AP2/ERF gene family searches and retrieval

A Hidden Markov Model (HMM) profile was constructed for the identification of new AP2/ERF genes. To build this profile, 595 AP2/ERF genes from six different species (C. reinhardtii, P. patens, S. moellendorffii, O. sativa subsp. Japonica, V. vinifera, and A. thaliana) were retrieved from The Plant Transcription Factor Database v3.0 [22]. A multiple sequence alignment was performed with Clustal Omega [23], and then the alignment was manually curated. The HMM model was constructed using the HMMER software package v3.1 [24], curated with the cut-off values and used to detect AP2/ERF genes in seven plant species and a green alga. The gene model of Arabidopsis thaliana was downloaded from the TAIR database [25]. The gene files of Carica papaya (ASGPBv0.4), Chlamydomonas reinhardtii (v5.5), Physcomitrella patens (v3.0), Selaginella moellendorffii (v1.0), and Solanum lycopersicum (iTAG2.3) were downloaded from Phytozome v9 [26]. The gene annotation of Musa acuminata (DH-Pahang, CIRAD) and Oryza sativa subsp. Japonica (IRGSP-1.0) were downloaded from EnsemblPlants (http://plants.ensembl.org/index.html, EMBL-EBI).

Sequence analysis and evolutionary reconstruction

The 832 retrieved amino acid sequences were subjected to a motif scan using the Pfam database v27.0 [27] to confirm the presence of the AP2/ERF domain. Then all the sequences were clustered using CD-HIT [28] with an identity cut-off of 0.9 to exclude isoforms and duplicated genes. Multiple sequence alignments of all of the AP2/ERF selected proteins were performed using Muscle [29]. The alignments were tested with the statistical package ProtTest 2.4 [30] to find the best evolutionary model to use in a maximum likelihood (ML) analysis. The phylogenetic reconstructions were carried out using the RAxML suite v.8.0.26 [31] with a LG + Γ substitution model and 1,000 bootstrap replicates. The AP2/ERF sequences of C. reinhardtii were used as the outgroup, based on the parsimony-based rooting [32, 33]. Groups formed in the phylogenetic analysis were named according to a previous study [6]. 27 sequences of the group I subgroup of RAP2.4 and 33 sequences of the group II subgroup of RAP2.1/2.9/2.10 were taken for a ML tree. The topology for the best-scoring trees was visualized using Figtree v1.4 (http://www.molecularevolution.org). The MEME software [34] was used to identify conserved motifs in the AP2/ERF designated clades based on the occurrence parameter for a single motif “one per sequence” and the maximum number of sites of each motif. The amino acid sequence similarities were obtained using Muscle [29] and visualized using BOXSHADE v3.31C (http://boxshade.sourceforge.net/).

Accession numbers

The sequence data from the genes isolated from C. papaya are available from the GenBank database under the following accession numbers: CpRAP2.4a (KU065114), CpRAP2.4b (KU065115), CpRAP2.1 (KU065116), and CpRAP2.10 (KU065117), CpDREB1A (KU065118), and CpHSP70 (KU065119).

Plants, growth conditions and stress treatments in papaya

Seeds from papaya (Carica papaya cv. Maradol) were surface-sterilized using 1.05% sodium hypochlorite and 200 μl of Tween-20 for one hour at 27°C. Subsequently, the papaya's seeds were shaken in 50 ml of 1.0-M KNO3 for 24 h at 27°C [35]. Floating seeds were discarded, and submerged seeds were shaken in 100 ml of sterile water at 32°C for three days or until the testae cracked [36]. Once the seeds germinated, seedlings were planted in a potting mixture of Horti Pearl, Sunshine, Vermiculite, and Peat moss (2:2:2:1). 180 plants were grown under a photoperiod of 16 h light/8 h dark at 25°C for six weeks. For the temperature stress treatments, three papaya plants from four different lines were selected based on size and health similarity prior the experiment to maintain a uniform start point. The plants were then incubated at either 4°C or 40°C for different exposure times.

Nucleic acid extraction

Total RNA from different plant tissues was isolated using the TRIzol Reagent (Ambion, http://www.ambion.com) according to the manufacturer´s instructions. Sap extraction was performed by transverse cutting of the stem of papaya seedlings, and the fluid sap was taken with a tip pre-loaded with 20 μl of TRIzol Reagent, avoiding the plant stem edges. For total DNA extraction, 5 g of leaf tissue was ground and mixed with 15 ml of extraction buffer [0.1-M Tris-HCl, pH 8.0; 1.0-M NaCl; 0.02-M EDTA, pH 8.0; 2% (w/v) CTAB; 2% (w/v) polyvinlypyrrolidone-40; 0.2% (v/v) 2-mercaptoethanol], and incubated for 1 h at 70°C. The solution was then twice extracted with equal volumes of chloroform:isoamyl alcohol (CIA, 24:1, v/v) and centrifuged at 15,000 × g, 4°C for 10 min. The aqueous phase was transferred to a 1.5 ml tube, and the DNA was precipitated with an equal volume of isopropanol, and kept at -80°C for 1 h. The DNA was then spooled out and washed with 80% ethanol-15-mM ammonium acetate, and a second wash was performed with 100% ethanol. Each washing step was performed for 20 min with gentle shaking. The DNA pellet was air dried and dissolved in 100 μl of Tris-EDTA buffer (10-mM Tris, pH 8.0; 1-mM EDTA, pH 8.0; 1-M NaCl) by incubating at 65°C for 1 h.

cDNA synthesis and semiquantitative reverse transcription PCR (RT-PCR)

For semi-quantitative RT-PCR, 1 μg of total RNA was used for the cDNA synthesis. Reverse transcription was performed by SMARTerTM PCR cDNA Synthesis Kit (Clontech, http://www.clontech.com), according to the manufacturer’s instructions. Each cDNA was diluted 1:5, and 2 μl of diluted sample was used in a 30 cycle PCR program with gene-specific primers as follows: The primers used: CpRap2.4a (forward) 5´- ATG CCT CAA CCT ATT TCA AAC GC-3´ and (reverse) 5´- TCA TGA CAA TAT GGA GGC CCA AT-3´); CpRap2.4b (forward) 5´- ATG GCA ACA GCT ATA GAT-3´ and (reverse) 5´- TTA ATA TTC GGA TAG TTT CCT TAT-3´); CpRap2.1 (forward) 5´- ATG GAA GGA GAG TGT TGT TCG ACG-3´ and (reverse) 5´- TCA GTC TTC ATC GGA AGT TTC CGG-3´; and CpRap2.10 (forward) 5´- ATG GAG GGT GGA GCG GAG-3´ and (reverse) 5´- TCA CTC GCC ATC CGA ACT ATC TG-3´). The housekeeping 18S gene was amplified from each sample to normalize the level of each test gene using 18S universal primers (forward, 5´-CGG CTA CCA CAT CCA AGG AA-3 and reverse, 5´-GCT GGA ATT ACC GCG GCT-3´). PCR products were run on a 2% agarose gel with ethidium bromide and visualized on a Molecular Imager® Gel Doc™ XR+ System.

Plasmid construction for plant transformation

PCR products of CpRap2.4a, CpRap2.4b, CpRap2.1, and CpRap2.10 were purified using a QIAprep Spin Miniprep Kit (QUIAGEN, https://www.qiagen.com) and then cloned into the pGem-T easy vector system (Promega, https://www.promega.com) according to the manufacturer's instructions. The ligations were transformed into E. coli XL1-Blue competent cells using a calcium protocol transformation [37], and sequenced by Sanger (Cinvestav-Langebio, Irapuato, Mexico). Subcloning of CpRap2.4a, CpRap2.4b, CpRap2.1 and CpRap2.10 were performed by using the following primers: CpRap2.4a (forward) 5´-AAA AAG CAG GCT TCA CCA TGC CTC AAC CTA TTT CAA ACG CG-3´ and (reverse) 5´-AGA AAG CTG GGT GTG ACA ATA TGG AGG CCC AAT CGA T-3´; CpRap2.4b (forward) 5´-AAA AAG CAG GCT TCA CCA TGG CAA CAG CTA TAG AT-3´ and (reverse) 5´-AGA AAG CTG GGT GAT ATT CGG ATA GTT TCC TTA TAG C-3´; CpRap2.1 (forward) 5´-AAA AAG CAG GCT TCA CCA TGG AAG GAG AGT GTT CG-3´ and (reverse) 5´-AGA AAG CTG GGT CTT CAT CGG AAG TTT CCG G-3´; and CpRap2.10 (forward) 5´-AAA AAG CAG GCT TCA CCA TGG AGG GTG GAG CGG AG-3´ and (reverse) 5´-AGA AAG CTG GGT GCT CGC CAT CCG AAC TAT CTG GGT C-3´. Gateway BP Clonase® (Invitrogen, http://www.invitrogen.com) was used to recombine the CpRap2.4a, CpRap2.4b, CpRap2.1, and CpRap2.10 genes with the flanking attB sites into the attP sites of the pDONR™221 vector (Invitrogen, http://www.invitrogen.com). Subsequently, the four CpRAP genes were transferred into the binary vector PK7FWG2.0 [38]; (http://gateway.psb.ugent.be/information), which contains the attR sites, using the Gateway LR Clonase® (Invitrogen, http://www.invitrogen.com). The binary vector PK7FWG2.0 contains the kanamycin resistance gene (nptII) under the control of the 35S promoter of cauliflower mosaic virus and the reporter gene for GFP.

Overexpression of papaya Rap2 genes in tobacco plants

The constructed binary vectors that contained specific RAP2 genes (CpRap2.4a::gfp, CpRap2.4b::gfp, CpRap2.1::gfp, CpRap2.10::gfp) were introduced into Agrobacterium tumefaciens EHA 105 cells. N. tabacum (Nicotiana tabacum L. cv. SR1) plants were transformed according to the method described previously [39]. A disk of leaves of N. tabacum was prepared using a sterile cork border (8 mm) and placed onto preculture medium (MS salts, 3% sucrose, 10-ml/l Vitamins B5, 1-mg/l Benzilaminopurine (BAP) and 3-g/l Gelzan. These explants were incubated at 25°C under a photoperiod of 16 h light/8 h dark for 24 h. The explants were transformed using the vacuum infiltration method and cocultived at 25°C in the dark for 3d and then transferred to selection medium (MS salts, 3% sucrose, 10-ml/l Vitamins B5, 1-mg/l Benzilaminopurine (BAP), 0.1-mg/l NAA, 3-g/l Gelzan, 200-mg/l Timetin and 150-mg/l kanamycin).

PCR analysis of putative transgenic plants

Genomic DNA of putative transformed plants (100 ng) was used for a PCR reaction to detect the presence of the nptII transgene using the primer pairs, nptII-forward (5´-ATG ATT GAA CAA GAT GGA TTG C-3´) and nptII-reverse (5´-TCA GAA CTC GTC AAG G-3). Specific RAP2 gene primers were tested on each transgenic plant to improve the quality of the analysis. PCR products were run on a 2% agarose gel with ethidium bromide and visualized on a Molecular Imager® Gel Doc™ XR+ System.

Stress tolerance tests of tobacco transgenic plants

Transgenic seeds from twelve transgenic lines N. tabacum were grown in selection medium (MS salts, 3% sucrose, 10-ml/l Vitamins B5, 200-mg/l Timentin, 150-mg/l kanamycin, and 3-g/l Gelzan) according to a previous study [39]. Transformed and non-transformed seedlings were transferred in a potting mixture of Horti Pearl, Sunshine, Vermiculite, and Peat moss (2:2:2:1) and then grown under a 16 h light/8 h dark photoperiod regime at 25°C for six weeks. Up to 45 days after germination (DAG) the plants were used for a temperature tolerance test. For cold stress treatment, the 45 DAG plants were transferred to the same potting mixture and incubated for 30 days at 4°C. In the case of high temperature stress treatment, 45 DAG plants were subjected to 40°C for 12 days. The control group was kept at room temperature (25°C).

Cellular localization of the green fluorescent signals in transgenic plants

GFP fluorescence (excitation filter 488 nm, emission filter band pass of 505–530 nm) was analyzed using a confocal laser-scanning FV100 Olympus microscope. DAPI staining of N. tabacum transgenic plants was performed in order to determine the location of the nuclei in the cells. Non-transformed plants were used as a GFP control of protein localization.

Histology and immunofluorescence microscopy

The plant tissue was fixed in tubes containing FAA with aspiration for 24 h. They were dehydrated through an ethyl alcohol series and embedded in paraffin (melting point 54–56°C) with a graded series of tertiary butyl alcohol. The paraffin blocks were sectioned serially at 5 μm thickness using a microtome. The deparaffinization was carried out with four washes with Histology grade Xylene for 2 min and by removal of xylene with absolute ethanol. Seventy percent ethanol followed by water for 1 min each. Tissue was permeabilized with 0.1% Triton X-100 in PBS for 15 min, respectively. After washes with PBST they were either incubated with anti-GFP from Anti-GFP antibody (ab6556) from Abcam. Secondary antibodies goat anti-rabbit IgG conjugated with Alexa 647 from Invitrogen (21245) using an excitation wavelength of 620 nm or 660 nm. After being washed for 30 min with PBST cells were mounted with moviol (DAPI-DABCO). Images were acquired using a confocal microscope FV100 Olympus with 60X (NA 1.4) oil immersion objective lens. In each experiment, the images of all samples were acquired with the same parameters of acquisition to ensure their comparability. To assess the distribution of all transgenic plants that expressed GFP fused proteins in the tissues. The GFP fused proteins were analyzed in a 500–530 nm emission filter in the image was color-coded in green. DAPI emission 425–475nm channels were color-coded in blue. For comparison all images were acquired with the same excitation and detection parameters without reaching signal saturation.

In vitro translation in the sap of Carica papaya L. cv. Maradol

We used GST-tagged PLCδ1PHD mRNA to test in vitro translation capability of the sap. Sap was extracted by cutting 1 to 2 mm of the apical bud, from C. papaya plants of six weeks old, suspending at storage buffer (24-mM HEPES buffer pH 7.4, 101-mM potassium acetate, 4.2-mM magnesium acetate, 11-mM DTT and 1-mM spermidine). The RNA was synthesized in vitro from GST-tagged PLCδ1PH (1–140; pGST3) a vector provided by Dr. Hitoshi Yagisawa [40]. 25 units of T7 RNA polymerase was incubated at 37°C for 1 hour in buffer containing 50 mM Tris-HCl pH 7.5, 15 mM MgCl, 2,5 mM dithiothreitol (DTT), 2 mM spermidine and 2mM dNTPs with 10 pmoles of vector. After incubation the RNA was purified by Qiagen RNeasy mini kit and verified by agarose gel electrophoresis. The translation reactions were performed with a 0.08 mM amino acid mixture, 75 mM potassium acetate, 1 μl of RNasin (40 u/μl), 0.5 μg/μl of GST-PLCδ1PHD RNA and 25 μl of C. papaya sap in a total volume of 50 μl with nuclease-free water. The reaction was incubated at 30°C for 2 hours. For Immunoblot analysis, the reaction was stopped with laemmli buffer and heated at 100°C for two minutes to denature the proteins and then loaded in a 10% SDS-PAGE gel. Bacterially expressed GST-PLCδ1PHD was used as a positive control. The SDS-PAGE was blotted into nitrocellulose paper block with 5% milk in PBST for one hour and incubated for 2 hours with anti-GST antibody (Abcam ab19256).three subsequent washes with PBST were carried out 10 min each and incubated for 2 hours with IRDye goat anti-rabbit antibodies and analyzed by Odyssey Infrared Imager 9120 (LI-COR Biosciences).

Nicotiana tabacum L. Grafting

Three independent lines Rap2.4a transgenic plants from the twelve lines of N. tabacum L. were grown in a greenhouse until the F1 generation was obtained. F1 seeds were germinated for 60 days. The grafts between the non-transformed and transformed plants were performed using the transgenic tobacco plants as rootstock and the wild-type tobacco plants as scion. For the grafting, straight cuts were made into the rootstock, and then the apical section of the tobacco wild-type plant was inserted until it fit snugly. The cut was wrapped with polythene strips for one week.

Photosynthesis measurements in transgenic plants

The photosynthetic net rate (Pn) and stomatal conductance (Gs) were measured using a portable infrared gas analyzer (Li-6400XT, Licor, Lincoln, NE, USA). High temperature (40°C) was measured at 0, 4, and 7 days. The analyzer was equipped with a clamp-on leaf cuvette that exposed 6 cm² of leaf area. Light, temperature, and humidity were 180 μmol m^-2 s^-1 (Li-6400-02B LED), 25± 1°C and 70%, respectively and a CO₂ concentration of 400 μmol mol^-1. The readings were determined within 60 seconds of having locked up the leaf chamber. Measurements were made on four randomly selected plants of each treatment.

Scanning electron microscopy

To study the promotion of stomatal closure by ABA, Nicotiana tabacum seeds were sown on ABA-free medium for 4 d after stratification, and then the tobacco in vitro plantlets were transferred into a hydroponic liquid MS medium (Murashige and Skoog, 1962), and after 4 d of acclimation, 50 μM of ABA was added to the hydroponic medium for 2 days of treatment. Stomatal aperture was observed from the central regions of leaves from the abaxial epidermis using Scanning Electron Microscopy (SEM). This experiment was carried out as follows: 1 cm of sample was fixed in 2.5% glutaraldehyde for 48 h, rinsed several times in buffer solution (0.2 M sodium phosphate, pH 7.2), and dehydrated in serial ethanol concentrations (v/v) of 30%, 50%, 70%, 85% and 96%. All these dehydrated rinses were sequentially followed by a wash in absolute ethanol for 60 min. The critical drying point was performed at 1072 psi/31°C (Samdri®-795 tousimis). The samples were then mounted on metallic stubs with carbon conductive adhesive tape (Electron Microscopy Science) and sputter coated with a 150 Å gold layer (Denton Vaccum Desk II). Stomatal aperture percentage was calculated for the abaxial epidermis at a magnification of 200×(0.1213 mm²). Length of the guard cells was also measured in five stomata per field. Counts and measurements were done in ten fields of each leaf. Sample analysis and image recording were made using a Scanning Electron Microscope (Jeol, JSM-6360LV).

Statistical analyses

For the photosynthesis measurements each bar represents the average measurement from three independent samples for each condition. Wild type-wild type and CpRap2.4a transgenic gene-wild type tobacco plants were taken and indicated by different letters depending on their differences. The data was analyzed by one-way analysis of variance (ANOVA) followed by the comparison of mean values using the Tukey's test at P<0.01 in Statgraphics Centurion (StatPoint Technologies, Inc., Warrenton, VA). Data were presented as mean ±SD error of three determinations per sample.

Genome-wide identification of AP2/ERF sequences and phylogenetic relationships

The retrieval of 832 AP2/ERF sequences from eight full-genomes (see Materials and Methods) by means of HMM was used to identify AP2/ERF homologs in papaya tree [41]. We detected 78 papaya AP2/ERF sequences, and we used the prefix CpERF to name the papaya AP2/ERF sequences. Two full-length cDNA sequences in papaya are closely related to AtRAP2.1 and AtRAP2.10, respectively, which are not described in the proteome file from the Phytozome database. These predicted sequences were obtained using the FGENESH+ suite [42] with a predicted coding sequence ranging from 157 to 181 amino acids, and they were named CpERF79 and CpERF80, respectively. These two new sequences bring the number of CpERF sequences to 80. No discrepancies were found between the PCR amplicons of these two genes and the FGENESH+-predicted coding sequences [42]. The complete list of the AP2/ERF TF family from papaya tree is available in S1 Table.

Phylogenetic trees of the retrieved AP2/ERF sequences were constructed from the AP2 domain, and they were grouped according to a previous study [6]. Four CpERF sequences were clustered in the group I, subgroup of RAP2.4 genes (CpERF31, CpERF33, CpERF45, and CpERF72), and three CpERF sequences were clustered in the group II, subgroup of RAP2.1/2.9/2.10 (CpERF70, CpERF79, and CpERF80; Fig 1A and 1B). We performed an amino acid analysis based on a sequence alignment for these two subgroups, RAP2.4 and RAP2.1/2.9/2.10, to determine the conserved motifs along the plant lineages (see Fig 1C). The four CMI motifs reported by Nakano et al. [6] were conserved in a RAP2.4 subgroup (S1 Fig), and two CMII-reported motifs were found in the RAP2.1/2.9/2.10 subgroup (S2 Fig).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Maximum likelihood phylogenetic trees of the RAP2.4 and RAP2.1/2.9/2.10 subgroup proteins of the AP2/ERF transcription factor family in eight plant species.

A) Phylogenetic reconstruction of the RAP2.4 subgroup of the group I AP2/ERF TFs. B) Phylogenetic reconstruction of the RAP2.1/2.9/2.10 subgroup of group II AP2/ERF TFs. Species names are color coded as follows: blue–Moss, pink–pseudofern, green–Arabidopsis, orange–papaya, red–tomato, yellow–banana, and cyan–rice. C) Schematic representation of the identified conserved motifs in each analyzed subgroup. Sequences of P. patens were used as the out-group. Black asterisks indicate the papaya AP2/ERF proteins analyzed in this work.

https://doi.org/10.1371/journal.pone.0165030.g001

Orthologous proteins of interest were calculated by means of reciprocal best Blast hits (BBH), [43] using the entire set of papaya AP2/ERF sequences against the Arabidopsis AP2/ERF retrieved sequences. For the RAP2.4 subgroup, CpERF31 protein is orthologous to AtRAP2.4 (AT1G78080), with three possible paralogous sequences (AT1G36060/ERF055, AT1G22190/RAP2-4b, and AT5G65130/ERF057), and CpERF33 protein is orthologous to ERF60 (AT4G39780), with two possible paralogous sequences (AT2G22200/ERF056 and AT4G13620/ERF062). For the RAP2.1/2.9/2.10 subgroup, CpERF80 protein is orthologous to AtERF10 (AT5G67190), with three possible paralogous sequences (AT4G36900/RAP2-10, AT3G50260/ERF011, and AT2G23340/DEAR3), and CpERF79 protein did not show reciprocal BBH, but was the closest related to the AtRAP2.1 protein.

CpERF31 and CpERF33 proteins were called CpRAP2.4a and CpRAP2.4b, respectively, due to their proximity to AtRAP2.4 (group I; see Fig 1). CpERF79 and CpERF80 were called CpRAP2.1 and CpRAP2.10, respectively, because of their similarity to the Arabidopsis proteins AtRAP2.1 and AtRAP2.10, from the AP2/ERF group II.

Expression analysis of CpRap2 genes in different plant tissue and sap under abiotic stress conditions

Molecular analysis of the expression of CpRap2 genes was performed to detect the expression of CpRap2.4a, CpRap2.4b, CpRap2.1 and CpRap2.10 genes, and the 18S gene was used as a reference. Fig 2 shows the RT-PCR levels of the CpRap2.4a, CpRap2.4b and CpRap2.10 genes obtained in the leaf and stem during different stress treatments of heat (40°C) and cold (4°C) (Fig 2A and 2B). The transcript level of these genes (CpRap2.4a, CpRap2.4b) were induced in the leaf and stem by heat (40°C) and cold (4°C) stress (Fig 2A, 2B and 2C) and S2 Table.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Semi-quantitative reverse transcription PCR (RT-PCR) analysis of the different CpRap genes exposed at extreme temperature stress conditions from Carica papaya tree.

A) RT-PCR analysis of CpRap genes exposed to 4°C and 40°C during different times of treatment in leaves of C. papaya. B) RT-PCR analysis of CpRap genes exposed to 4°C and 40°C during different times of treatment in stems of C. papaya. C) RT-PCR analysis of CpRap genes exposed at 4°C and 40°C during different times of treatment in roots of C. papaya. The quantification values of CpRap genes were normalized with that of the 18S gene. All analyses were performed with three biological replicates.

https://doi.org/10.1371/journal.pone.0165030.g002

Previous studies found that the AtDreb1A (dehydration-responsive element-binding protein 1A) gene is expressed in response to drought and low temperature, and the AtHsp70 (70-kilodalton heat shock proteins) gene is expressed in response to high temperature. Therefore, we isolated the orthologous genes from C. papaya var. Maradol and used them as positive controls for the extreme temperature experiments. The CpDreb1A gene in the leaf and stem is differentially expressed in response to extreme temperatures (Fig 2A and 2B). CpDreb1A-type gene transcript accumulated as exposure time increases to stress unlike the behavior of this transcript expression in roots (Fig 2C). In comparison the transcript expression profile of CpHsp70-type gene clearly shows a higher yield in roots than in other tissues of the papaya plants (Fig 2C). The expression of both CpHsp70 and CpDreb1A-type genes decreases when the plants have reached at 120 minutes of exposure to stress. This decrease in expression occurs when seedlings were greatly affected already by stress. It is important to note that this does not happen with the CpDreb1A-type transcription in root. The transcript accumulation of the CpDreb1A-type gene was diminished in leaf and stem, but not in root of the papaya seedlings plant. The expression of the CpHsp70 gene was also differential accumulated in the root of the papaya seedlings (Fig 2A, 2B and 2C).

We carried out PCR and RT-PCR and verify that no DNA contamination responsible for the amplifications from our Total RNA or RNA form sap extractions (Fig 3A). We detected the RNA of CpRap2.4a, CpRap2.4b, CpRap2.1 and CpRap2.10-type gene in the sap as well as in the complete tissue of the plant. CpRap2.4a, CpRap2.4b and CpRap2.1 gene expression increased in both sap and tissues, while other genes, such as CpDreb1A and CpHsp70, were only found in the tissue, also suggesting a lack of contamination of RNA from the tissue into the sap (Fig 3B and S3 Table). Transcript accumulation in control seedlings have a basal expression, however in response to stress at 4°C or at 40°C, there is a transcript accumulation, suggesting that CpRAP2.1, CpRAP2.4a and CpRAP2.4b are involved in setting the signals for plant survival at extreme temperatures. The gene specificity of RT-PCR was confirmed by sequencing all of the RT-PCR products. The results for the CpRap2.1 gene were different in sap as compared to the whole plant indicating a selective mechanism between the RNA that is send to the sap. Furthermore upon checking the RNA extracted from sap we found ribosomal RNA in the extracts from sap as seen in Fig 3C. Therefore we tested the idea that protein translation could occur in the sap and carried out in vitro translation reactions using sap to create a foreign protein GST PLCd1PHD (Fig 3D). Surprisingly we were able to translate a protein using sap of C. papaya from an in vitro synthetized RNA that codifies for GST PLCd1PH D, Other results from the in vitro translation reactions from sap included translation and purification of 6his-fibrilarin a nucleolar protein S3 Fig and S1 File. Furthermore, added mRNA of gfp-Rap2.4a to wound embryonic also showed some cells translating the foreign mRNA and incorporating it in the cells S4 Fig and S1 File.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Transcriptional expression of different genes in the sap of Carica papaya and in vitro translation from sap.

A) PCR from Genomic DNA, Total RNA, and cDNA of leaf and sap in the absence or presence of DNase I. Nomenclature: 1. 1 Kb ladder (Promega); 2. PCR of DNase I treated Genomic DNA; 3. PCR of Genomic DNA; 4. PCR of DNase I treated total RNA; 5. PCR of total RNA; 6. PCR of cDNA synthesized from DNase I treated total RNA; 7. PCR of DNase I treated Sap RNA; 8. PCR of Sap RNA; 9. PCR of cDNA synthesized from DNase I treated RNA sap. B) RT-PCR analysis of CpRap genes in sap or the whole plant of C. papaya exposed to 4°C and 40°C. Quantification values of CpRap genes were normalized with the 18S gene. All analyses were performed with three biological replicates. C) Total RNA, comparison of tissue vs sap separated on a 1% agarose gel after electrophoresis with ethidium bromide. 28 and 18 S rRNA are mark by the arrows. D) Immunoblot analysis after in vitro translation of GST-PLCd1PHD with sap of C. papaya. Recombinant GST-PLCd1PHD was loaded as a control (C), 20 μl of the in vitro translation reactions were loaded, minus RNA added to the sap, with RNA and sap and RNA only.

https://doi.org/10.1371/journal.pone.0165030.g003

Stress tolerance assay of overexpressing CpRAP2 proteins in tobacco plants

The phenotype analysis of F1 transgenic and non-transformed tobacco plants under the stress of extreme temperatures (4°C or 40°C) is shown in Fig 4, where a single plant is a typical representative from each population. We used 12 plants for each experiment from 3 selected lines for each transgene. All plants were exposed at 4°C for 30-day. Fig 4A shows that non-transformed plants did not survived, while transgenic tobacco plants overexpressing the CpRap2.1 gene had a survival rate of 16.6%, and the CpRap2.10, CpRap2.4a and CpRap2.4b genes showed survival rates of 66.6% (Fig 4A). High temperature stress treatments (40°C exposure for 12 days) resulted in the lack of survival of the non-transformed plants, while transgenic tobacco plants carrying the CpRap2.1, CpRap2.10, CpRap2.4a and CpRap2.4b genes had a survival rate of 16.6, 33.3, 100 and 50%, respectively (Fig 4B). Tobacco plants that overexpressed CpRAP2.4a or CpRAP2.4b showed the particular phenotype of all of the adult leaves being slightly marked by a green and chlorotic (yellow green) color after the treatment at 4°C or 40°C (Fig 4A and 4B); they also began to flower after 12 days of high temperature treatment. Tobacco plants that overexpressed CpRAP2.4a or CpRAP2.4b showed a normal plant growth, however they presented some wilting or chlorosis. While under treatment their stems did not showed a signs of death tissue and maintained their vigor after 30 days treatment at 4°C. When the plants were incubated at 40°C for 12 days the tobacco plants that overexpressed CpRAP2.4a or CpRAP2.4b showed a reduced plant growth, with an abnormal curling and crinkling of some leaves, including the reduction expansion of leaves, wilting, chlorosis and their stems showed some loss of vigor.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Extreme temperature tolerance of tobacco transgenic plants that carry in their genome different CpRap genes controlled by the 35S promoter.

A) Phenotypes are shown for 12 transgenic tobacco plants with the CpRap2.1, CpRap2.10, CpRap2.4a, and CpRap2.4b gene under low temperature stress (4°C) or at room temperature (25°C). B) Phenotypes are shown for 12 transgenic tobacco plants with the CpRap2.1, CpRap2.10, CpRap2.4a and CpRap2.4b gene under high temperature stress (40°C) or at room temperature (25°C). Wild-type tobacco plants were used as a control.

https://doi.org/10.1371/journal.pone.0165030.g004

Furthermore we compared seed germination of the wild-type (Wt), CpRAP2.4A and CpRAP2.4B overexpression transgenic lines in the presence and absence of Abscisic acid (ABA). ABA is a phytohormone critical for plant growth and development and plays an important role in integrating various stress signals and controlling downstream stress responses. The results are shown in S5 Fig, where the expression of both genes in tobacco allows their germination under ABA presence.

Localization of the CpRAP2 proteins in transgenic tobacco seedlings

In order to identify the sub-cellular localization of the CpRAP2.1, CpRAP2.10, CpRAP2.4a and CpRAP2.4b proteins, they were fused with a green fluorescent protein (GFP). Each construct was expressed constitutively under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco seedlings overexpressing the chimeric CpRAP2.1::GFP, CpRAP2.10::GFP, CpRAP2.4a::GFP or CpRAP2.4::GFP proteins were analyzed for GFP fluorescence with a confocal microscope (Fig 5, larger files can be observed in S6 Fig). The nuclear location of CpRAP2.1, CpRAP2.10, CpRAP2.4a, and CpRAP2.4b protein was confirmed by GFP and DAPI merged images showing a complete match (Fig 5). GFP was observed in the leaves of transgenic tobacco with CpRAP2.1, CpRAP2.10, CpRAP2.4a or CpRAP2.4b in the sieve elements (SE), which are part of the phloem.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Subcellular accumulation patterns of CpRAP proteins fused to GFP in Nicotiana tabacum plants.

The leaves or roots of tobacco seedlings after 8 days of germination were taken under different light sources Transmitted light is observed in the horizontal first line, DAPI staining is observed in the second horizontal line and GFP is observed in the third horizontal line. DAPI staining or GFP fluorescence of leaf or root is shown for tobacco transformed plants that they carry different CpRAP genes (the CpRap2.1, CpRap2.10, CpRap2.4a and CpRap2.4b gene) in their genome. The fluorescence of GFP and DAPI staining were taken at 40X on an Olympus FV1000 confocal microscope. Wild-type tobacco plants were used as a control.

https://doi.org/10.1371/journal.pone.0165030.g005

Micrographs taken from transmitted light (bright-field microscopy) in the leaves and roots of different lines of tobacco seedlings (control, CpRAP2.1, CpRAP2.10, CpRAP2.4a and CpRAP2.4b) localized to the veins of the leaves. Only the transgenic tobacco expressing CpRAP2.4a::GFP shows a strong GFP signal in phloem tissue where it can distinguish the sieve elements. Micrographs of the root apices show a different level of accumulation of green fluorescent protein in transgenic tobacco. The accumulation of GFP in CpRAP2.4a was very strong, and the accumulation of GFP protein was observed in the nuclei and the cell membranes of the roots. For transgenic tobacco seedlings with CpRAP2.1 or CpRAP2.10, the accumulation of GFP protein was restricted to around the cell membrane and was not present in the nuclei of the cells. These results and the in silico analysis suggests to us that CpRAP2.4a is a nuclear protein that possibly serves as a transcription factor.

Long-distance transport assay of CpRap2.4 mRNA by tobacco grafting

Movement of CpRAP2.4a::GFP was investigated by grafting experiments with tobacco transformed plants with the constructs CpRAP2.4a::GFP as a rootstock and non-transformed tobacco plants as scion (Fig 6A). Genomic DNA from tobacco transformed and wild-type plants were used for PCR for CpRAP2.4a gene. Wild-type tobacco plant did not contain the amplicon for the neomycin phosphotransferase II (nptII) gene, which is contained in the T-DNA that was inherent to the transgenic explants (Fig 6A). Total RNA from the leaves of each graft was obtained and RT-PCR was performed using the specific primer for the CpRAP2.4a gene. We observed that the tobacco transgenic plant with the construct CpRAP2.4a::GFP in the rootstock transferred the CpRAP2.4a::GFP RNA through the vascular conduits of the plant to the wild-type scion.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Long-distance transport assays of CpRap2.4a by tobacco grafting.

A) Genomic DNA (left section) was isolated from wild-type (upper section; Scion) or transgenic plants (below section; Rootstock) for amplification using PCR. The nptII primers were used to identify the neomycin phosphotransferase II (nptII) that is the selection marker of plants contained in the T-DNA. Total RNA (right section) was isolated from the wild-type tobacco (upper section; Scion) or from the transgenic tobacco plant (below section; Rootstock) for amplification using RT-PCR. CpRap2.4a primers were used to identify the accumulation of the CpRap2.4 transcript. Quantification values of nptII or the CpRap2.4a gene were normalized with the 18S gene. B) Subcellular accumulation patterns of CpRAP2.4a proteins fused to GFP in a tobacco plant graft. Leaf sections of a wild-type tobacco (horizontal first line) were taken under different light sources (DAPI staining is observed in the first vertical line, GFP is observed in the second vertical line and transmitted light is observed in the third vertical line). DAPI staining or GFP fluorescence in the leaf is shown for transformed tobacco plants (second horizontal line) that carry the CpRap2.4a gene in their genome. The fluorescence of the GFP and DAPI staining was taken at 40X by an Olympus FV1000 confocal microscope. Wild-type tobacco plants were used as the control (third horizontal line). C) Phenotypes of the tobacco plant grafts with the CpRap2.4a gene under high temperature stress (40°C) or at room temperature (25°C). Wild-type tobacco plants were used as a control. D) and E) Physiological effects of CpRap2.4a rootstock overexpressing tobacco plants under high temperature treatments in the photosynthetic activity and stomatal conductance from leaves of the wild type tobacco plants. N. tabacum transgenic (Rootstock) and wild type tobacco (Scion) plants were treated at 40°C during different times. Data was analyzed by one-way analysis of variance (ANOVA) followed by the comparison of mean values using the Tukey's test at P<0.01. Data were presented as mean ±SD error of at least three independent determinations per sample. Different letters indicate significant differences. Units: [photosynthesis = μmol.CO2.m^-2.s^-1; Stomatal conductance = mmol.m^-2.s^-1].

https://doi.org/10.1371/journal.pone.0165030.g006

The CpRAP2.4a protein was also localized in the nucleus of the wild-type scion (Fig 6B). DAPI was used to verify that it was in the nucleus, and both the transformed tobacco leaf with the CpRap2.4a and the wild-type leaf from the scion tobacco leaf showed nuclear localization. Although the wild-type scion signals were clearly lower. Control wild-type leaves did not show GFP fluorescence (Fig 6B). Graft tobacco plants were exposed to heat-induced stress (40°C), where non-transformed tobacco was affixed to the transformed plants (rootstock, Fig 6C); we observed that the rootstocks confer a capacity of the grafts to tolerate heat stress in conditions that wild-type tobacco plants did not tolerate (Fig 6C). Also the phenotype of the grafted plants included the presence of yellowish-green leaves, and their stems were evenly turgid despite the stress treatment. Control non-transformed tobacco plants show the leaves and stems were necrotic with a brown color, with a high loss of turgor during the stress treatment. Their leaves showed an abnormal curling and crinkling, which may lead to necrosis and their stems showed a progressive death, with loss of vigor. Wild type and CpRAP2.4 transgenic plants showed the same photosynthesis and stomatal conductance when no stress was added to the plants. After 4 days wild type plants had a negative photosynthetic rate and a very low stomatal conductance while the wild-type scion with the CpRAP2.4a showed a small reduction in photosynthetic rate and no significant difference in stomatal conductance (Fig 6D). This trend was maintained up to the 7 days of the experiment indicating the CpRAP2.4a rootstock those provide the wild type scion a change in physiological response to high temperature (40°C).

The stomata of wild-type plants had smaller guard cells and the aperture percentage were approximately 66% lower than those in 35S::CpRap2.4a plants in ABA treatment (Fig 7). Furthermore, the size of stomata were 36% larger in 35S::CpRap2.4a plants. Wild type plant under the presence of ABA showed to be very tightly closed in a round configuration marked with yellow arrows which was not seen in the in 35S::CpRap2.4a plants under the same treatment. Therefore, CpRAP2.4a functions as a negative regulator of ABA-mediated stomatal closure. Different results were obtained by Lin, Park [12] where the RAP2.4-ox plants and rap2.4–1 mutant plants responded normally to ABA inhibition of seed germination and root elongation. These results suggest that enhanced extreme temperatures tolerance of the CpRAP2.4a plants is likely a combination of functions from the CpRAP2.4a as a transcription factor and having a role in maintain stomata open upon signaling like that of ABA pathway. Consistent with this notion, the expression of CpRap2.4a is up-regulated by extreme temperature treatment.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. RAP2.4a localization in leaf cells and its effect on stomatal aperture.

A) Immunocolocalization studies show the localization of GFP-35S::CpRAP2.4a in transgenic plants in nucleus and sieve elements. Lignin from sieve elements shows autofluorescence at 480nm but not at 620 nm. B) SEM of stomatal aperture in wild type tobacco plants versus transformed tobacco plants (35S::CpRap2.4a). Arrows show stomata in blue (open) and red (close) for wild type and GFP-35S::CpRAP2.4a in transgenic plants in presence or absence of ABA. Yellow arrows indicate rounded closed stomata only seen in wild type plants in the presence of 50 μM ABA. C) Phenotype of Stomata of leaves from the abaxial epidermis of the wild type tobacco plants (Wt) versus transformed tobacco plants (35S::CpRap2.4a) in presence or absence of ABA.

https://doi.org/10.1371/journal.pone.0165030.g007