g at 4°C and the supernatant was transferred to a new (72% amino acid identity with A. thaliana class IV chiti- microtube. The fluorogenic reaction was carried out in 2 nase, GenBank accession number: NP_191010). Two of ml volume containing 1 mM 4-methyl umbelliferyl glu- the stress response genes are similar to dehydrins: C254 curonide (MUG) in an extraction buffer supplemented (97% amino acid identity with P. persica Ppdhn1, Gen- with a 50 L aliquot of the protein extract supernatants. Bank accession number: AAC49658) and C304, 100% The protein quantity of the sample extracts was deter- amino acid identity with P. persica type II SK2 dehydrin mined as described previously [29], using bovine serum Ppdhn3 (Genbank accession number: AAZ83586). albumin (BSA) as a standard. Cold-induced expression of Ppbec1, Ppxero2 and Pptha1 We evaluated the expression levels of three cold-induced Results Identification of peach cold-regulated genes by digital candidate genes by RT-PCR: a basic endochitinase expression analyses of EST datasets (C2131, Ppbec1), a dehydrin (C254, Ppxero2) and a thau- Coordinated gene expression analyses of peach fruit ESTs matin-like protein (C2317, Pptha1). These genes were datasets revealed 10 major hierarchical clusters (Addi- chosen due to the high number of ESTs in cold-stored tional File 1), containing unique contigs. We identified fruits (E3), as revealed by the digital expression analyses 164 contigs with preferential expression in fruits stored at (Figure 2). The expression level of a contig similar to 4°C (E3: non-ripe; long term cold storage). Table 2 con- lipoxygenase (C3336, Pplox1) that does not express pref- tains a complete list of these contigs together with their erentially in cold stored fruits (E3) as well as the expres- annotations, GO biological process annotations and the sion level of a contig (C407, Ppact7) that does not origin of the ESTs in each contig. Contigs with statistically significantly change expression under the different post- differential expression, in E3 compared to the other stages harvest conditions, were analyzed (Figure 2). Interest- are also indicated. ingly, all five genes analyzed showed an expression pat- tern significantly similar to the ones predicted by the Approximately 95% of the 164 cold-induced peach genes digital expression analyses (Figure 2). The genes Ppbec1, share significant identity with sequences in Arabidopsis, Ppxero2 and Pptha1 have an increased expression in cold- Page 5 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Table 2: Putative function of 164 genes preferentially expressed in cold stored peach fruits. 1 2 Contig E3 E1+E2+E4 AC test Putative Function;Arabidopsis ortholog Biological process unknown (GO:0000004) C517 10 6 E4 NC domain-containing protein (located in mitochondrion); At5g06370 C675 12 4 E2; E4 Expressed protein; At3g03870 C774* 11 4 E2; E4 Novel gene C2089 20 0 E1; E2; E4 Expressed protein (located in endomembrane system); At5g64820 C2112 31 2 E1; E2; E4 Cupin family protein (nutrient reservoir activity); At1g07750 C2139 12 0 E1; E2; E4 Novel gene C4065 13 8 E2 Expressed protein; At5g52870 C273 5 2 Expressed protein; At5g24660 C477 7 6 Expressed protein (located in endomembrane system); At5g64510 C1207* 8 7 Novel gene C2134 3 2 Expressed protein; At1g71080 C2148 4 1 Novel gene C2155 4 1 Expressed protein; At5g11730 C2167 3 2 RWD domain-containing protein; At1g51730 C2173 7 1 Expressed protein (located in mitochondrion);At5g60680 C2193 3 2 Novel gene C2211 8 1 Ankyrin repeat family protein (protein binding); At2g28840 C2241 6 2 Expressed protein (located in mitochondrion); At5g51040 C2267 7 0 Integral membrane family protein; At4g15610 C2315 5 3 Expressed protein; At1g70780 C2318 3 2 Ribosome associated membrane protein RAMP4; At1g27350 C2343 9 9 Novel gene C2560 6 1 Expressed protein; At3g27880 C2591 6 1 Expressed protein (located in mitochondrion); At5g24600 C2682* 4 2 N-methyl-D-aspartate receptor-associated protein; At4g15470 C2713 4 1 Glycine-rich protein; At4g22740 C2778 12 7 Zinc finger (AN1-like) family (DNA and zinc ion binding); At3g52800 C2806 8 2 C2 domain-containing protein; At1g22610 C3094 3 2 Reticulon family protein (located in ER and mitochondrion); At3g10260 Cell homeostasis (GO:0019725) C2265 91 38 E1; E2; E4 Metallothionein-like protein; At5g02380 C2202* 5 1 Metallothionein-like protein; NSM Cell organization and biogenesis (GO:0016043) C734 17 9 E2; E4 Proline-rich/extensin family; At2g27380 C1240 62 20 E1; E2; E4 Proline-rich/extensin family; At1g54215 C2494* 10 3 E2 Actin-depolymerizing factor 4; At5g59890 C2831 20 6 E1; E2; E4 Leucine-rich repeat/extensin family; At4g13340 C3041 12 5 E2; E4 Leucine-rich repeat/extensin family; At4g13340 C831 4 2 BON1-associated protein (BAP2); At2g45760 C1062 4 1 Invertase/pectin methylesterase inhibitor family; At5g62360 C2060 7 3 Expansin family; At4g38400 C2086* 6 1 Arabinogalactan-protein; At5g64310 C2073 6 2 Zinc finger protein (CYO1); At3g19220 C2574 7 3 Invertase/pectin methylesterase inhibitor family; At2g01610 C2762* 4 1 Profilin 4; At2g19770 C2815 4 1 Phytochelatin synthetase; At4g16120 Cellular protein metabolism (GO:0044267) C228* 112 51 E1; E2; E4 DJ-1 family protein/protease-related; At3g02720 C379* 50 21 E1; E2; E4 DJ-1 family protein/protease-related; At3g02720 C1027* 47 46 E1; E2; E4 Heat shock cognate 70 kDa protein 1; At5g02500 C1660 51 25 E1; E2; E4 Cysteine proteinase inhibitor-related; At2g31980 C2099* 13 1 E1; E2; E4 DJ-1 family protein/protease-related; At3g02720 C2436 17 3 E1; E2; E4 Rhomboid family protein; At1g63120 C2715 41 21 E1; E2; E4 Luminal binding protein 1 (BiP-1); At5g28540 C2066* 3 2 60S ribosomal protein L23A; At3g55280 Cellular protein metabolism (GO:0044267) C2072* 6 2 DNAJ heat shock protein; At3g44110 C2217* 7 3 20S proteasome beta subunit A; At4g31300 C2308* 9 0 Heat shock protein 70; At3g12580 C2345* 4 2 Ubiquitin carrier protein E2; At2g02760 C2364 5 2 Phosphatase-related (SGT1B); At4g11260 C2388 5 3 F-box family protein (AtSKP2;2); At1g77000 Page 6 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Table 2: Putative function of 164 genes preferentially expressed in cold stored peach fruits. (Continued) C2593 4 1 C3HC4-type RING finger family protein; At1g26800 C2597 6 2 26S proteasome regulatory subunit S3; At1g20200 C2691 7 6 C3HC4-type RING finger family protein; At5g47610 C2360 10 7 Structural constituent of ribosome; At5g15260 C2735 9 4 40S ribosomal protein S9; At5g39850 C3022 6 2 Translation initiation factor IF5; At1g36730 C3051* 5 2 DJ-1 family protein/protease-related; At3g02720 C3520 4 1 60S ribosomal protein L36; At3g53740 C3551* 11 4 Cysteine proteinase inhibitor; At3g12490 C3656 6 4 40S ribosomal protein S26; At3g56340 C4131 3 2 C3HC4-type RING finger family protein; At5g48655 Development (GO:0007275) C2802 10 2 E1 Senescence-associated protein; At1g78020 C2919 10 1 E1; E2 Senescence-associated protein; At5g20700 C1113 6 3 Auxin-responsive protein; At3g25290 C3887* 4 1 Maternal effect embryo arrest 60; At5g05950 C3942 6 4 SIAMESE, cyclin binding protein; At5g04470 C2457 6 0 Nodulin MtN3 family protein; At5g13170 Generation of precursor metabolites and energy (GO:0006091) C2304 7 1 NADH dehydrogenase; At4g05020 C2541 8 1 Uclacyanin I; At2g32300 C2552 5 0 Flavin-containing monooxygenase family protein; At1g48910 Metabolism (GO:0008152) C1017 15 9 E2 Xyloglucan endotransglycosylase; At4g25810 (carbohydrate) C1258* 19 2 E1; E2; E4 Phosphoesterase family protein; At3g03520 (phospholipid) C2373 15 8 E2; E4 -alanine-pyruvate aminotransferase; At2g38400 (amino acid) C2397* 27 9 E1; E2; E4 S-adenosylmethionine decarboxylase; At3g02470 (polyamine) C2554* 17 3 E1; E2; E4 UDP-glucoronosyl/UDP-glucosyl transferase; At5g65550 (anthocyanin) C2957 11 0 E1; E2; E4 Glycosyl hydrolase family 3; At5g49360 (carbohydrate) C2669 61 28 E1; E2; E4 Phosphoserine aminotransferase; At4g35630 (amino acid) C656 4 3 Nucleoside diphosphate kinase 3; At4g11010 (nucleotide) C821* 4 1 UDP-glucoronosyl/UDP-glucosyl transferase; At5g49690 (anthocyanin) C926* 7 6 (1-4)--mannan endohydrolase; At5g66460 (carbohydrate) C1000* 8 2 Alkaline alpha galactosidase; At1g55740 (carbohydrate) C1693 9 3 Haloacid dehalogenase-like hydrolase; At5g02230 C1943 4 3 2-oxoglutarate-dependent dioxygenase; At1g06620 (ethylene) C2424 5 0 -amylase; At4g17090 (starch) C2495 8 1 Cinnamoyl-CoA reductase; At4g30470 (lignin) C2522 11 8 Glycosyl hydrolase family 5; At1g13130 (carbohydrate) C2569 7 1 Short-chain dehydrogenase/reductase family; At3g61220 C2602 5 0 Short-chain dehydrogenase/reductase family; At4g13250 C2610 5 0 Galactinol synthase; At3g28340 (carbohydrate) C2222 6 0 Carboxyesterase 5; At1g49660 C2635 6 4 GNS1/SUR4 membrane family protein; At4g36830 (fatty acid) C2705 7 4 DSBA oxidoreductase family protein; At5g38900 (organic acid) C669 4 2 Dehydrogenase; At5g10730 C2936 4 1 Pyruvate decarboxylase; At5g17380 (glycolisis) C2940 4 1 Farnesyl pyrophosphate synthetase 1; At5g47770 (lipid) C2976 6 1 Aminoalcoholphosphotransferase; At1g13560 (phospholipid) C3047* 7 4 Dienelactone hydrolase; At3g23600 (alkene) Metabolism (GO:0008152) C3058* 5 1 Cellulose synthase; At4g39350 (cellulose) C3152 8 3 Purple acid phosphatase; At3g52820 (phosphate) C3225 4 1 Acyl-activating enzyme 12; At1g65890 (phospholipid) C4127 6 2 -3fatty acid desaturase; At5g05580 (fatty acid) C86 6 3 Embryo-abundant protein; At2g41380 C677 4 2 Cyclic phosphodiesterase; At4g18930 (RNA) C802 4 3 RNA recognition motif-containing protein; At5g04600 (RNA) C2798 3 2 Small nuclear ribonucleoprotein G; At2g23930 (RNA) Response to stress (GO:0006950) C30 57 27 E1; E2; E4 Cold acclimation WCOR413-like protein; At3g50830 C254 71 10 E1; E2; E4 Dehydrin Xero2; At3g50970 C304* 189 124 E1; E2; E4 Type II dehydrin SKII; (ERD14) At1g76180 C910 126 38 E1; E2; E4 Class III acidic endochitinase; At5g24090 C1479 96 25 E1; E2; E4 Harpin inducing protein; At5g06320 C1708 30 12 E1; E2; E4 Thaumatin-like protein; At1g20030 C2131 65 2 E1; E2; E4 Class Ib basic endochitinase; At3g12500 Page 7 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Table 2: Putative function of 164 genes preferentially expressed in cold stored peach fruits. (Continued) C2177 15 4 E1; E4 Thaumatin-like protein; At1g20030 C2317 67 6 E1; E2; E4 Thaumatin-like protein; At1g20030 C2514* 20 15 E2 Glutathione peroxidase; At4g11600 C2528 22 7 E1; E2; E4 Hevein-like protein; At3g04720 C2655* 10 6 E4 DREPP plasma membrane polypeptide; At4g20260 C2988* 37 6 E1; E2; E4 Polygalacturonase inhibiting protein; At5g06860 C2473* 10 0 E1; E2; E4 Major allergen Pru p 1; At1g24020 C2147 8 0 Thaumatin-like protein; At1g20030 C2441 8 1 Class IV chitinase; At3g54420 C2507 5 2 Pyridoxine biosynthesis protein; At5g01410 C2556 5 0 4-aminobutyrate aminotransferase; At3g22200 C2578 3 2 Aldehyde dehydrogenase; At1g44170 C2926 7 2 Wounding stress inducimg protein; At4g24220 C3613* 3 2 Harpin inducing protein; At3g11660 C1889* 5 4 Major allergen Pru p 1; At1g24020 C3858* 4 2 Late embryogenesis abundant protein 3; At4g02380 Signal transduction (GO:0007165) C815 9 1 Leucine-rich repeat family protein; At3g49750 C1192* 6 5 CBL-interacting protein kinase 12; At4g18700 C2205 5 4 Ser/Thr kinase; At2g47060 C2312* 8 3 Touch-responsive/calmodulin-related protein 3; At2g41100 C2430* 6 6 Remorin family protein; At5g23750 C2548 10 6 Fringe-related protein; At4g00300 C2829* 3 2 Protein kinase, 41K; At5g66880 C2853 5 3 GTP-binding protein Rab2; At4g17170 C3690* 10 8 Ser/Thr kinase; At4g40010 Transcription (GO:0006350) C452 4 2 Myb family; At5g45420 C2742* 5 1 DREB subfamily A-6; At1g78080 C3420* 8 4 MADS-box protein (AGL9); At1g24260 C3812 3 2 WRKY family; At4g31550 Transport (GO:0006810) C716 13 5 E2; E4 Proton-dependent oligopeptide transport family; At5g62680 C1846 15 10 E4 Auxin efflux carrier family protein; At2g17500 C2091 18 0 E1; E2; E4 Protease inhibitor/seed storage/lipid transfer family; At1g62790 C163 4 1 Vesicle-associated membrane protein; At1g08820 C208 9 2 GTP-binding secretory factor SAR1A; At4g02080 C235 5 4 Sugar transporter; At1g54730 C484 11 6 Porin; At5g67500 C1526 5 4 emp24/gp25L/p24 protein; At3g22845 Transport (GO:0006810) C2062 3 2 Ripening-responsive protein; At1g47530 C2236 3 2 Ras-related GTP-binding protein; At4g35860 C2476 9 1 Bet1 gene family; At4g14450 C2679 5 0 Sulfate transporter ST1; At3g51895 C3063 4 2 Amino acid carrier; At1g77380 C3066 4 1 Sulfate transporter; At3g15990 C3099 3 2 Ras-related GTP-binding protein; At1g52280 Statistically significant cold-induced contigs detected with the Audic and Claverie test (p < 0.01) vs. E1, E2 or E4 cDNA libraries. The column shows the cDNA library with differences to E3. The column described the locus identifier (id) of the Arabidopsis most similar protein. The locus ids with  [37] are the Arabidopsis cold response genes similarly up-regulated; the locus ids with  [31] are the genes with opposite response, down-regulated in Arabidopsis (ColdArrayDB; http:// cold.stanford.edu/cgi-bin/data.cgi). Between parentheses: the principal subcategory of the biological process "metabolism" associated to the annotation. 4 -10 NSM: Not significant match (E value < 10 ) with A. thaliana sequences. -10 * Contigs that shown significant sequence homology (e value > 10 ) with contigs from others hierarchical clusters. Page 8 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 A Figure 1 nnotation frequency comparison of cold-induced, cold-repressed or unrelated to cold-induction contigs Annotation frequency comparison of cold-induced, cold-repressed or unrelated to cold-induction contigs. The frequency of contigs that are associated with a specific Gene Ontology are expressed as the percentage of the total annota- tions for each analyzed group (164 for the cold-induced, 138 for the cold-repressed and 1,238 for unrelated to cold-induction). The numbers of contigs in each group, belonging to each biological process classification, are show at the top of each bar. The category "others process" are: cell adhesion (GO: 0007155, 1 contig); cell communication (GO: 0007154, 1 contig); cell cycle (GO: 0007049, 5 contigs); cell death (GO: 0008219, 1 contig); cell homeostasis (GO: 0019725, 4 contigs); organism physiolog- ical process (GO: 0050874; 1 contig); regulation of GTPase activity (GO: 0043087; 1 contig); response to stimulus (GO: 0050896; 10 contigs) and viral life cycle (GO: 0016032; 1 contig). stored fruits, whereas the Pplox1 gene increased expression Cis-element regulatory motifs related to cold gene expres- in woolly fruits rather than cold-stored fruits. sion regulation such as ABRE [13], MYCR [31,32], MYBR [31,33] and DRE/CRT [34] were identified in all three pro- Identification of conserved motifs in the promoters of cold- moters of these cold-inducible genes (Figure 3). In addi- inducible genes Ppbec1, Ppxero2 and Pptha1 tion, three statistically significant predicted motifs were We cloned 826 bp, 1,348 bp and 1,559 bp fragments cor- present in the promoters of these cold-inducible genes responding to the regions upstream of the translation start (TACGTSGS, TGTGTGYS and CTAGAASY (Figure 3). codons of Ppbec1, Ppxero2 and Pptha1, respectively. The These motifs were not found in the Pplox1 promoter iden- sequences of these promoter regions as well as the cDNA tified in this work (Additional File 5). of their corresponding genes are shown in the Additional Files 2, 3 and 4. Cold-induced Ppbec1 and Ppxero2 promoters in transiently transformed peach fruits and stably transformed Arabidopsis The high sequence identity between the Ppxero2 contig with the coding region of Ppdhn1[30] was also observed Transient transformation assays of peach fruits revealed within the promoter sequences of these two genes. Only that all three cloned promoters (pBIPpbec1, pBIPxero2 one nucleotide difference at position -469 was found, sug- and pBIPptha1) were able to activate GUS (uidA) expres- gesting that Ppxero2 and Ppdhn1 may be the same gene sion (Figure 4). However, only the pBIPpbec1 and (Additional File 3). However, the promoter isolated in pBIPxero2 promoter constructs showed cold-inducible this work is about 230 bp longer (at the 5' end) than the increases in GUS activity (Figure 4). The pBIPtha1 con- previously published promoter [30]. struct was expressed at both 20°C and 4°C. Comparable Page 9 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 All three constructs were able to activate GUS expression, but only the Ppbec1 and Ppxero2 promoters (pBIPpbec1 and pBIPxero2, respectively) induced expression in response to cold (Figure 5). As observed with the fruit transient transformation assays, the Pptha1 promoter (pBIPtha1) expressed GUS under all conditions analyzed. Discussion and Conclusion Digital expression analyses of EST datasets have permitted us to identify a large diversity of cold-inducible genes in peach fruits, three of which were chosen for further anal- yses (Ppbec1, Ppxero2 y Pptha1). Both digital expression analyses and RT-PCR suggest that the Ppbec1, Ppxero2 and Pptha1 are cold-inducible genes. The promoters of these cold-inducible genes were isolated and characterized using both transient transformation assays in peach fruits and stable transformation in Arabidopsis. These analyses have revealed that the isolated Ppbec1 and Ppxero2 pro- moters are cold-inducible promoters, whereas the isolated Pptha1 promoter was not cold-inducible. These results, therefore, demonstrate that the isolated Ppbec1 and Ppxero2 promoters are sufficient for cold-induced gene expression. Furthermore, these results suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. Plants respond to cold temperatures by modifying the E tern Figure 2 valu s of selected ation of the accuracy of genes by RT-PCR the predicted expression pat- transcription and translation levels of hundreds of genes Evaluation of the accuracy of the predicted expres- [35,36]. These acute molecular changes are related to sion patterns of selected genes by RT-PCR. (A) RT- plant cell physiological and biochemical modifications PCR analysis of RNA expression of three cold-induced genes: (cold acclimation) that lead to stress tolerance and cold Ppbec1, Ppxero2, and Pptha1 under different post-harvest adaptation (a chronic response). In peach fruits, cold tem- conditions. These post-harvest conditions include: fruits peratures induce chilling injury, possibly due to global processed in a packing plant (E1: non-ripe; no long term cold transcriptome changes [37]. With the exception of studies storage); packing followed by a shelf-life at 20°C for 2-6 days in the model organism A. thaliana [4] and work published (E2: Ripe; no long term cold storage; juicy fruits); packing fol- recently [17,38], little is known about the peach global lowed by cold storage at 4°C for 21 days (E3: non-ripe; long transcriptional response to cold. Using the Pearson corre- term cold storage) and packing followed by cold storage at 4°C for 21 days and shelf-life at 20°C for 2-6 days (E4: Ripe; lation coefficient, we analyze the coordinated gene expres- long term cold storage; woolly fruits). The expression level sion of 1,402 contigs. This analysis revealed 164 genes of Pplox1 was analyzed as a control for genes that do not preferentially expressed in peach fruits, of which digital express preferentially in cold stored fruits (E3). Ppact7 was expression analyses [18] revealed 45 of these genes (27%) analyzed as a control for genes that do not significantly with statistically significant cold-induction. A large pro- change expression levels between the four post-harvest con- portion of the contigs preferentially expressed at 4°C ditions analyzed. The two arrows associated with each gel (around 74% of the total) do not exhibited significant represent 500 bp (upper) and 300 bp (lower). The number of -10 sequence homology (e-value < e ) with the rest of the ESTs associated with each contig and library source is indi- analyzed contigs (Table 2). This result could suggest that cated. (B) Densitometry quantification of the expression these contigs represent genes with non-redundant func- level obtained by RT-PCR, the figure shows the bands inten- tions that will have a special importance during the expo- sities for each gene relative to Ppact7 intensity. sure of the fruits to low temperatures. results were seen in fruits from three different peach vari- Among the highly expressed genes in cold stored fruits, we eties (data not shown). found genes related to stress response in plants, including three dehydrins (C30, C254 and C304), three chitinases Similar results were seen when these promoter-GUS con- (C910, C2131 and C2441), four thaumatin-like proteins structs were analyzed in stably transformed Arabidopsis. (C1708, C2177, C2317 and C2147), and polygalacturo- Page 10 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Putative Figure 3 cis-regulatory elements identified in Ppbec1, Ppxero2 and Pptha1 promoter sequences Putative cis-regulatory elements identified in Ppbec1, Ppxero2 and Pptha1 promoter sequences. Topologies of the Ppbec1 (A), Ppxero2 (B) and Pptha1 (C) promoters are shown. The promoters are draw proportionally (the bar correspond to 100 bp). Boxed regions: predicted 5' UTR region. Black arrow shows the position of different cis-regulatory elements related to low temperature responses: ABRE, DRE/CRT, MYBR and MYCR. The putative cis-regulatory elements identified by the motif prediction program YMF3.0 are shown as grey triangle, black circle and asterisk. The sequences, the symbol and the sig- nificance score (Zscore) of the motifs, are shown in the upper left corner. The degenerate bases allowed in the motifs are S (C or G) and Y (C or T). Note: in order to ensure at the legibility of the figure, not all cis-elements are marked in (B) and (C). However, the complete sequences of these promoters are available in Additional Files 3 and 4. Cold-inducible peach Figure 4 Ppbec1 and Ppxero2 promoters in transiently transformed peach fruits Cold-inducible peach Ppbec1 and Ppxero2 promoters in transiently transformed peach fruits. (A) Structure of the binary vector constructs used for functional analysis of the Ppbec1, Ppxero2 and Pptha1 promoter-uidA fusions. LB and RB: left and right T-DNA border. (B) Histochemical GUS staining of fruit slices from agro-infiltrated peaches stored at 20°C for 5 days post-inoculation or 4°C for 10 days. These images correspond to the transient transformation of O'Henry variety fruits. How- ever, similar results were seen in all varieties assayed (data not shown). Page 11 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 Figure 5 Conserved heterologous regulation of the cold-inducible peach Ppbec1 and Ppxero2 promoters in transgenic Arabidopsis plants Conserved heterologous regulation of the cold-inducible peach Ppbec1 and Ppxero2 promoters in transgenic Arabidopsis plants. The upper panel shows histochemical GUS staining of representative transgenic Arabidopsis lines carry- ing the Ppbec1 promoter-uidA fusion, Ppxero2 promoter-uidA fusion and Pptha1 promoter-uidA fusion. The lower panel shows the results of fluorometric GUS-assays of three independent Arabidopsis transgenic lines (L1, L2 and L3) containing the Ppbec1 promoter-uidA fusion, Ppxero2 promoter-uidA fusion or Pptha1 promoter-uidA fusion. Homozygous T3 plants were grown for 14 days in MS plates with 0.8% agar at 24°C (white bars) and then transfer to 4°C for 7 days (blacks bars). The aster- isk above each bar represents those samples that have a statistically significant increase in GUS activity in the cold treated plants when compared to the untreated plants. Bars represent the mean ± standard deviation, n = 5. t-student * p < 0.01. nase inhibiting protein (C2988), similar to what was We also found some genes related to protein folding and reported by Ogundiwin et al [38]. Dehydrins are degradation, such as heat shock proteins, BiP-1 and DJ-1 hydrophilic proteins that belong to the subgroup D-11 of family proteins (Table 2). These processes are very active the LEA ("late-embryogenesis-abundant") proteins [39]. when plants face low temperatures, chemical and oxida- There is some evidence that suggests that dehydrins pro- tive stress. These proteins participate in the prevention tect macromolecules such as membranes and proteins and repair of damage produced by cold, through the sta- against the damages associated with water deficiency [40- bilization of protein structure and the degradation of pro- 42]. In peach, these genes are induced during cold accli- teins that are not folded correctly [50,51]. mation and in cold-stored fruits [30,38]. It has been observed that pathogenesis-related (PR) proteins such as In this work we were interested in isolating and function- chitinases and thaumatins are accumulated in the apo- ally characterizing promoters of cold-inducible peach plastic space in winter rye during cold acclimation. These genes. To date, only a few inducible promoters have been proteins also may have antifreeze properties that will pro- identified in crop plants. The Pptha1, Ppbec1 and Ppxero2 tect the integrity of the plant cell avoiding the formation genes were chosen for promoter cloning and characteriza- of ice [43,44]. It has also been observed that these types of tion based on the up-regulation that these genes showed proteins retain their enzymatic activity under low temper- in the in silico analysis and RT-PCR. The promoter atures, and may form part of a general response mecha- sequences of these genes contain several cis-regulatory ele- nism associated with unfavorable conditions, by ments such as DRE/CRT, ABRE, MYCR (MYC recognition providing protection from opportunist pathogen attack site) and MYBR (MYB recognition site) [13,31-34] that are whilst the plant is in a weakened state [45-47]. A similar related to stress response, specifically to cold/dehydra- role is shared by polygalacturonase inhibiting proteins in tion. These cis-regulatory elements are conserved in sev- different plants models [48,49]. eral plant species [52]. The presence of these conserved Page 12 of 15 (page number not for citation purposes) BMC Plant Biology 2009, 9:121 http://www.biomedcentral.com/1471-2229/9/121 motifs suggests that these promoters may respond to the Additional file 2 cold. Using transient transformation in peach fruit we Sequence of the Ppbec1 promoter and open reading frame. The data confirmed that the promoters isolated from Ppbec1 and provided represents the sequences of the Ppbec1 promoter and open read- Ppxero2 are induced during low temperature storage, but ing frame. not at room temperature. On the other hand, the Pptha1 Click here for file promoter is active under all the temperatures analyzed. [http://www.biomedcentral.com/content/supplementary/1471- This could indicate that the Pptha1 promoter sequence 2229-9-121-S2.DOC] might not contain all the elements needed to regulate Additional file 3 expression in a cold-inducible manner. Alternatively, the Sequence of the Ppxero2 promoter and open reading frame. The data agro-infiltration technique may induce stress signals that provided represents the sequences of the Ppxero2 promoter and open read- will activate this promoter. However, this last possibility ing frame. is not likely because the activation of the Pptha1 promoter Click here for file at all analyzed temperatures is also seen in the stably [http://www.biomedcentral.com/content/supplementary/1471- transformed transgenic Arabidopsis plants. The promot- 2229-9-121-S3.DOC] ers Ppbec1 and Ppxero2, however, are cold-induced both in Arabidopsis transgenic plants as well as transient express- Additional file 4 Sequence of the Pptha1 promoter and open reading frame. The data ing fruits, suggesting that the Ppbec1 and Ppxero2 promot- provided represents the sequences of the Pptha1 promoter and open read- ers are cold-inducible peach promoters. The cold- ing frame. inducibility of these promoters in A. thaliana also suggests Click here for file that this model plant may be used to functionally analyze [http://www.biomedcentral.com/content/supplementary/1471- peach cold-induced genes as well as their corresponding 2229-9-121-S4.DOC] cis-elements and trans-acting factors. Additional file 5 The identification of these fruit tree cold-inducible pro- Sequence of the Pplox1 promoter and open reading frame. The data provided represents the sequences of the Pplox1 promoter and open read- moters as well as the conserved heterologous regulation of ing frame. these promoters in peach and Arabidopsis, demonstrates Click here for file that these two transformation assays may be used to [http://www.biomedcentral.com/content/supplementary/1471- molecularly define the cis-elements and trans-acting regu- 2229-9-121-S5.DOC] latory factors that are associated with cold-responsive genes. By better understanding the regulatory mecha- nisms associated with cold-responsive genes, we may bet- ter understand the molecular differences and similarities Acknowledgements between cold acclimation and chilling injury as well as the This work was supported by ICM P06-065-F; FDI G02P1001 (Chilean Genome Initiative) with funding from the Chilean government as well as role these processes play in fruit tree growth and fruit ASOEX (Asociación de Exportadores de Chile A.G.), FDF (Fundación para quality. el Desarrollo Frutícola) and Fundación Chile; Proyecto Consorcio BIOF- RUTALES S.A.; PBCT R11 and CONICYT Fellowship D-21080654 to AM. Authors' contributions AT: identified and cloned the promoters. AT, MS, LM and References HS drafted the manuscript. AT and MS: performed the dig- 1. Thomashow MF: So what's new in the field of plant cold accli- ital expression analysis. AM and AT: performed the con- mation? Lots! Plant Physiol 2001, 125(1):89-93. 2. 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