JXB Advance Access originally published online on January 8, 2007
Journal of Experimental Botany 2007 58(3):673-686; doi:10.1093/jxb/erl242
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RESEARCH PAPER |
Members of the ethylene signalling pathway are regulated in sugarcane during the association with nitrogen-fixing endophytic bacteria
1Instituto de Bioquímica Médica, UFRJ, 21941-590, Rio de Janeiro, RJ, Brazil
2Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas, do Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915, 22460-030, Rio de Janeiro, RJ, Brazil
3CNPAB/EMBRAPA, BR465, Km47 23851-970, Seropédica, RJ, Brazil
To whom correspondence should be addressed. E-mail: hemerly{at}bioqmed.ufrj.br or hemerly{at}cshl.edu
Received 31 March 2006; Revised 18 October 2006 Accepted 24 October 2006
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Abstract |
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Nitrogen-fixing bacteria have been isolated from sugarcane in an endophytic and beneficial interaction that promotes plant growth. In this work, for the first time, the involvement of ethylene signalling in this interaction was investigated by molecular characterizing members of this pathway in sugarcane. The expression pattern of a putative ethylene receptor (SCER1) and two putative ERF transcription factors (SCERF1 and SCERF2) show exclusive modulation in plants inoculated with the diazotrophic endophytes. The gene expression profile of SCER1, SCERF1, and SCERF2 is differentially regulated in sugarcane genotypes that can establish efficient or inefficient associations with diazotrophic micro-organisms, exhibiting high or low biological nitrogen fixation (BNF) rates, respectively. In addition, SCER1, SCERF1, and SCERF2 expression is different in response to interactions with pathogenic and beneficial micro-organisms. Taken together, that data suggest that SCER1, SCERF1, and SCERF2 might participate in specific ethylene signalling cascade(s) that can identify a beneficial endophytic association, modulating sugarcane responses toward the diazotrophic endophytes.
Key words: Biological nitrogen fixation, diazotrophic endophytes, ERF, ethylene-responsive factor, ethylene signalling, plant–microbe interaction, receptor, sugarcane
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Introduction |
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Endophytic nitrogen-fixing bacteria have been isolated from sugarcane (Saccharum spp.) tissues, including Gluconacetobacter diazotrophicus (Döbereiner et al., 1988), Herbaspirillum seropedicae (Baldani et al., 1986), and H. rubrisubalbicans (Baldani et al., 1996). The association seems to be beneficial for the plant, as bacteria promote plant growth when inoculated in sugarcane plantlets (Sevilla et al., 2001; Oliveira et al., 2002). This effect on plants can possibly be mediated by nitrogen fixation and/or hormone production, such as auxin (Fuentes-Ramirez et al., 1993) and gibberellin (Bastian et al., 1998) by the bacteria. Interestingly, in this particular type of interaction, bacteria colonize the intercellular spaces and vascular tissues of most plant organs, without the plant exhibiting any disease symptoms (Baldani et al., 1997; Reinhold-Hurek and Hurek, 1998). The plant mechanisms that allow bacterial colonization in an endophytic and non-pathogenic manner, establishing a beneficial association, are not clear yet. The fact that distinct sugarcane genotypes have different rates of biological nitrogen fixation (BNF) suggests that plant genetic factors might be controlling the process of bacteria recognition, colonization, and/or nitrogen fixation (Urquiaga et al., 1992).
In previous work, the SUCEST (Sugarcane Expression Sequence Tags Sequencing Project) database was explored to investigate the plant machinery implicated in signalling during the first stages of the association with diazotrophic and plant hormone-producing endophytes. Clusters, exclusively or preferentially expressed in cDNA libraries from plants inoculated with G. diazotrophicus or H. rubrisubalbicans, and that represent candidate genes involved in the association, were reported (Nogueira et al., 2001). Several of the genes identified are possibly involved in different processes of plant/bacteria communication, indicating that signalling pathways are probably being triggered by the endophytic bacteria in the initial steps of plant colonization (Vargas et al., 2003). Members of the ethylene-signalling pathway, a putative ethylene receptor and putative members of the ERF family of transcription factors, were found to be present in the data sets of ESTs differentially expressed during the association, suggesting a possible role for this hormone in the interaction of sugarcane with endophytic diazotrophic bacteria.
Ethylene is a phytohormone that takes part in various plant developmental processes (Abeles et al., 1992). It also plays a role signalling plant–bacteria interactions, such as in nodule formation in the rhizobia/leguminosae symbiosis (Penmetsa and Cook, 1997) and in pathogen defence (Ohme-Takagi et al., 2000). The diversity of ethylene roles and its specificity of action imply a complexity in its signalling pathway (Chen et al., 2005). Ethylene is perceived by a family of integral membrane receptors similar to bacterial two-component histidine kinases. In Arabidopsis thaliana there are at least five ethylene receptor members: ethylene receptor 1 (ETR1), ETR2, ethylene response sensor 1 (ERS1), ERS2, and ethylene insensitive 4 (EIN4) (reviewed in Guo and Ecker 2004). Homologues have been reported in a number of other plants including some monocots, such as rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum) (Chen et al., 2005). A transcriptional cascade works downstream in ethylene response signalling pathways, leading to differential gene expression (Solano et al., 1998). Ethylene-responsive gene (ERF1) is an ethylene-inducible transcription factor that belongs to the ERF family of plant-specific regulators. ERFs contain one copy of the AP2/ERF DNA binding domain that is the common feature of this protein family (Hao et al., 1998). ERF1 directly regulates transcription of a wide-variety of ethylene-responsive genes by specifically binding to a 11 bp cis-acting responsive element referred to as a GCC box.
This report describes the regulation of members of the ethylene-signalling pathway during the association between sugarcane and endophytic diazotrophic bacteria. In addition, it characterizes for the first time a putative ethylene receptor (SCER1) and two putative ethylene responsive factors (SCERF1 and SCERF2) from sugarcane. The expression pattern of these genes was investigated in response to the association with diazotrophic endophytes, in plant genotypes with different BNF contributions and in sugarcane plants infected with pathogenic micro-organisms. SCER1, SCERF1, and SCERF2 expression responses to beneficial and pathogenic micro-organisms suggest that these genes have a role in plant defence signalling. Taken together, the data suggests that ethylene signalling pathways might play a role in the establishment of the association between sugarcane and endophytic diazotrophic bacteria.
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Materials and methods |
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Computer analysis
ESTs from 21 cDNA libraries of the SUCEST database (http://sucest.cbmeg.unicamp.br/public/) were analysed in this work. The cDNA libraries with <5000 ESTs were normalized or were excluded from the analysis. AM1, AM2, FL1, FL3, FL4, FL5, LB1, LB2, LR1, RT1, RT2, RT3, RZ2, RZ3, SB1, SD1, SD2, ST1, and ST3 represent distinct tissues/organs of sugarcane plants (Vettore et al., 2001). AD1 and HR1 represent tissues of the root–stem transition region of in vitro-grown plantlets infected with G. diazotrophicus strain PAL5 (AD1) or H. rubrisubalbicans strain HCC103 (HR1), respectively.
Homologues to SCSBAD1086C02.g, SCCCRT1002D01.g, and SCACHR1038.D01.g ESTs were searched by sequence homology using the BLAST program (Altschul et al., 1997) or by key words. To estimate gene expression of the putative ethylene receptors and ERFs, the number of EST reads (sequenced from 5' end) corresponding to a given cluster was divided by the total number of reads (sequenced from 5' end) in a given cDNA library. It results in an absolute estimate of mRNA abundance, or the frequency estimates in the cDNA libraries.
SCER1, SCERF1, and SCERF2 cDNA sequences were translated into hypothetical proteins, whose theoretical characteristics were obtained from several databases using the BLAST program in the NCBI (www.ncbi.nlm.nih.gov/BLAST). Protein sequences were entered into MotifScan (pattern searches), ProDOM (protein domain identification), and Interpro (protein domain and pattern search identification). Alignments of ScER1 and ScERF2 deduced amino acid sequences from sugarcane and its homologues in A. thaliana and other plant species were carried out using the ClustalW algorithm. Phylogenetic analyses were performed with the Mega 2.1 program (Kumar et al., 2001) and bootstrap analyses using the Neighbor–Joining method with 2000 replicates.
Plant growth and micro-organism inoculation
The sugarcane genotypes used in this work were: SP70-1143 (high inputs of N from BNF), Chunee (low inputs of N from BNF), and SP70-3370 (susceptible to Leifsonia xyli subsp. xyli). Plants of SP70-1143 and Chunee genotypes were propagated by cuttings of the stalk containing one bud. The stalk cuttings were grown in a sterile mixture of sand:vermiculite (2:1 v/v) in a greenhouse. Plants were watered daily and supplied with 50 ml Hoagland's solution weekly. Following treatment, roots and shoots were frozen in liquid N2 and stored at –80 °C. Mature leaves, number +5, were collected from three-year-old plants growing in the field.
In vitro-grown plants, free of micro-organisms, were obtained by sterile meristem culture. Hormone treatments were performed with SP70-1143 in vitro-grown plants, free of micro-organisms. Plants were cultivated for 10 h in MS medium with an addition of either 20 µM of ethephon or 100 µM jasmonic acid, or both hormones together. Control plants were incubated in the same medium, without hormones.
In vitro-grown sugarcane SP70-1143 and SP70-3370 genotypes were micropropagated according to the method of Hendre et al. (1983). After culturing the plantlets in a rooting and shooting medium (a modified MS liquid medium; Murashige and Skoog, 1962) for 50–60 d, the plantlets were transferred to 20 ml of the same medium with the concentration of nutrients and sucrose reduced 10-fold and without hormones. In vitro-grown SP70-1143 plantlets were inoculated as described in James et al. (1994). 0.1 ml of suspensions containing 10–6 to 10–7 endophytic diazotyrophic bacteria G. diazotrophicus (PAL5), H. seropedicae (HRC54), and H. rubrisubalbicans (HCC103) were added to the plant growth medium. In mock-inoculated plants, the same volumes of bacterial growth medium were added to the plant growth medium. The pathogenic bacteria Leifsonia xyli subsp. xyli (CTC B07 strain) was inoculated into in vitro-grown plantlets of the SP70-3370 genotype. Controls were mock-inoculated with bacterial growth medium. Seven days after the inoculation, plants were harvested and examined for endophytic diazotrophic bacterial colonization by the Most Probable Number (MPN) estimation, according to the methods of Reis et al. (1994). All plants were maintained at 30 °C with an irradiance of 60 mmol photons m–2 s–1 for 12 h d–1. Stalks of field-grown sugarcane plants of the SP86-155 genotype, with and without mosaic virus disease, were germinated in a greenhouse and shoots were harvested 15 d later.
RNA extraction and cDNA synthesis
For each expression analysis experiment, three to five plantlets were pooled and total RNA was extracted according to Logeman et al. (1987). For the conventional semi-quantitative RT-PCR reactions, the first-strand cDNA was synthesized using the ‘First-Strand cDNA Synthesis Pharmacia Kit’, using Not-dT as primer, according to the manufacturer's instructions. The TaqMan® Kit (Applied Biosystems) was used for semi-quantitative real-time RT-PCR reactions, using random hexamers as primer, according to the manufacturer's instructions.
Conventional RT-PCR
Conventional RT-PCR reactions of 50 µl contained 5 µl of the first-strand cDNA reaction diluted four times, 200 ng of the specific oligonucleotides er1a (5'-AGTCCCCAGACTCCTTCGAAT-3') and er1b (5'-ATGCCACCTGTAAGAAGACCA-3'), or er2a (5'-CTCGAGGATGGAAGCCTTGAA-3') and er2b (5'-TACTTGGCGCTATGCCGTCAA-3'), 200 mM of dNTP, and 5 U of Taq polymerase, in the enzyme buffer. The PCR was performed as follows: 5 min denaturation at 94 °C, 30 cycles of 1 min denaturation at 94 °C, 1 min annealing at 50 °C, and 1 min polymerization at 72 °C, followed by 5 min extension at 72 °C. The polyubiquitin constitutive gene was used as a control in PCR reactions. PCR reactions of 50 µl contained 5 µl of the first-strand cDNA reactions diluted four times, 200 ng of the specific oligonucleotides ubi1 (5'-ATGCAGATCTTTGTGAAGAC-3') and ubi2 (5'-TTACTGACCACCACGAAGAC-3'), 200 mM of dNTP, and 5 U of Taq polymerase, in the enzyme buffer. The PCR was performed as follows: 5 min denaturation at 94 °C, 23 cycles of 1 min denaturation at 94 °C, 1 min annealing at 55 °C, and 1 min polymerization at 72 °C, followed by 5 min extension at 72 °C. Products of the PCR reactions were eletrophoretically separated on 1% agarose gel, transferred onto a nylon membrane, and hybridized with a cDNA fragment from sugarcane er1 or er2, or a ubiquitin cDNA fragment from A. thaliana. Quantification of PCR amplification was performed using the ImageQuant version 5.2 Copyright© 1999 Molecular Dynamics program. The data from RT-PCR are the result of at least two experiments repeated twice.
Real-time RT-PCR
Primers used for real-time RT-PCR were designed on the 3'-UTR sequences of SCERF1 and SCERF2 genes using the Primer3 software: Scerf1a.fwr (5'-TCGACGGCTCTGTGTTCTGA-3') and Scerf1a.rev (5'-AACCAATAAACAGAGTAACCGAGTAGAAG-3'); Scerf2a.fwr (5'-CTGCTGTACTTAGCAGCAAGC-3') and Scerf2a.rev (5'-TGTGACACCCGACCTAACAC-3'). The 28S constitutive gene was used as a control in PCR reactions; 28S. Fwr (5'-GCGAAGCCAGAGGAAACT-3') and 28S. rev (5'-GACGAACGATTTGCACGTC-3'). cDNA was amplified using the SYBR-Green® PCR Master kit (Perkin-Elmer Applied Biosystems) on the GeneAmp 9600 thermocycler (Perkin-Elmer Applied Biosystems), under standard conditions. The analysis of RT-PCR output data followed the manufacturer-suggested 
Ct method (Perkin-Elmer Applied Biosystems). The data from real-time PCR are the result of at least two experiments repeated twice.
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SCER1 encodes a putative ERS1-type ethylene receptor from sugarcane
Five ESTs encoding putative ethylene receptors were identified in the SUCEST database. Their representation in the SUCEST cDNA libraries was investigated by computer analysis of EST frequencies. The EST SCSBAD1086C02.g, called SCER1, is exclusively represented in the AD1 library, constructed from plants inoculated with G. diazotrophicus. None of the other four genes shows any regulation during the plant association with the nitrogen-fixing bacteria in the electronic analysis (Fig. 1A). The full-length Scer1 cDNA was fully sequenced, revealing an open reading frame 1899 bp long that encodes a putative protein of 632 amino acids (Fig. 2A). The closest SCER1 homologue in SUCEST (SCEZLR1031D12.g) was named SCER2 and it was also included in our studies as control. The SCER2 full-length cDNA sequence was available in the database. It contains an open reading frame with 1899 bp, which encodes a putative protein 632 amino acids long. The other three ESTs found in SUCEST (SCJFRT1007A12.g, SCJLRZ1019D02.g, and SCAGLB1069B10.g) named SCER3, SCER4, and SCER5, respectively, were not fully sequenced, therefore it was not possible to study their DNA and protein sequence features in more detail.
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According to structure and amino acid sequence similarities, the ethylene receptor gene family can be classified into two subfamilies (Hua et al., 1998). In the subfamily I, ETR1 and ERS1 contain three hydrophobic domains in the amino terminus, and well-conserved motifs in the histidine–kinase domain; by contrast, ETR2, ERS2, and EIN4 (subfamily II) have four stretches of hydrophobic amino acids, and lack most of the functional hallmarks of the bacterial histidine kinases. One member of each subfamily (ERS1 and ERS2) lacks a receiver domain. Domains in the predicted ScER1 and ScER2 proteins were searched to determine to which subfamily of ethylene receptors they belong (Fig. 2B). Three transmembrane domains could be detected in the N-terminal ethylene-binding region. They also contain the residues Ala31, Ile62, Cys65, and Ala102, conserved between ethylene receptors and thought to be important to their normal functions (Bleecker and Schaller, 1996), and the amino acids Cys4 and Cys6, which are required for the receptor dimmerization (Schaller and Bleecker, 1995). ScER1 and ScER2 polypeptides also include a well-conserved histidine kinase domain with all of their motifs, H, N, G1, F, G2 (Fig. 2A). In between the ethylene binding and histidine kinase domains, there is a region with sequence similarity to the GAF domain. Both ScER1 and ScER2 proteins lack the receiver (or response regulator) domain, as the ERS1 ethylene receptors from A. thaliana. Comparison of the deduced amino acid sequence with ethylene receptors from other plant species revealed that ScER1 and ScER2 show the highest level of identity with the ethylene receptors OsERS2 (91%) and OsERS1 (96%) from rice, respectively, that are members of subfamily I (Fig. 2C). These data indicate that the SCER1 and SCER2 genes encode putative ERS1-like ethylene receptors.
SCERF1 and SCERF2 are members of the ERF class of transcription factors
Searching the SUCEST database, 98 ESTs encoding putative members of the ERF family could be identified. The electronic expression pattern of the putative ERFs detected two ESTs (SCCCRT1002D01.g and SCACHR1038D01.g) that are highly expressed in infected libraries and roots, respectively, named SCERF1 and SCERF2. Both of them are more represented in the HR library, infected with H. rubrisubalbicans (Fig. 1B). The other putative ERFs are represented in almost all libraries from SUCEST, such as SCCCLR2001E02.g and SCCCLR1067G09.g, respectively named SCERF3 and SCERF4, that are displayed in Fig. 1B. SCERF1 and SCERF2 were selected for further characterization.
The full-length SCERF1 and SCERF2 cDNAs contain open reading frames of 1020 bp and 849 bp long and encode putative proteins of 339 and 282 amino acids, respectively (Fig. 3A). ERF proteins comprise one of the largest families of transcription factors in plants, with 122 members present in the A. thaliana genome, according to the last classification reported by Nakano et al. (2006). Based on this classification, the ERF family includes proteins with one AP2/ERF DNA binding domain comprising the completely conserved residues Gly-4, Arg-6, Glu-16, Trp-28, Leu-29, Gly-30, and Ala-38. Computational analysis revealed that the deduced ScERF1 and ScERF2 amino acid sequences contain only one conserved AP2/ERF domain, containing all the seven conserved residues (Fig. 3A). In addition, both ScERF1 and ScERF2 contain the residues Arg-8, Gly-11, Ile-17, Arg-18, Arg-26, Ala-39, Asp-43, and Asn-57 that are conserved in 95% of the ERF family members (Nakano et al., 2006). Therefore, the amino acid features of the AP2/ERF domain of ScERF1 and ScERF2 indicate that they are members of the ERF family of sugarcane.
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A phylogenetic tree was generated based on the alignment of AP2/ERF domains of ScERF1, ScERF2, and members of the 12 distinct ERF groups designated in Arabidopsis (Nakano et al., 2006). ScERF1 and ScERF2 formed a well-supported clade with proteins of group VII, indicating that both sugarcane genes belong to this group (Fig. 3B). Conserved motifs outside the AP2/ERF domain were identified in the various ERF groups (Nakano et al., 2006). The motifs found in group VII proteins were searched in ScERF1 and ScERF2 (Fig. 3A). The N-terminal CMVII-1 motif, MCGGAI(I/L), a typical feature of group VII proteins, is completely conserved in ScERF1 and has one amino acid substitution in ScERF2 (Fig. 3A). In addition, both sugarcane ERFs contain the motifs CMVII-5 and CMVII-6. ScERF1 also has CMVII-4, CMVII-7, and CMVII-8 motifs.
Searches at the NCBI database were performed to identify protein sequences closely related to the ERF/AP2 domain and the entire ScERF1 and ScERF2 predicted proteins. The analyses revealed that the closer relative to ScERF1 is one ERF protein from Oryza sativa (GenBank accession no. BAD35280), which has 363 amino acids and shows 98% identity and 98% similarity with ScERF1. The closest homologue of ScERF2 is also a rice protein (GenBank accession no. NP_908602) which contains 207 amino acids. The entire ScERF2 and rice ERF homologue predicted proteins share only 54% identity and 62% similarity, but they exhibit 85% identity and 92% similarity between their ERF domains.
To obtain clues about the possible biological roles of ScERF1 and ScERF2, another phylogenetic tree was generated based on entire predicted proteins of already described plant ERFs involved in development, hormone response, and host defence (Fig. 3C). ScERF1 formed a well-supported branch (bootstrap value of 98%) with the JERF3 protein of Lycopersicon esculentum and CaPF1 protein of Capsicum annuum. JERF3 (Jasmonate and Ethylene Response factor 3) is induced by ethylene, JA, cold, salt, and abscicic acid, while CaPF1 is also involved in processes of disease resistance, in addition to being induced by several abiotic stresses (Wang et al., 2004; Yi et al., 2004). ScERF2 is more distantly related in this branch, forming a group together with C. annuum protein CaERFLP1 (bootstrap value of 65%), also reported to be involved in salt tolerance and disease resistance (Lee et al., 2004).
SCERF1 and SCERF2 expression is responsive to ethylene
Members of the ERF family were first identified as ethylene-responsive element-binding proteins. Nevertheless, the characterization of different ERF genes have shown that not all members of these large family may necessarily act downstream of ethylene. In order to address that, the responsiveness of SCERF1 and SCERF2 to ethylene was investigated (Fig. 4).
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In vitro-growing plants of the SP70-1143 variety, free of micro-organisms, were cultivated for 10 h in MS medium with an addition of 20 µM of ethephon (ET), a compound that releases ethylene in culture medium. Since cross-talk between ethylene and jasmonate signalling pathways can involve the transcriptional activation of ERF genes (Lorenzo et al., 2003), plants were also treated for 10 h with 100 µM jasmonic acid (JAS) and with both ethephon and jasmonic acid (ET/JAS). As a control, plants were incubated in medium without hormones. Figure 4 shows that SCERF2 expression is 9–10-fold induced by treatment with ethephon and jasmonic acid separately. In addition, the treatment with both hormones had a synergistic effect on the stimulation of SCERF2 expression that increases almost 20-fold. Although SCERF1 expression is not highly induced by ethephon or jasmonic acid alone in the tested conditions, a higher induction is observed in plants treated with both hormones together. The data indicate that both SCERF1 and SCERF2 are ethylene/jasmonate–responsive genes, suggesting that they might play a role on the ethylene signalling pathway.
SCER1, SCERF1, and SCERF2 expression is early regulated by endophytic diazotrophs
The computer analysis of EST frequencies of the sugarcane putative ethylene receptor SCER1 and the putative ERFs SCERF1 and SCERF2 suggested a differential expression of these genes in sugarcane plants inoculated with the endophytic diazotrophic bacteria. The proper control for these analyses would be a dataset of ESTs expressed in mock-inoculated in vitro-growing plants. However, this cDNA library was not generated in the SUCEST Project. To determine whether SCER1, SCERF1, and SCERF2 expression is responsive to bacteria inoculation, the mRNA abundance of these genes was investigated in plants growing in vitro and inoculated with G. diazotrophicus and a mixture of Herbaspirillum spp species. These experiments were performed by semi-quantitative conventional RT-PCR (SCER1) or real-time RT-PCR (SCERF1 and SCERF2). The genotype SP70-1143 was used in these analyses, since it establishes an efficient association with the endophytes, showing high BNF contributions (Urquiaga et al., 1992). In all the experiments, plant colonization by the inoculated bacteria was confirmed by the Most Probable Number (MPN) estimation (Materials and methods). Rates of colonization always ranged between log 4–6 cfu g–1 FW (data not shown).
Seven days after inoculation, a basal level of expression was found for all the genes in mock-inoculated control plants, free of micro-organisms (Fig. 5A). A strong induction of SCER1 expression (about 5–6-fold higher) was observed in plants 7 d after inoculation with G. diazotrophicus or Herbaspirillum spp mRNA levels of SCER2, the closest homologue of SCER1, did not change under the same inoculation conditions, suggesting that bacterial inoculation does not trigger a general response of the ethylene receptors. Interestingly, the association with the endophytic diazotrophs led to different outcomes on the expression of the sugarcane ERF genes. SCERF2 expression was induced, around 3–4-fold, in plants infected with Herbaspirillum spp, and G. diazotrophicus, respectively. By contrast, SCERF1 expression was up to 2-fold lower in plants infected with Herbaspirillum spp, by contrast with control plants (Fig. 5A). Altogether, the data show that SCER1, SCERF1, and SCERF2 expression respond to the sugarcane/nitrogen-fixing bacteria association, indicating a possible role of the ethylene signalling pathway in this particular type of interaction.
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In order to investigate if the modulation of SCER1, SCERF1, and SCERF2 expression in plants colonized with the diazotrophic endophytes is an early response during the establishment of the interaction, the expression pattern of these genes was analysed in plants 24 h after inoculation with G. diazotrophicus and Herbaspirillum spp (Fig. 5B). SCER1 was 5-fold induced 24 h after plants were inoculated with both G. diazotrophicus and Herbaspirillum spp, suggesting that it might be an early responsive gene to bacteria inoculation. The data obtained with the sugarcane ERFs show different responses depending on the bacterial species. SCERF2 was 10-fold induced by G. diazotrophicus inoculation, but did not respond to Herbaspirillum spp under this experimental condition. By contrast, SCERF1 was 2-fold repressed only in plants inoculated with Herbaspirillum spp, and G. diazotrophicus did not trigger any detectable response on SCERF1 expression 24 h after inoculation. The results obtained 7 d after inoculation already pointed toward higher responses of SCERF1 to Herbaspirillum spp and of SCERF2 to G. diazotrophicus. Therefore, the data suggest that SCERF1 and SCERF2 expression can respond early to the bacterial inoculation, nevertheless the early response seems to be dependent on the bacterial species.
SCER1, SCERF1, and SCERF2 expression is developmentally regulated and dependent on the plant genotype
To determine the expression profile of SCER1, SCERF1, and SCERF2 in plant organs, the mRNA levels were investigated by RT-PCR (SCERF1) or real-time PCR (SCERF1 and SCERF2) in plants of the SP70-1143 genotype growing in soil in different developmental stages. SCER1 showed a basal level of expression in roots and leaves of 15-d-old plants, without a preferential expression for any plant organ (Fig. 6A). During development, SCER1 mRNA levels increased in leaves of 30-d-old plants and decreased in mature leaves. SCERF2 mRNA levels were high in roots and leaves of 15 and 30-d-old plants, drastically decreasing in mature leaves. By contrast, SCERF1 expression was preferential in roots, and very low mRNA levels were detected in leaves. As shown in Fig. 6A, the expression of SCERF1 was gradually reduced in leaves from 15 and 30-d-old plants, reaching very low levels in mature leaves.
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The influence of plant genotype in the regulation of SCER1, SCERF1, and SCERF2 expression was investigated by quantifying mRNA levels of these three genes during development of plants of the Chunee genotype. Differing from the profile observed in SP70-1143 plants, SCER1 expression did not change in response to the leaf developmental stage in Chunee plants, remaining at similar levels in leaves of 30-d-old plants and in mature leaves (Fig. 6B). Remarkably, by contrast with SP70-1143, SCERF2 exhibited much lower expression levels in all organs and developmental stages analysed in Chunee plants. By contrast, SCERF1 expression was significantly higher (over 100-fold) in roots of Chunee plants, than in SP70-1143. SCER2 was also analysed in SP70-1143 and Chunee genotypes, showing constitutive and comparable levels of expression in both genotypes (data not shown). The data suggest that plant genotype might regulate, to a certain extent, expression of SCER1, SCERF1, and SCERF2 genes during development.
SCER1, SCERF1, and SCERF2 expression is modulated in pathogenic interactions
In order to determine if the SCER1, SCERF1 and SCERF2 responses are associated only with beneficial endophytic diazotrophic bacteria or with any other micro-organism interaction, gene expression was also analysed in pathogenic interactions. Inoculations were carried out with Leifsonia xyli subsp. xyli (LX) that is a pathogenic bacterium responsible for the ratoon stunting sugarcane disease, with colonization properties similar to those seen in endophytic bacteria (Harrison and Davis, 1986). Modulation of expression in shoots from plants with the Sugarcane Mosaic Virus, SCMV (MV), responsible for the Sugarcane Mosaic Virus Disease (Comstock and Lentini, 2002), was also analysed to investigate sugarcane interaction with a micro-organism other than bacteria.
In contrast to the enhanced SCER1 expression observed in plants associated with the endophytic diazotrophic bacteria, SCER1 mRNA levels strongly decreased, 10-fold, in the pathogenic interactions (Fig. 7). SCERF2 gene expression did not respond to the pathogenic interaction with L. xyli subsp xyli and it was 5-fold repressed in plants infected with the Sugarcane Mosaic Virus, contrary to the induction of expression observed in an efficient association with the endophytes. Remarkably, SCERF1 expression was highly induced, more than 5-fold and 4-fold, in plants infected with the Sugarcane Mosaic Virus or L. xyli subsp. xyli, respectively (Fig. 7). Taken together, the data show a differential regulation of SCER1, SCERF1, and SCERF2 gene expression in beneficial and pathogenic interactions, supporting the hypothesis that these genes might play a specific role in beneficial associations between sugarcane and endophytic nitrogen-fixing bacteria.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Sugarcane lives in a particular type of interaction with nitrogen-fixing bacteria. Endophytic diazotrophic bacteria colonize the intercellular spaces and vascular tissues of sugarcane organs, without triggering any disease in the plant. The signalling mechanisms that underlie this particular type of plant/bacteria association are poorly understood. This paper presents the first report on the molecular characterization of members of the ethylene signal transduction cascade in sugarcane. The data suggest that this signalling pathway might play a role in the establishment of the association between sugarcane and endophytic diazotrophic bacteria.
In this work, a putative ethylene receptor, SCER1, and two putative ERF transcription factors, SCERF1 and SCERF2 have been analysed. Five putative ethylene receptors were identified in the SUCEST database, indicating that, similarly to other plant species, ethylene signalling is also triggered by a family of receptors in sugarcane. ScER1 features indicate that it is an ERS1-type of receptor that has all residues important for the function of ethylene receptors (Bleecker and Schaller, 1996; Schaller and Bleecker, 1995), implying that it might be functional in sugarcane. However, further investigation of biological activity is required. Analysis of ScERF1 and ScERF2 domains classified the two proteins as members of the ERF family of transcription factors, which belong to group VII (Nakano et al., 2006). Gene expression response to ethylene and jasmonate indicates that both SCERF1 and SCERF2 are ethylene/jasmonate–responsive genes, suggesting that they might act downstream of the ethylene-response signalling pathway. It still remains to be investigated if SCERF1 and SCERF2 expression is responsive to abscicic acid (ABA), another plant hormone reported to regulate ERF mRNA levels (Yang et al., 2005).
The expression of SCER1, SCERF1, and SCERF2 in SP70-1143 plants grown in vitro respond to inoculation with different species of endophytic nitrogen-fixing bacteria species. SCER1 and SCERF2 mRNA levels increase after inoculation with G. diazotrophicus or Herbaspirillum spp, compared with non-inoculated control plants. By contrast, SCERF1 expression is slightly repressed in SP70-1143-inoculated plants. Interestingly, only a few members of the ethylene receptor and ERF families respond to the association with the endophytic diazotrophs. It suggests that the putative SCER1 ethylene receptor and SCERF1 and SCERF2 transcription factors may be part of specific ethylene signalling cascades that modulate sugarcane responses toward the diazotrophic endophytes. Further investigation on other putative ERFs identified in the SUCEST database would be helpful to clarify the ethylene responses in this type of association. Curiously, these studies revealed differences in the modulation of gene expression triggered by sugarcane colonization with different bacterial species. Differences in gene expression levels among independent inoculation events have been observed by our group (data not shown). In addition, it is also possible that different bacterial species lead to different responses by the plant.
Taking together the expression profiles of SCER1, SCERF1, and SCERF2, it can be speculated that these genes may not only respond to the association with endophytic diazotrophic bacteria, but might also be able to discriminate between beneficial and pathogenic interactions. This hypothesis is supported by the observation that SCER1, SCERF1, and SCERF2 expression is modulated by interactions with pathogenic micro-organisms in a different manner as that observed in the beneficial associations. To strengthen this idea, studies using other symbionts and pathogens should be performed. In addition, differences in SCER1, SCERF1, and SCERF2 expression patterns were observed in different sugarcane genotypes. Although the analysis of plants growing in non-sterile soil may be influenced by microbial levels within the soil and/or plant, and the BNF contributions could not be measured in the samples, the data show a positive correlation between response of SCER1, SCERF1, and SCERF2 expression in inoculated plants and levels of expression in genotypes with different BNF efficiencies. It is more striking for the ERF genes. SCERF2 expression is induced in SP70-1143 plants (high BNF) inoculated with the endophytic diazotrophic bacteria; in correlation, SP70-1143 plants growing in soil exhibit much higher SCERF2 mRNA levels than Chunee plants (low BNF). On the other hand, SCERF1 is repressed in SP70-1143-inoculated plants, and shows high levels of expression in roots of Chunee plants. These data are in agreement with macroarray studies performed by our group that showed higher SCERF1 mRNA levels in Chunee than in SP70-1143 plants (JJV Cavalcante, unpublished results).
The dynamics and tissue localization of SCER1, SCERF1, and SCERF2 expression during plant growth remain to be determined in order to investigate if they are synchronized with sites of bacterial colonization. It has been demonstrated that G. diazotrophicus and Herbaspirillum spp colonize both roots and leaves of sugarcane, that exhibit high levels of bacterial counts during the earlier stages of development (Cavalcante and Dobereiner, 1988; Baldani et al., 1986, 1996; Olivares et al., 1996). In general, higher SCER1 and SCERF2 mRNA levels were found in roots and younger leaves, decreasing in mature leaves of SP70-1143 plants. By contrast, SCERF1 showed preferential expression in roots (Fig. 6), in agreement with the electronic northern results that show a high level of SCERF1 expression in root libraries (Fig. 1B). It has already been described that ERS1-type ethylene receptors are differentially expressed depending on the plant developmental stage. RNA in situ hybridization analysis revealed that AtERS1 is ubiquitously expressed in several A. thaliana tissues, with apparently stronger signals in the younger cells than in the older ones (Hua et al., 1998). ERF genes are also differentially expressed in flower, leaf, inflorescence stem, and root (Okamuro et al., 1997). Therefore, a differential control of expression in plant tissues could explain some differences observed in SCER1, SCERF1, and SCERF2 mRNA levels during sugarcane development. It can be speculated that SCER1, SCERF1, and SCERF2 expression could also be, to some extent, correlated with the association with the endophytes, reflecting ethylene signalling cascades activated and/or repressed in the newly formed organs during colonization by the endophytes and establishment of the association.
All together, the data strongly indicate that ethylene signalling has a role in the sugarcane association with endophytic diazotrophic bacteria. In legume symbiosis, it is well known that ethylene plays mostly a negative role, possibly by limiting bacteria infection and nodule number. Several observations come from studies with inhibitors of ethylene biosynthesis and the application of exogenous ethylene (Ligero et al., 1987; Peters and Crist-Estes, 1989; Lee and LaRue, 1992); and plant mutants defective in ethylene synthesis or transduction (Penmetsa and Cook, 1997; Guinel and Sloetjes, 2000; Ooki et al., 2005). Plants with endophytic interactions need to have the number of endophytes under stringent control, in order to confirm the non-pathogenic aspect of the association (Iniguez et al., 2005). The balance of sugarcane responses leading to defence and permission to the beneficial endophytic bacteria colonization might be controlled by an intricate signalling mechanism that possibly differs from the ones operating in legumes, considering that the diazotrophic bacteria colonizes most sugarcane tissues with colonization properties similar to those seen in some pathogenic bacteria, such as L. xyli subsp. xyli. As already discussed, it is conceivable that specific ethylene signalling cascade(s) might be part of the signalling machinery that can identify a beneficial endophytic association, modulating sugarcane responses toward the diazotrophic endophytes. Recently, the involvement of the ethylene signalling response in plant associations with beneficial endophytic and root-colonizing bacteria has been described (Iniguez et al., 2005; Léon-Kloosterziel et al., 2005), in agreement with our findings.
SCER1, SCERF1, and SCERF2 expression responses to beneficial and pathogenic micro-organisms suggest a role of these genes in plant defence signalling. An important issue now is to understand the function of ScER1, ScERF1, and ScERF2 in the establishment of the sugarcane/diazotrophic endophytes interaction. Ethylene receptors are negative regulators of the pathway (Ciardi et al., 2000). Some evidence indicates that increased levels of receptors reduce ethylene sensitivity and, consequently, defence responses in plant/microbe interactions. Lotus japonicus over-expressing the ethylene receptor gene ers1 from muskmelon showed an increased number of infection threads and nodule primordia when inoculated with Mesorhizobium loti (Nukui et al., 2004). Transgenic tomato plants over-expressing the ERS1-like NR ethylene receptor showed a reduction in defence responses after infection with the virulent bacteria Xanthomonas campestris pv. vesicatoria (Ciardi et al., 2000). The strong and specific induction of SCER1 expression during sugarcane association with endophytic diazotrophic bacteria, and its drastic repression in pathogenic interactions, suggests that increased ScER1 levels might be dependent on a beneficial association. Therefore, we can speculate that increased levels of the ScER1 receptor in beneficial associations might be reducing ethylene sensitivity and, consequently, plant defence against the diazotrophic endophytes. This mechanism would make the plant host more tolerant to specific diazotrophic micro-organisms, allowing the establishment of the endophytic interaction.
The role of SCERF1 and SCERF2 in this proposed ethylene pathway is less clear. Several ERF family members, including tomato Pti4, Pti5, Pti6, and A. thaliana AtERF1, AtERF2, and AtERF5, play a positive role against pathogens (Fujimoto et al., 2000; Gu et al., 2002). Over-expression of these genes enhances defence gene expression and plant disease resistance to bacteria (Berrocal-Lobo et al., 2002; Gu et al., 2002). However, it has already been described that some ERF proteins could act by repressing the GCC box-mediated gene expression (Fujimoto et al., 2000). It is important to characterize if ScERF1 and ScERF2 have positive or negative roles on transcriptional regulation of defence genes, in order to be able to distinguish between a function on the activation or on the repression of plant defences. Also, SCERF1 and SCERF2 are close homologues, members of group VII of the ERF family, and they respond in different ways to bacterial colonization. It is still conceivable that they participate in different ethylene signalling pathways, with positive and negative roles on the association, in order to balance bacterial levels inside the plant. Genetic manipulation of ScERF1 and ScERF2 levels in sugarcane plants are currently being done to investigate this hypothesis. The possibility cannot be discarded that these ethylene signalling members may also act in other physiological processes, such as plant growth and development. The closest homologues of ScERF1 and ScERF2, CaPF1 and CaERFLP1 from C. annuum, and JERF3 from L. esculentum, are proteins that are also involved in the response to abiotic stresses (Lee et al., 2004; Wang et al., 2004; Yi et al., 2004). SCERF1 and SCERF2 are not induced by wounding (JJV Cavalcante, unpublished results), and the response to other abiotic stresses should be investigated in order to define SCERF1 and SCERF2 roles in sugarcane.
The understanding of the signalling pathways involved in the establishment of sugarcane association with beneficial endophytic diazotrophic bacteria is the first step in order to be able to obtain a more efficient association in this culture and, eventually, to extend it to other Gramineae that are important food sources for the human diet. This work has contributed to identifying the sugarcane genes that might be involved in the initial steps of plant colonization by the endophytes. In future, such genes could be used as markers to select the best models of sugarcane/diazotrophic endophytes association. In addition, the identification of the plant genes which are crucial for the association will provide tools for future genetic manipulation of sugarcane to increase the efficiency of BNF, making this culture more economic, and minimizing the environmental problems resulting from the use of the nitrogen fertilizers.
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Acknowledgements |
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We are grateful to Leonardo Mega França and Ana Cláudia de Jesus for technical assistance in DNA sequencing and plant culture, respectively. We are also grateful to Copersucar for providing sugarcane genotypes and plant material infected with mosaic virus disease; to Gonçalo Apolinário da Silva for providing plant material infected with L. xyli subsp. xyli, and to Embrapa/Agrobiologia for providing the endophytic diazotrophic bacteria strains. CV, EMN, FV, and KS are indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and JJVC is indebted to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for graduate fellowships. ASH receives support from a CNPq research grant. The study was partially supported by the project PronexII/CNPq and PADCT III.
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* These authors contributed equally to this work.
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