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J Exp Bot. Mar 2009; 60(3): 907–922.
PMCID: PMC2652048

Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening

Abstract

Seven ERF cDNAs were cloned from two Japanese plum (Prunus salicina L.) cultivars, ‘Early Golden’ (EG) and ‘Shiro’ (SH). Based on the sequence characterization, these Ps-ERFs could be classified into three of the four known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Functional nuclear localization signal analyses of two Ps-ERF proteins (Ps-ERF1a and -1b) were carried out using confocal microscopy. Expression analyses of Ps-ERF mRNAs were studied in the two plum cultivars in order to determine the role of this gene family in fruit development and ripening. The seven Ps-ERFs displayed differential expression pattern and levels throughout the various stages of flower and fruit development. The diversity in Ps-ERFs accumulation was largely due to the differences in their responses to the levels of ethylene production. However, other plant hormones such as cytokinin and auxin, which accumulate strongly throughout the various developmental stages, also influence the Ps-ERFs expression. The effect of the plant hormones, gibberellin, cytokinin, auxin, and ethylene in regulating the different Ps-ERF transcripts was investigated. A model was proposed in which the role played by the plant hormone auxin is as important as that of ethylene in initiating and determining the date and rate of ripening in Japanese plums.

Keywords: Double sigmoid curve growth pattern, ethylene-responsive factor (ERF), plant hormones, plum fruit development and ripening

Introduction

Ethylene, a gaseous phytohormone, mediates diverse developmental and physiological processes throughout the entire life cycle of plants (Abeles et al., 1992). The involvement of ethylene in the ripening of climacteric fruit is a symbolic incident of ethylene regulation. However, the developmental processes and the signal transduction mechanisms involved in such a phenomenon are less understood (Giovannoni, 2001). The expression of ethylene-related genes is induced through transduction of the ethylene signal from receptors to dedicated transcription factors (Giovannoni, 2004). Ethylene-responsive factors (ERFs) are uniquely present in the plant kingdom and belong to the AP2/EREBP-type transcription factors, which function as trans-acting factors at the last step of transduction (Ohme-Takagi and Shinshi, 1995). The ERF transcription factors contain a highly conserved DNA-binding domain (Ohme-Takagi and Shinshi, 1995) that interacts monomerically with the target DNA (Allen et al., 1998). Many ERF proteins have been shown specifically to bind the so-called GCC box with a strictly conserved GCCGCC core domain to modulate transcription of a wide variety of ethylene-responsive genes, indicating that a transcriptional cascade is involved in ethylene signalling (Ohme-Takagi and Shinshi, 1995; Büttner and Singh, 1997; Zhou et al., 1997; Solano et al., 1998; Gu et al., 2002). Some ERFs function as activators of GCC box-dependent transcription and others act as active repressors that down-regulate, not only basal transcription levels of a reporter gene, but also the transactivation of other transcription factors (Fujimoto et al., 2000). Although Arabidopsis has ~122 predicted ERF genes (Nakano et al., 2006), only a few have been characterized so far (Sakuma et al., 2002). In fact, only in tomato and apple, the role of ERFs in fruit ripening has been reported (Tournier et al., 2003; Wang et al., 2007).

The molecular mechanisms involved in fruit ripening have been studied to gain a broader insight into the factors that contribute to the differences in the date and rate of ripening among Prunus spp., including plum cultivars (Abdi et al., 1997; Tonutti et al., 1997; El-Sharkawy et al., 2007, 2008; Trainotti et al., 2007; Ziliotto et al., 2008). The difference in ethylene production rate observed among several plum cultivars (Abdi et al., 1997) could partially be due to the differences in the accumulation levels and/or pattern of various ethylene biosynthesis, perception, and signal transduction elements, which could be further related to ‘allelotype’ or ‘genotype’ variance (El-Sharkawy et al., 2007, 2008). However, allelic form alone still does not explain all the significant phenotypic disparity in fruit ripening. Furthermore, significant accumulation of certain ripening-related genes in fruits treated with the ethylene antagonist 1-methylcyclopropene (1-MCP) suggests that ethylene signalling is not the only essential signal that contributes to fruit ripening and indicates the possibility of more signalling pathways that are as crucial as that of ethylene (El-Sharkawy et al., 2007, 2008; Ziliotto et al., 2008).

The objective of this study was to understand the role of ERF gene family members in fruit development and ripening in Japanese plums. The ethylene evolution and the accumulation profile of seven Ps-ERF mRNAs were studied during fruit development and ripening in early ‘EG’ and late ‘SH’ cultivars. The aim was to determine whether there are dissimilarities that could account for the diversity in ripening behaviour. Finally, the localization of two of Ps-ERF proteins and the function of their putative NLS sites were visualized by confocal microscopy.

Materials and methods

Plant material and post-harvest treatments

Fruits from two Japanese plum (Prunus salicina L.) cultivars ‘Early Golden’ (‘EG’) and ‘Shiro’ (‘SH’) were harvested and treated as described previously (El-Sharkawy et al., 2007). Other tissues such as flowers and early developmental stages were collected from ‘EG’ cultivar. All plant material was frozen in liquid nitrogen and stored at –80 °C.

Hormones and hydrogen peroxide treatments

‘EG’ leaves were transferred to 250 ml flasks containing 100 ml of appropriate treatment. The flasks were incubated on a rotary shaker (100 rpm) for 2 h at room temperature. The treatments include gibberellin–GA3, cytokinin–BA, and auxin–IAA (all 0.4, 4, and 40 μM), ethylene as ethephon (1, 2, and 4 mM), and hydrogen peroxide-H2O2 (0.5, 1, and 2 mM). At the end, all the treated-leaves were removed, briefly blotted dry, immediately frozen in liquid nitrogen, and stored at –80 °C for RNA analysis. Leaves without any treatment were used as control.

RNA isolation

Total RNA from fruit samples was extracted using the methods described by Boss et al. (1996). For vegetative tissues and flowers, total RNA was extracted using the Plant Total RNA Purification kit (Norgen, Thorold, ON, Canada), as per the manufacturer's instructions. All RNA extracts were treated with DNase I (Promega, Madison, WI, USA) then cleaned up with the RNeasy mini kit (Qiagen, Mississauga, ON, Canada).

Isolation and in silico analysis of plum cDNA sequences

For the isolation of plum ethylene-responsive factors homologues (Ps-ERFs), first strand cDNA synthesis was carried out using 20 μg of total DNase-treated RNA in a 50 μl aliquot. One μl of cDNA was used in a PCR with the appropriate degenerate primers. Several sets of degenerate primers (Tournier et al., 2003) were used to isolate Ps-ERF clones that shared the structural characteristics associated with functional ERF transcription factors. The isolated fragments were cloned in pGEM-T easy vector (Promega), sequenced, and compared with database sequences using the BLAST program (Altschul et al., 1997). Extension of the partial cDNA clones was carried out using the 3′- and 5′- RACE kit (Invitrogen, Burlington, ON, Canada). Full-length amplification of cDNA sequences, designated Ps-ERF1a, Ps-ERF1b, Ps-ERF2a, Ps-ERF2b, Ps-ERF3a, Ps-ERF3b, and Ps-ERF12, was carried out using the Platinum Taq DNA Polymerase High Fidelity following the instructions provided by the manufacturer (Invitrogen). Alignments of the predicted protein sequences were performed with ClustalX (Jeanmougin et al., 1998) and GeneDoc (Nicholas and Nicholas, 1997). The Neighbor–Joining tree was constructed with PAUP* 4.0b3. Bootstrap values from 1000 replicates were obtained. The tree was visualized with the TreeView program (Page, 1996).

Real-time quantitative RT-PCR

DNase-treated RNA (5 μg) was reverse transcribed in a total volume of 50 μl using SuperScript III Reverse Transcriptase (Invitrogen). Real-time quantitative PCR (QPCR) was performed using 20 ng of total RNA in a 20 μl reaction volume using Sybr Green PCR MasterMix (Qiagen) on a M×4000® multiplex Quantitative PCR system (Stratagene, La Jolla, CA, USA). M×4000® v 4.20 software (Stratagene, La Jolla, CA, USA) was used to design gene-specific primers (see Supplementary Table S1 at JXB online). Other details are well described in the supplemental online materials and methods part.

Protoplast isolation and transient expression of Ps-ERF::GFP fusion proteins

The coding sequences of Ps-ERF1a and -1b were cloned as a C-terminal fusion in-frame with the GFP into the pGreen vector (Hellens et al., 2000) and expressed under the control of the 35S promoter. A high fidelity PCR system was used to amplify the full-length Ps-ERF1a and -1b clones. The corresponding ORFs were cloned using the BamHI restriction site of the pGreen vector. Protoplasts used for transfection were obtained from suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells according to the method described by Leclercq et al. (2005). Protoplasts were transfected by a modified polyethylene glycol method as described by Abel and Theologis (1994). Typically, 0.2 ml of protoplast suspension (0.5×106) was transfected with 50 μg of shared salmon sperm carrier DNA and 30 μg of either 35S::GFP (control), or 35S/Ps-ERF1a::GFP, or 35S/Ps-ERF1b::GFP plasmid DNA. Transfected protoplasts were incubated 16 h at 25 °C and analysed for GFP fluorescence by confocal microscopy (Leclercq et al., 2005). All transient expression assays were repeated at least three times.

The resulting plasmids, Ps-ERF1a::GFP and Ps-ERF1b::GFP, were engineered further by replacing amino acid residues, as well as by removing either one or the two clusters of the basic charged residues of the putative NLS from the full-length Ps-ERF1a and -1b sequences using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene, San Diego, CA, USA). The protoplast suspension was transfected with the different mutant versions as described previously.

Results

Molecular characteristics and organization of Ps-ERFs

Seven novel putative ERF clones were isolated from plum using an RT-PCR approach in order to investigate their involvement in fruit development and ripening. Ps-ERF1a, -1b, -2a, -2b, -3a, -3b, and -12 predicted to encode proteins of 282, 261, 381, 327, 231, 235, and 172 amino acid residues (Table 1), with calculated molecular weights of 30.2, 29.4, 42.1, 36, 25.1, 25.5, and 18.3 kDa, respectively. The relationships between the predicted amino acid sequences, as indicated by percentage similarity over the whole sequence, are presented in Table 1. Although the various Ps-ERF cDNAs exhibited low sequence homology between them (13% identity, 20% similarity), several signature elements were detected. Multiple alignments of full-length predicted Ps-ERF proteins with other reported ERF sequences (see Supplementary Fig. S1 at JXB online) highlighted a number of conserved motifs and structural similarities that are commonly associated with the ERF/AP2 family of plant transcription factors (Fujimoto et al., 2000; Sakuma et al., 2002; Tournier et al., 2003). The deduced amino acid sequences of Ps-ERFs comprise a conserved DNA-binding ERF/AP2 domain (ranging from 58 to 59 aa), which is characteristic of the plant ERF gene family (see Supplementary Fig. S1 at JXB online). The ERF domain of Ps-ERF proteins and other plant ERFs showed high sequence homology (67–94% identity and 81–100% similarity). By contrast, full-length Ps-ERFs exhibited a considerable divergence, not only to other characterized ERFs (15–66% similarity), including Arabidopsis, tomato, and other plant species, but also among themselves (Table 1). Such low overall sequence similarity is largely due to the low level of homology outside the ERF domain. Furthermore, the ERF domain in all plum sequences includes two key amino acid residues, 14th A (Ala) and the 19th D (Asp), believed to contribute a functional GCC box-binding activity in many ERFs (Sakuma et al., 2002). In addition, all the seven cDNAs possess a basic region in their predicted proteins that might function as a nuclear localization signal (NLS) (Raikhel, 1992), and all, except Ps-ERF12, hold an acidic domain that might act as an activation domain (AD) for transcription (Yang et al., 2002).

Table 1.
Amino acid sequence comparison between the predicted full length ethylene responses factors (Ps-ERF) cDNAs

Comparison of the amino acid sequence and phylogenetic analysis of 34 characterized ERFs (Fig. 1), from various plant species, revealed that Ps-ERF sequences belong to three out of the four classes of ERF proteins (Fujimoto et al., 2000; Tournier et al., 2003). Predicted Ps-ERF1a and -1b proteins are classified as members of ‘B-3’/‘Class I’ ERFs (Sakuma et al., 2002; Tournier et al., 2003) (Fig. 1). Both sequences shared 39–63% similarity to their homologues in Arabidopsis, tomato, tobacco, and apple (see Supplementary Fig. S1a at JXB online). Like all ‘Class I’ gene members, Ps-ERF1a and -1b predicted proteins possess a putative NLS motif near the C-terminal region, hold an ERF domain consisting of 59 aa located near the middle of the sequences and comprise an acidic activator domain placed in the N-terminal region (see Supplementary Fig. S1a at JXB online; Fig. 2) (Zhou et al., 1997; Fujimoto et al., 2000; Tournier et al., 2003). Despite the presence of all these common features, phylogenetic analysis pointed out the presence of at least two clearly separated branches among ‘Class I’ ERFs (Fig. 1). The first branch, Group A, includes Ps-ERF1a, At-ERF1, and At-ERF2 that exhibited a relatively high sequence similarity (59–64%) and characterized by the presence a short (25 aa) acidic domain (see Supplementary Fig. S1a at JXB online). The second branch gene members, Group B, are structurally similar in their long acidic domain, but displayed considerable sequence divergence (41–51% similarity). However, the acidic domain in Ps-ERF1b and MdERF2 sequences are much longer (70–130 aa) when compared to other group B members (53–55 aa) and occupy almost all of the N-terminal region (see Supplementary Fig. S1c at JXB online; Fig. 2). The previous observations suggest that Ps-ERF1b and MdERF2 sequences could represent a new clade under ‘Class I’ ERFs.

Fig. 2.
Structure of Ps-ERF proteins (Ps-ERF1a, -1b, -2a, -2b, -3a, -3b, and -12). Black and hatched boxes represent the ERF and acidic domains, respectively. The putative nuclear localization signals (NLS) are marked with filled or open arrows within the boxes. ...

Ps-ERF2a and -2b encode for proteins that belong to the recently identified ‘B-2’/‘Class IV’ ERFs (Fig. 1) (Tournier et al., 2003). Ps-ERF2a, -2b and their putative homologues from other species in this class are well conserved even outside the ERF domain (76–86% similarity) (see Supplementary Fig. S1b at JXB online). Consistent with other ‘Class IV’ ERFs, Ps-ERF2a and -2b have a conserved ERF domain (58 aa) near the N-terminus, a putative NLS [KRKRK/106–110 aa and KRARK/71–75 aa for Ps-ERF2a and -2b, respectively] located just before the ERF domain (see Supplementary Fig. S1b at JXB online; Fig. 2). In addition, a highly conserved N-terminal signature sequence of unknown function [MCGGAII/L] (Tournier et al., 2003) was also identified in both clones (see Supplementary Fig. S1b at JXB online). However, sequence analysis of different ERFs from ‘Class IV’ revealed that gene members of this class could be further divided into two groups (Fig. 1) based on the position of the acidic domain (Fig. 2). Group A includes Ps-ERF2a and its closest homologues (Fig. 1), which exhibit a long amino acid sequence (369–381 aa; see Supplementary Fig. S1b at JXB online) with an acidic domain (22–24 aa) near the N-terminus (Fig. 2) (Yi et al., 2004; Zhang et al., 2004). Conversely, Ps-ERF2b and its closest homologues of group B (Fig. 1), exhibits a shorter amino acid sequence (260–327 aa; see Supplementary Fig. S1b at JXB online) with a slightly longer acidic domain (26–32 aa) near the C-terminal end (Fig. 2) (Tournier et al., 2003; Lee et al., 2004; Mizuno et al., 2006).

Ps-ERF3a, -3b, and -12 predicted proteins assign to ‘B-1’/‘Class II’ (Sakuma et al., 2002; Tournier et al., 2003) (Fig. 1) that includes all ERFs possess the conserved EAR repressor motif (ERF-associated amphiphilic repression) in the C-terminal end (Ohta et al., 2001) (see Supplementary Fig. S1c at JXB online; Fig. 2). The ERF domain for ‘Class II’ gene members is located close to the N-terminal region and consists of 58 aa (Fig. 2). A putative NLS motif was identified within the ERF domain region (see Supplementary Fig. S1c at JXB online) (Tournier et al., 2003; Long-Guo and Jin-Yuan, 2008). Interestingly, a second putative NLS motif near C-terminal region has been identified in Ps-ERF3a (see Supplementary Fig. S1c at JXB online). However, sequence analysis of ‘Class II’ ERFs revealed that this class could also be divided into two groups (Fig. 1). Group A members include Ps-ERF3a, Ps-ERF3b, AtERF3, AtERF4, SlERF3, and NtERF3, which are characterized by a long amino acid sequence (221–235 aa) (see Supplementary Fig. S1c at JXB online). Moreover, Ps-ERF3a and -3b hold an acidic domain of 27 aa and 28 aa, respectively, just before the EAR motif. On the other hand, Ps-ERF12 orthologues was only found in Arabidopsis, AtERF12. Plum and Arabidopsis ERF12, which are so far the only members of the ‘Class II’/group B (Fig. 1), are characterized by having a short N-terminal end and an overall short amino acid sequence length (172–189 aa) (see Supplementary Fig. S1c at JXB online). Both plum and Arabidopsis ERF12 predicted proteins lack the acidic domain.

Expression of Ps-ERFs during fruit development and ripening

To understand the possible role of the various ethylene responsive factors in fruit physiology, the expression of the seven isolated Ps-ERF mRNAs was quantified by real-time PCR to determine their accumulation patterns throughout fruit development and ripening.

Figure 3 shows the expression profile of the different Ps-ERFs during flowering and early fruit development (0–15 DAB). The isolated sequences were differentially expressed throughout this stage of development. Out of the seven studied cDNAs only Ps-ERF1a and -12 were greatly expressed in flowers. Although Ps-ERF1b and -3a were expressed in lower levels, but in the same time their accumulation throughout this stage represented the highest levels among the whole experiment. The accumulation of the different Ps-ERF mRNAs were considerably stimulated at bloom (~4 DAB for Ps-ERF1a, -1b, and -3b) or after fertilization (~ 7 DAB for Ps-ERF2a, -2b, -3a, and -12), and decreased gradually afterwards in initiated fruit (10–15 DAB).

Fig. 3.
Steady-state transcript levels of Ps-ERFs mRNA assessed by real-time quantitative PCR during flowering and early fruit development of the ‘EG’ cultivar. The fertilized flowers stage is marked with an asterisk. The experiments were carried ...

Stone fruit development period (22–77 DAB) could be divided into three different stages (S1–S3) (Tonutti et al., 1997; El-Sharkawy et al., 2007). The first stage (S1, 22–37 DAB) is illustrated by intense cell division and differentiation, and rapid growth. Low levels of ethylene production, ranging from 0.8–2 nl g−1 h−1 ±0.2, have been detected during S1 of fruit development (data not shown). Throughout S1 (Fig. 4; Stage 1), six out of the seven Ps-ERF transcripts steadily increased to a peak early, ~27 DAB (for Ps-ERF1a, -1b, -2a, and -2b) or later, ~32 DAB (for Ps-ERF3b and –12) and declined thereafter. The drop pattern in the case of early peaked transcripts, ~27 DAB, was much sharper than in later accumulated ones, ~32 DAB. Ps-ERF3a was expressed at high constant levels during the same period.

Fig. 4.
Steady-state transcript levels of Ps-ERFs mRNA assessed by real-time quantitative PCR during S1 and S2 of fruit development using the whole ‘EG’ fruit. Throughout S3 of fruit development, expression was studied in the pulp (black filled ...

In the second stage (S2, 42–52 DAB), there is hardly any increase in fruit size but the endocarp hardens to form a solid stone (pit hardening). Ps-ERF1a, -2a, and -2b mRNAs steadily decreased with the progress of fruit age to reach their basal levels at the end of this stage, ~52 DAB, while those of Ps-ERF3b were weakly accumulated (Fig. 4; Stage 2). By contrast, Ps-ERF1b, -3a, and -12 transcript signals were strongly detected, but their accumulation patterns were different. Ps-ERF1b and -3a reached their relative maximal levels ~47 DAB, while those of Ps-ERF12 reached it earlier, ~42 DAB. The three transcripts subsequently dropped to their normal low levels (Ps-ERF3a and -12) or were almost undetectable (Ps-ERF1b) at the end of this stage, ~52 DAB.

The third stage (S3, 57–77 DAB) is accompanied by rapid cell division resulting in a significant increase in fruit size. During the S3 stage when the pulp (mesocarp) separates from the seed (endocarp+embryo), Ps-ERFs expression profile in the pulp was, generally, parallel to those in the seed (Fig. 4; Stage 3). However, markedly higher accumulation of Ps-ERF1b, -2a, -2b, and -3a mRNAs (2–11-fold) were detected in the seed compared with the pulp. Ps-ERF3b and -12 transcripts were uniformly accumulated in fruit pulp and seed. Ps-ERF1a accumulation was slightly higher in the pulp than in the seed. In the pulp, transcripts of Ps-ERF2b, -3a, -3b, and -12 seem to be constitutively expressed throughout S3, although those of Ps-ERF12 were present at much higher levels. A slight stimulation in Ps-ERF1a, -1b, and -2a transcription levels were detected (~62 DAB) and steadily declined thereafter. In the seed, Ps-ERF1a, -1b, -2a, -2b, and -3a clones displayed very similar expression patterns. Their mRNAs were strongly accumulated ~62 DAB and decreased afterwards, reaching a basal level at the end of this stage, ~ 77 DAB. However, those of Ps-ERF3b and -12 were induced at low and high constant levels, respectively.

The last stage of fruit development (S4) denotes the fruit ripening or climacteric stage. Throughout this stage the fruit starts to enhance ripening in an ethylene-dependent manner (El-Sharkawy et al., 2007, 2008). ‘EG’ fruit displayed an early, rapid ripening, and short and rapid (maximal 5 d) ethylene production profile (see Supplementary Fig. S2 at JXB online). A dramatic increase in Ps-ERFs transcription levels has been accelerated throughout ‘EG’ fruit ripening, 78–83 DAB (Fig. 5). Their transcripts were barely detectable at the non-climacteric stage, ~78 DAB. As ripening progressed and higher levels of autocatalytic ethylene produced, their expression levels increased in abundance. However, their patterns of transcript accumulation were different. In pulp, Ps-ERF1a, -1b, -3b, and -12 increased to a peak ~82 DAB, and declined afterward at the post-climacteric stage, ~83 DAB (Fig. 5). Their accumulation pattern correlated well with the evolution of ethylene production and their maximal transcript levels coincided with the climacteric ethylene peak (see Supplementary Fig S2 at JXB online; Fig. 5), whereas, those of Ps-ERF2b and -3a transcripts were gradually augmented along with the progression of fruit ripening and continued to increase past the climacteric peak, reaching their maximal levels in post-climacteric fruit, ~83 DAB. Ps-ERF2a peaked early at the pre-climacteric stage, ~80 DAB, and declined thereafter. All Ps-ERFs, except for the Ps-ERF2b, expression pattern in seed had a similar trend as in pulp, but at much lower levels. The Ps-ERF2b accumulation profile, in ‘EG’ seed, mimics that of Ps-ERF2a, and they reached their highest levels in the pre-climacteric stage, ~80 DAB (Fig. 5).

Fig. 5.
Steady-state Ps-ERFs transcript levels assessed by real-time quantitative PCR throughout ‘EG’ fruit ripening, non-treated (left panel) and treated (right panel) with 1-MCP. The expression was studied in pulp (black filled bars) and in ...

The association of Ps-ERF transcripts induction with fruit ripening was further investigated using 1-MCP, which completely abolished the ethylene burst and ripening in the pretreated fruits (data not shown). Surprisingly, the increases of Ps-ERF1b and -2a mRNAs during ‘EG’ fruit ripening were not found to be ethylene-dependent, but seemed to be ripening-related, since their transcripts were still present in considerable amounts in MCP-treated fruits (Fig. 5). MCP completely inhibited the ethylene-associated accumulation of Ps-ERF2b, -3a, and -12 in the whole fruit. However, Ps-ERF1a and -3b mRNAs accumulation were fully inhibited in the pulp and did not significantly respond to 1-MCP treatment in the seed.

The differences in ripening behaviour between various plum cultivars might be due to the differences in their capacity to produce and respond to ethylene (El-Sharkawy et al., 2007, 2008). In order to determine the involvement of the different Ps-ERFs in ethylene sensitivity and, subsequently, in the capacity of the fruit to ripen, their expression was investigated during ‘SH’ fruit ripening. ‘SH’ fruit exhibited a suppressed climacteric pattern and ripened later than ‘EG’ (see Supplementary Fig. S2 at JXB online).

Throughout ripening of ‘SH’ fruit (90–105 DAB), the expression pattern of Ps-ERF1a, -1b, -2a, and -2b in the pulp was stimulated in parallel with the evolution of autocatalytic ethylene production (see Supplementary Fig. S2 at JXB online; Fig. 6), however, their accumulation levels were obviously lower than in case of ‘EG’ fruit (compare Figs 5 and and6).6). The accumulation of Ps-ERF3a and -3b transcripts remained temporally constant and low; however, those of Ps-ERF12 were totally absent. Only Ps-ERF2a and -2b transcripts were detected in the seed. Ps-ERF2a and -2b accumulated in ‘SH’ seeds in the same manner as in pulp but at slight lower levels (Fig. 6).

Fig. 6.
Steady-state Ps-ERFs transcript levels assessed by real-time quantitative PCR throughout ‘SH’ fruit ripening, non-treated (left panel) and treated (right panel) with 1-MCP. The expression was studied in pulp (black filled bars) and in ...

MCP treatment completely eliminated the ethylene burst and ripening in the treated ‘SH’ fruits (data not shown). MCP-‘SH’ fruit strongly repressed the ethylene-related induction of Ps-ERF1a and -3a, and, contrary to the situation in the ‘EG’ cultivar, also for Ps-ERF1b and -2b. Although, Ps-ERF2a and -3b transcripts accumulation were totally inhibited in the pulp, their expression was significantly enhanced in the seed. Similar to the situation during ‘SH’ fruit ripening, Ps-ERF12 transcripts were totally missing in MCP-‘SH’ treated fruit (Fig. 6).

Effect of different hormones and hydrogen peroxide treatments on the expression of the Ps-ERFs

In view of the inducible nature of Ps-ERFs during fruit development and ripening, the question was raised whether any of the Ps-ERFs are regulated by one or more of the various hormones and/or enzymes that are associated with fruit development in plum. In particular, that significant Ps-ERFs transcript levels were accumulated in non-ethylene-related tissues such as young fruit, seed, and MCP-treated fruits. To address this hypothesis, the expression profile of the seven transcripts was assessed in leaves exposed to different treatments (gibberellin, cytokinin, auxin, ethylene, and hydrogen peroxide).

None of the Ps-ERFs significantly respond to gibberellin treatment at least under the conditions used in this study (data not shown).

Only Ps-ERF1a, -1b, -3a, and -12 transcription levels were accelerated in response to cytokinin application (Fig. 7). Ps-ERF1a, -1b, and -3a mRNAs were steadily augmented with increasing cytokinin concentrations, while Ps-ERF12 decreased. The effect of cytokinin treatment on the induction of the four Ps-ERF transcripts was significantly higher for Ps-ERF1b and -3a (~26-fold) than those of Ps-ERF1a and -12 (~8-fold). However, Ps-ERF2a and -3b were negatively regulated by the treatment and Ps-ERF2b did not significantly respond.

Fig. 7.
Steady-state Ps-ERFs transcript levels assessed by real-time quantitative PCR in ‘EG’ leaves treated with cytokinin (BA), auxin (IAA), ethylene (ethephon), and hydrogen peroxide (H2O2) using the indicated concentrations. The experiments ...

Leaves treated with auxin exhibited a remarkable increase of all the studied Ps-ERFs. Ps-ERF1a and -1b transcripts were strongly accumulated but only with low auxin concentration (0.4 μM) and gradually decreased with higher concentrations. The other five Ps-ERF mRNAs were largely and selectively detected in leaves treated with 4 μM auxin concentration (Fig. 7).

Ethylene treatment accelerates the transcription of the various Ps-ERFs, excluding Ps-ERF2a that was not significantly altered by the treatment (Fig. 7). Ps-ERF1a, -1b, and -12 expressions were slightly induced by low ethylene concentration (1 mM) and strongly released thereafter to reach a maximal level with higher concentrations. Contrary, Ps-ERF2b signal was greatly detected with low ethylene concentration and declined afterward, however, those of Ps-ERF3a and -3b increased to a peak with 2 mM ethylene concentration.

Accumulation of all Ps-ERFs mRNAs, excluding Ps-ERF1a, was significantly enhanced in H2O2-treated leaves (Fig. 7). Expression of Ps-ERF1b and -2b was strongly detected in leaves treated with a low H2O2 concentration (0.5 mM) and declined gradually with higher concentrations. Ps-ERF2a, -3a, -3b, and -12 transcripts clearly showed the same manner of accumulation when they were selectively peaked with 1 mM H2O2 concentration.

Ps-ERF1a and -1b encoded proteins are targeted to the nucleus

Inspection of the amino acid sequences of all ‘Class I’ ERFs characterized so far revealed that each ERF member belonging to this class holds typical nuclear localization sequences (NLSs) of the bipartite class with two clusters of basic residues separated by 12–18 amino acids (see Supplementary Fig. S1a at JXB online). The two NLS clusters in ‘Class I’/Group A ERFs (Fig. 1) consist of the basic amino acids (KR) and (KK/RRR/KK); however, there are (KRR/K) and (KRRRK) in Group B (see Supplementary Fig. S1a at JXB online). Although Ps-ERF1b and MdERF2 belong to Group B, they appear to exhibit a particular putative NLS sequence. The amino acid residues ‘Ser-Ala’ and ‘Thr-Ala-Ala-Thr’ located in the middle of the second NLS cluster of Ps-ERF1b and MdERF2, respectively, (see Supplementary Fig. S1a at JXB online), are unique to these clones while all other ‘Class I’ ERFs, including Ps-ERF1a, have the basic amino acids ‘Lys’ and/or ‘Arg’. It was checked that this modification in the Ps-ERF1b NLS sequence was not caused by a cloning or sequencing error by sequencing several clones from three independent PCRs and using a high fidelity DNA polymerase. Due to the strong difference between these amino acids in terms of charge, polarity, and hydrophobicity, it was decided to examine the subcellular localization of Ps-ERF1b and compare it with its closest homologue in plum Ps-ERF1a. Their coding regions were fused to the GFP tag and were transiently expressed in tobacco protoplasts. Fluorescence microscopy analysis demonstrated that control cells transformed with the GFP gene alone displayed fluorescence spread throughout the cytoplasm and nucleus, in accordance with the expected cytosolic localization of the GFP proteins (Fig. 8A). By contrast, Ps-ERF1a and Ps-ERF1b::GFP fusions were localized exclusively in the nucleus, indicating that both Ps-ERF1s were fully able to redirect the GFP from the cytosol to the nucleus.

Fig. 8.
Ps-ERF1a, and -1b proteins and their modified versions were fused to the GFP tag. N. tabacum protoplasts were transfected with the following constructs: (A) the control 35S::GFP, Ps-ERF1a::GFP and Ps-ERF1b::GFP; (B) the site-directed mutagenesis versions ...

To investigate the structural role of the two amino acid residues ‘Ser’ and ‘Ala’ located at the Ps-ERF1b NLS site, the two amino acids ‘Arg’ found in the putative NLS site of Ps-ERF1a were changed to ‘Ser’ and ‘Ala’ by site-directed mutagenesis, MuERF1a/SA. Similarly, the two amino acids ‘Ser’ and ‘Ala’ in Ps-ERF1b were modified into ‘Arg’, MuERF1b/RR. Surprisingly, protoplasts transfected with a MuERF1a/SA::GFP fusion showed green fluorescence through the entire cytoplasm and nucleus. By contrast, MuERF1b/RR::GFP fusion still localized exclusively to the nucleus (Fig. 8B). Taken together, these data strongly suggest that the second NLS cluster of Ps-ERF1a is essential for proper nuclear localization.

To examine this domain further, the two clusters of the basic charged residues of the NLS from the full-length Ps-ERF1a (ΔERF1a-KR/KRRKK) and -1b (ΔERF1b-KRK/KSAKR) sequences were deleted, and the resulting mutants were transfected with tobacco protoplasts. This deletion caused the loss of nuclear localization of both Ps-ERF1s, and the green fluorescence was uniformly distributed throughout the cytoplasm and nucleus (Fig. 8C). Therefore, this bipartite domain region is the sole NLS necessary to target such proteins into the nucleus.

To characterize the bipartite NLS domain of both Ps-ERF1s further, the amino acid residues KRRKK and KSAKR were removed from the full-length Ps-ERF1a (ΔERF1a-KRRKK) and -1b (ΔERF1b-KSAKR), respectively. As shown in Fig. 8D, this elimination dramatically altered the normal targeting of the protein via the nucleus, but only for Ps-ERF1a. The Ps-ERF1b localization was not significantly affected. These results confirm again the importance of the ‘KRRKK’ sequence in the correct Ps-ERF1a nuclear localization and that the ‘KSAKR’ domain in Ps-ERF1b plays only a minor role in protein localization.

After that, the amino acid residues KR and KRK were deleted from the full-length Ps-ERF1a (ΔERF1a-KR) and -1b (ΔERF1b-KRK), respectively, in order to discover their role in protein nuclear localization. ΔERF1a-KR was exclusively localized to the nucleus, while the ΔERF1b-KRK protein was visualized in the nucleus as well as in the cytoplasm (Fig. 8E). Therefore, it seems that ‘KRK’ domain in Ps-ERF1b play the major role to target the protein via the nucleus.

Discussion

ERF proteins were first identified as transcription factors, which possess GCC box binding activity (Ohme-Takagi and Shinshi, 1995; Büttner and Singh, 1997). Although sequence identity can be as low as 13% among the different ERFs, all have highly conserved signature elements associated with the ERF/AP2 transcription factors (Fujimoto et al., 2000; Ohta et al., 2001; Sakuma et al., 2002; Tournier et al., 2003; Cao et al., 2006), including the presence of a typical ERF domain that is responsible for the DNA binding activity, the acidic activator domain (AD), and a basic amino acid reach region putatively functions as a nuclear localization signal (NLS). However, variations within the ERFs are reasonable enough to divide them into four main classes based on sequence structure and similarities (Fujimoto et al., 2000; Sakuma et al.; 2002; Tournier et al., 2003). These variant characteristics include the length and position of the ERF domain. Moreover, the presence of the highly conserved C-terminal EAR repressor and N-terminal (MCGGAII/L) motifs in all ERF proteins belong to Class ‘II’ and ‘IV’, respectively (Ohta et al., 2001; Tournier et al., 2003). Further, the NLS motif is located near the C-terminal end for ‘Class I’, just before the ERF domain for ‘Class II’, and within the ERF domain for ‘Class IV’ ERFs. Some other minor features that might distinguish between ERF proteins within the same class have been identified, such as the length, the position, and the presence of the acidic domain for Class ‘I’, ‘II’, and ‘IV’ ERFs, respectively. Despite the extensive structural differences between them, all are functional ERF transcription factors, as defined by their DNA binding ability and their function as transcriptional activators or repressors (Fujimoto et al., 2000; Tournier et al., 2003).

In this study, seven Ps-ERFs were isolated. Based on their structural organizations and dendrogram analysis, a well-defined branch, ‘Class III’, has Arabidopsis, tomato, and other plant species sequences but lack plum sequences, suggesting that there are likely to be as yet unidentified ERF genes within the plum genome. In species where multiple ERFs have been characterized, each protein appears to have a unique mode of function (Fujimoto et al., 2000). Accordingly, the Ps-ERFs fall into three out of the four different classes of the previously characterized ERF proteins (Fujimoto et al., 2000; Tournier et al., 2003). Ps-ERF1a and -1b belong to ‘Class I’ ERFs that have been shown to function as activators of transcription (Zhou et al., 1997; Solano et al., 1998; Fujimoto et al., 2000; Ohta et al., 2000; Tournier et al., 2003; Wang et al., 2007). Ps-ERF3a, -3b, and -12 represent members of ‘Class II’ ERFs. Several members of this class have been demonstrated to be active repressors of transcription (Fujimoto et al., 2000; Ohta et al., 2000, 2001). Ps-ERF2a and -2b are categorized as ‘Class IV’ ERFs based on the location of the putative NLS site and the presence of the conserved N-terminal motif (Tournier et al., 2003). The function of this motif (MCGGAII/L) is unknown yet, but it is unlikely to be required for nuclear localization or for binding to the GCC box (Tournier et al., 2003). Moreover, the function of the ‘Class IV’ ERFs is also unclear.

As expected for transcription factors, Ps-ERF1a and -1b were localized in the nucleus of tobacco cells. All ‘Class I’ ERF gene members contain typical bipartite NLSs with two clusters of basic residues. However, functionality of either of these NLSs (Ps-ERF1b and MdERF2 or Ps-ERF1a, At-ERF1, and At-ERF2) is often sufficient to target the protein via the nucleus, although in some instances (Sl-ERF1, Pti4, Nt-ERF1, and Nt-ERF2) both are active (Gu et al., 2002).

Presuming that the sequences isolated in this study encode functional ethylene-responsive factors, their expression profile was studied in different plant tissues and under various conditions in order to determine their role in fruit development in terms of a double sigmoid growth pattern, their involvement in ethylene sensitivity, and the capacity of the fruit to ripen, and their hormone-dependent accumulation.

The difference in the Ps-ERFs accumulation profile throughout flower development suggested the contribution of different plant hormones, strongly occurring during this stage, in the regulation of the various Ps-ERFs. Based on the times of transcripts induction in flowers and the expression of Ps-ERFs post-application of the different hormones, it appears that Ps-ERF1a, -1b, and -3b mRNAs are induced in opened flowers, before fertilization (~4 DAB), in an ethylene-dependent manner. Interestingly, the expression pattern of these three transcripts was entirely similar to the previously characterized ethylene biosynthesis elements (ACC synthesis, Ps-ACS), which supports the possible role of ethylene in their accumulation (El-Sharkawy et al., 2008). After fertilization, large amounts of auxin and cytokinin might be accelerated in response (Miller et al., 1987; Hartmann et al., 2002), which leads to the up-regulation of different transcripts associated with the presence of these two hormones. The large accumulation of Ps-ERF3a and -12 post-fertilization (~7 DAB) might be enhanced in auxin- and/or cytokinin-dependent manners. However, those of Ps-ERF2a and -2b accumulate in an auxin-dependent manner since cytokinin has no effect in their expression. Importantly, the up-regulation of Ps-ERF2a, -2b, -3a, and -12, post-fertilization, coincided with the complete inhibition of the different Ps-ACS transcripts, which eliminate any feasible role played by ethylene in their accumulation (El-Sharkawy et al., 2008).

The increase of the various Ps-ERF transcripts during the S1 stage corresponds to a strong accumulation of Ps-ACS mRNAs, low levels of ethylene production, high ACC and MACC content, and massive levels of IAA and cytokinin content, all of which are important for cell division during early embryogenesis (Miller et al., 1987; Hartmann et al., 2002; DeDios et al., 2006; El-Sharkawy et al., 2008). Mantiri et al. (2008) clearly showed that Medicago ERF (MtSERF1) is essential for somatic embryogenesis and is induced by auxin and cytokinin that are both accelerated during embryo development. Thus, Ps-ERFs accumulation may be enhanced in an ethylene-, auxin-, and/or a cytokinin-dependent manner throughout this stage.

Ps-ERF1b, -2b, -3a, and -12 transcripts were detected in considerably high concentrations during the S2 stage. To determine the role of these four transcripts, it was essential to understand clearly the physiological aspects that characterize the S2 stage. As mentioned previously, during this stage, there is hardly any increase in fruit size which coincided with a significant reduction in auxin content (Miller et al., 1987; Trainotti et al., 2007), indicating a minor role for auxin. Furthermore, the evaluation of different ethylene biosynthesis and perception elements in plum fruits (i.e. the same developmental stages studied in the present work) revealed the absence of all these ethylene elements throughout this stage, and also suggesting an insignificant role for ethylene (El-Sharkawy et al., 2007, 2008). The only fruit development process accelerated during this stage was the lignification of the endocarp in a H2O2-dependent manner to form a solid stone (seed). The role of H2O2 in the lignification of the cell wall was clearly determined by Ros Barceló (2005), and Kärkönen and Fry (2006). These results led to the belief that the transcription of these four selected Ps-ERFs could be altered due to the high levels of endogenous H2O2. Interestingly, application of H2O2 activated the accumulation not only of these four transcripts but also of Ps-ERF2a and -3b.

Although there is hardly any ethylene emission detected throughout S3 of fruit development, Ps-ERF transcripts were generally abundant in young fruit at this stage. On the other hand, S3 seed exhibited much greater Ps-ERFs transcription contents than those found in the pulp. The seed in this stage is highly lignified and dry, and ethylene synthesis is usually inhibited (Rodrìguez-Gacio and Matilla, 2001), which eliminate any possible ethylene regulation.

During S3, the fruit recovered its activity by strongly accelerated cell division and expansion, which results in a significant increase in fruit size in an auxin-dependent manner (Abel and Theologis, 1996; Christian et al., 2006; Trainotti et al., 2007). Furthermore, seed from this stage exhibited the relatively high auxin contents necessary for embryogenesis (Miller et al., 1987). Finally, our data showed that application of auxin significantly enhanced the transcription levels of all Ps-ERFs. Consequently, it seems that auxin is the logic hormone, which controls the transcription of Ps-ERFs throughout the S3 stage.

It is almost certain that the series of modifications that transform a mature green fruit into a ripe fruit occur during S3 (El-Sharkawy et al., 2007, 2008; Trainotti et al., 2007) and involve many different metabolic pathways. Therefore, these ethylene-independent factors that control the transition of a fruit from the end of growth to the onset of ripening are of primary importance. Previous studies showed that auxin content steadily increased in peach fruits during the S3 stage and throughout fruit ripening (Miller et al., 1987; Ohmyia, 2000; Trainotti et al., 2007), which strengthens the idea that auxin is actively involved in the fruit ripening process.

Expression analysis during ‘EG’ fruit ripening revealed that all Ps-ERFs accumulated greatly in the whole fruit (pulp and seed). Such high levels of accumulation could be cytokinin-, ethylene-, and/or auxin-dependent. Cytokinin can increase ethylene biosynthesis via a post-transcriptional mechanism that increases the ACS protein stability (Chae and Kieber, 2005). Similarly, auxin can affect ethylene levels through an increase in ACS transcription levels (Abel et al., 1995; Trainotti et al., 2007; El-Sharkawy et al., 2008). Ethylene also can affect its own biosynthesis, either by increasing (autocatalysis) or decreasing (autoinhibition) its rate of production (Nakatsuka et al., 1998). Furthermore, the three hormones could accelerate the transcription of either all (as in case of auxin) or some (as in the case of cytokinin and ethylene) plum ERFs characterized in this study.

Despite the important role played by cytokinin in enhancing ethylene production, as well as its role in the induction of several Ps-ERF transcripts, this hormone could not be present in such adult tissue and, consequently, could not participate in any fruit ripening process (Hartmann et al., 2002). However, Ps-ERFs and, accordingly, the ripening process might be enhanced during fruit ripening in both ethylene- and auxin-dependent manners.

The differences in the accumulation levels and/or pattern of the various ethylene elements throughout ripening of early and late fruits (El-Sharkawy et al., 2007, 2008; this paper) might be largely due to the variation in the levels of auxin content and ethylene produced among the two plum cultivars. Such variations affect the capacity of the fruit to produce and respond to ethylene, which results in the differentiation in ripening behaviour thereafter (Miller et al., 1987; Ohmyia, 2000). If this is the case, auxin might be accumulated rapidly and in much higher levels in early cultivars during S3 (Miller et al., 1987), which leads to the up-regulation of different transcripts and proteins associated with auxin, including different ethylene synthesis, perception, and signal transduction elements (El-Sharkawy et al., 2007, 2008; this paper). When such high levels of ethylene-related proteins accumulate during S3, the mature green fruit transit into the S4 ripening stage early (~78 DAB). The ripening process in fruit during the non-climacteric stage (before the onset of autocatalytic ethylene) progresses only in an auxin-dependent manner (Miller et al., 1987; Ohmyia, 2000; Trainotti et al., 2007). Once the fruit initiates autocatalytic ethylene (pre-climacteric), the ripening process will be enhanced in both auxin- and ethylene-dependent manners, since transcripts of some ethylene elements still present in considerable levels in non-ethylene associated tissues such as seed and MCP-treated fruits (Miller et al., 1987; Trainotti et al., 2007; El-Sharkawy et al., 2008; Ziliotto et al., 2008; this paper). Therefore, the possibility of regulating many ethylene-related genes by auxin is the best alternative that explains the significant accumulation of such transcripts during fruit ripening in an ethylene-independent manner.

Late cultivars, including ‘SH’, seem to produce insufficient quantities of auxin to co-ordinate the transition into ripening stage. The low level of auxin throughout the S3 stage results in minimal accumulation of ethylene-related proteins and, consequently, the fruit reaches the S4 stage late (~90 DAB) accompanied by a slow ripening process thereafter. Ethylene production in ‘EG’ fruit reached a maximum (33.3 nl g−1 h−1 ±1.0) after approximately 5 d post-harvest, however, those of ‘SH’ fruit reached its maximum (5.7 nl g−1 h−1 ±0.7) after approximately 11 d post-harvest. On the other hand, treatment of such late varieties with exogenous auxin or ethylene restored the typical climacteric pattern (Abdi et al., 1997; Ohmyia, 2000). Taken together, it seems that auxin is at least one of the most important factors, if not the only factor, which commands the transition of a fruit from the S3 growth stage to the S4 ripening stage. The role of auxin continues thereafter in association with ethylene in enhancing fruit ripening.

In this investigation, as well as, in our previous reports (El-Sharkawy et al., 2007, 2008) many ethylene related genes were characterized. These ethylene-related proteins can accumulate either in an ethylene-dependent, or auxin-dependent, or both ethylene- and auxin-dependent manners. However, the role played by auxin is more significant in controlling the ripening behaviour. The importance of auxin comes from its being essential in initiating the ripening phenomenon itself, and after that, in concert with ethylene, in accelerating the ripening process. Any genetic and/or environmental factors that negatively affect the accumulation of auxin in the early developmental stages (before ripening) resulted in the significant delay in fruit ripening. Characterization of many auxin-related elements will be our next step in order to establish the role of auxin in fruit ripening and to investigate the existence of any cross-talk between ethylene and auxin through controlling the regulation of auxin-related genes by ethylene.

Supplementary data

The supplementary data available at JXB online consists of a detailed description of the real-time PCR condition and the method used for expression levels calculation.

Supplementary Table S1. The real-time PCR primers used in this study.

Supplementary Fig. S1. The amino acid sequence alignment of ‘Class I’ (a), ‘Class IV’ (b), and ‘Class II’ (c) ERFs.

Supplementary Fig. S2. The ethylene production (nl g−1 h−1) of five fruits during ripening of early ‘EG’ and late ‘SH’ plum cultivars.

Supplementary Material

[Supplementary Data]

Glossary

Abbreviations

AD
acidic activator domain
aa
amino acids
BA
benzyladenine
DAB
days after bloom
EAR
ERF-associated amphiphilic repression
‘EG’
‘Early Golden’
ERF
ethylene-responsive factor
GFP
green fluorescent protein
IAA
indole acetic acid
NLS
nuclear localization signal
1-MCP
1-methylcyclopropene
‘SH’
‘Shiro’
S1, S2, S3, S4
the four stages of plum fruit development

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