The Hevea brasiliensis AP 2 / ERF superfamily : from ethylene signalling to latex harvesting and physiological disease response

Etilen merupakan hormon yang dikenal karena perannya dalam proses penyadapan lateks di tanaman karet (Hevea brasiliensis). Hormon tersebut memfasilitasi aliran lateks melalui aktivasi metabolisme endogen dalam sel lateks beranastomosis yang disebut latisifer. Etilen memiliki peran ganda yaitu menguntungkan karena memacu produksi lateks dan tidak menguntungkan pada tingkat tertentu sehingga menyebabkan munculnya penyakit fisiologis yang disebut sebagai kering alur sadap (KAS). Beberapa penelitian mendalam telah dilakukan untuk mengungkap aktor molekuler dalam biosintesis dan sinyalisasi etilen pada Hevea brasiliensis. Salah satu superfamili yang penting dan terlibat sebagai faktor transkripsi terakhir yang diketahui pada sinyalisasi etilen adalah APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF). Saat ini, 114 sekuen unik yang menyandi superfamili AP2/ERF pada Hevea telah diidentifikasi dan dikarakterisasi. Karakterisasi spesifik pada saat kondisi stres penyadapan dan kejadian KAS telah berhasil mengidentifikasi 36 marka ekspresi gen (GEMs). Delapan belas GEMs diprediksi memiliki ortologi dengan 19 gen AP2/ERF pada Arabidopsis. Meskipun karakterisasi ini difokuskan pada regulasi tingkat transkripsi, regulasi pasca-transkripsi dan pascatranslasi potensial dari HbAP2/ERFs juga diprediksi. Dikarenakan akumulasi transkrip yang tinggi pada latisifer dan dalam respon terhadap multi-stres abiotik, tiga grup HbERF (HbERFVII, HbERF-VIII dan HbERF-IX) diduga memiliki peran penting dalam toleransi Hevea selama produksi lateks. Untuk beberapa gen kunci, analisis fungsional lanjut perlu dilakukan untuk sepenuhnya memahami regulasi dari HbAP2/ERFs. Akhirnya, penanda molekuler dalam kaitannya untuk pemuliaan tanaman karet kemungkinan dapat dikembangkan dari superfamili ini.


Abstract
Ethylene is a hormone known for its involvement in the process of latex harvesting in Hevea brasiliensis.It facilitates latex flow by activation of endogenous metabolism in the anastomosed latex cells called laticifers.In regard to its ambivalent role, ethylene is both favourable to the latex production and unfavourable to a certain level, to the apparition of a physiological disease termed as tapping panel dryness (TPD).Comprehensive researches have been carried out to reveal the molecular actors in ethylene biosynthesis and signalling pathways in Hevea brasiliensis.One of the most important superfamily implicated as the last transcription factor known in plant ethylene signalling is the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF).Currently, 114 unique sequences related to the Hevea AP2/ERF superfamily have been identified and characterized.Specific characterizations under the condition of harvesting stress and the occurrence of TPD have identified 36 gene expression markers (GEMs).Eighteen of these GEMs were predicted as ortholog with 19 Arabidopsis AP2/ERF genes.The characterization was mainly focused on transcriptional regulation, whilst potential posttranscriptional and post-translational regulations of HbAP2/ERF genes were formerly predicted.Three HbERF groups (HbERF-VII, HbERF-VIII and HbERF-IX) were hypothesized to have an important role in Hevea tolerance during latex production as they highly accumulated in laticifers and in response to multiple abiotic stresses.Further functional analysis of several key genes is suggested in order to fully understand the regulation of HbAP2/ERFs.Finally, the molecular markers for future Hevea breeding could be possibly developed from this superfamily.

Introduction
Ethylene is a hormone known for its involvement in the process of latex harvesting in Hevea brasiliensis.It facilitates latex flow by activation of endogenous metabolism in the anastomosed latex cells called laticifers (Pujade-Renaud et al., 1994).Latex regeneration disrupts the metabolism homeostasis in laticifers and triggers the production of ROS in particular the O2 -form by lutoidic NADPH oxidase.In extreme cases of stress, this leads to an oxidative burst provoking coagulation of the rubber particles (d'Auzac et al., 1993).This condition will cause the onset of a physiological disease known as tapping panel dryness (TPD).Thus, the ethylene is subjected to an ambivalent role, both favourable for the latex production and unfavourable in certain level leading to the apparition of TPD.For several years, comprehensive researches have been carried out to reveal the molecular actors in ethylene biosynthesis and signalling pathways in Hevea brasiliensis.
One of the most important superfamily implicated as the last actor known in plant ethylene signalling is the APETALA2/ ETHYLENE RESPONSE FACTOR (AP2/ERF).Members of the AP2/ERF superfamily encode transcription factors involved in the regulation of biological processes, including floral and ovum development, responses to phytohormones, and adaptation to biotic and abiotic stress (Mizoi et al., 2012;Licausi et al., 2013a, Li et al., 2012;Xu et al., 2011).These transcription factors were characterized by a conserved DNA-binding AP2 domain of about 60 amino acids (Ohme-Takagi & Shinshi, 1995).The AP2 domain was able to interact specifically with GCC-box (Ohme-Takagi & Shinshi, 1995) or dehydration-responsive element/C-repeat (DRE/CRT) cis-acting elements in the promoter region of target genes (Ito et al., 2006;Sakuma et al., 2006).The AP2/ERF superfamily was classified according to the number of AP2 domain.The AP2 family consists of proteins with a double domain of AP2 in repeated tandem (Okamuro et al., 1997).The ERF family encodes transcription factors containing a single AP2 domain.Finally, the Related to AB1/VP1 (RAV) family encodes proteins with a single AP2 and an additional domain of B3-type (Kagaya et al., 1999).There are different classifications of the superfamily.Sakuma et al. (2002) have described five subfamilies consisting of the APETALA2 (AP2), Related to AB1/VP1 (RAV), Dehydration Responsive Element Binding Proteins (DREB) and Ethylene-Responsive Element Binding Proteins (EREBP) also called the Ethylene Response Factor (ERF).By contrast, Nakano et al. (2006) have categorized these proteins in three families consisting of the AP2, ERF and RAV.
The current state-of-the-art of ethylene research in Hevea brasiliensis especially in relation to the characterization of AP2/ERF superfamily involved in diverse physiological mechanisms of Hevea brasiliensis is reviewed in this paper.The main ideas discussed are consisted of (1) characterization of the ethylene biosynthesis and signalling pathways in rubber tree, (2) identification of AP2/ERF gene expression markers related with potential function in rubber tree, (3) potential post-transcriptional and post-translational regulations of Hevea AP2/ERF genes, (4) potential important role of Hevea AP2/ERF genes in reversible tapping panel dryness generated by oxidative stress, and (5) future perspective for functional analysis of AP2/ERF candidate genes.

Characterization of the ethylene biosynthesis and signalling pathways in rubber tree
The ethylene biosynthesis may be induced by the presence of endogenous or exogenous ethylene in Hevea brasiliensis.The aminocyclopropane-1-carboxylic acid (ACC) synthase activity or ACS and the ACC oxydase or ACO are the last two enzymes of the ethylene biosynthetic pathway (Yang & Hoffman, 1984).The genes involved in the ethylene biosynthesis pathway have been identified and partially characterized (Kuswanhadi, 2006).Three HbACO genes (HbACO1, HbACO2, and HbACO3) and three HbACS genes (HbACS1, HbACS2, and HbACS3) have been identified (Kuswanhadi et al. 2007;Kuswanhadi et al., 2010).The transcript of HbACO1 was the most abundant among three HbACOs in bark tissue of 3-month-old budded plants.However, it was reduced upon stimulation by ethylene in leaves and bark (Kuswanhadi, 2006).The HbACO2 and HbACO3 genes were transiently induced in response to ethylene.The application of an ethylene inhibitor, i.e. 1-MCP, abolished the induction of ethylene in both of the mentioned genes demonstrating the positive feedback regulation.All HbACOs genes were expressed at all developmental stages.All these works have suggested that HbACO1 gene was responsible for the ethylene biosynthesis regulating at the basal level while the HbACO2 and HbACO3 genes were regulated in response to external factors (Kuswanhadi et al., 2010, Kuswanhadi et al., 2007).
The molecular actors involved in ethylene perception and signalling were also studied in Hevea brasiliensis (Duan et al., 2010).Two genes of ethylene perception, Hevea brasiliensis Ethylene Receptor 2 (HbETR2) and Hevea brasiliensis Ethylene-insensitive 2 (HbEIN2), and a gene of ethylene signalling (HbEIN3) were differentially regulated by ethylene treatment.The transcript of HbETR2 gene was rapidly accumulated upon stimulation of ethylene while the transcripts of HbEIN2 and HbEIN3 genes were significantly reduced.This early induction HbETR2 was suppressed by an ethylene inhibitor, 1-MCP.In this work, Duan et al. (2010) have shown the effect of ethylene and wounding treatments on the downstream-response genes of ethylene signalling pathway.Thus, it opened the perspective of crosstalk signalling between phytohormones in Hevea brasiliensis.In search of transcription factors involved in this mechanism, the Hevea AP2/ERF superfamily has been the focus subject of study.Based on known previous knowledge in model plants such as Arabidopsis thaliana and Oryza sativa, these transcription factors were involved in the hormonal response of various biotic and abiotic aspects.Duan et al. (2013) have classified 142 members of Hevea AP2/ERF superfamily based on the AP2 domain sequence from full length transcripts of RNA sequencing from rubber clone PB 260.The authors have classified HbERF family into 10 functional groups.The transcriptomic database was generated from somatic embryo tissue, leaves, bark, latex, and roots.The work of Duan and colleagues has further focused on the study of transcription factors involved in the ethylene and jasmonate signalling.Among those Ethylene Response Factors, two Arabidopsis genes (ERF1 and ORA59) were found at the intersection of both hormonal signalling (Huang et al., 2015, Pre et al., 2008).In Hevea, the HbERF-IXc4 and HbERF-IXc5 genes were the orthologue to ERF1 while the HbERF-IXc6 gene was the orthologue of ORA59 (Duan, 2011).A further study revealed that HbERF-IXc4 and HbERF-IXc5 genes behaved like the Arabidopsis ERF1.Both of genes were induced by ethylene and methyl jasmonate (MeJA).When all the treatments such as MeJA and ethylene were combined, the transcript abundance has multiplied.A preliminary functional study using trans-activity and subcellular localization experiments was performed for HbERF IXc4 and HbERF IXc5 genes.The HbERF-IXc4 and HbERF-IXc5 proteins were able to bind to the promoter of AtPDF1.2 gene and were localized in the nucleus (Duan, 2011).
The Hevea brasiliensis AP2/ERF super-family was further characterized by Piyatrakul and colleagues in 2012.Firstly, the work has focused in the role of AP2/ERF transcription factors in the developmental aspect of rubber tree.The transcript accumulation of AP2/ERF genes was analyzed during the process of somatic embryogenesis from callus lines with different regeneration capacity in various vegetative and reproductive tissues (Piyatrakul et al., 2012).Secondly, Piyatrakul et al. (2014) focused the experiment on the search for ERF family having an important role in the laticifers related to the latex production.The transcriptomic database was improved by supplementing RNA samples from reproductive tissues (immature and mature male flowers, immature and mature female flowers, and zygotic embryos).The conducted work has confirmed 114 contigs encoding Hevea AP2/ERF genes.These transcription factors were further confirmed by in silico analysis of genomic sequences from rubber clone CATAS-7-33-97.The transcriptomic and genomic database showed a difference in the number of AP2/ERF genes.Potential isoforms of the genes were hypothesized since the clones used for sequence comparison were unlike.In addition, the absence of certain tissues in the transcriptomic database (such as cambium and xylem) could explain the absence of several Hevea AP2/ERF genes.However, the work has identified the Hevea ERF Group VII as gene expression markers in latex suggesting a potential role in the hypoxia regulation in laticifers.Functional analysis by trans-activity and subcellular localization has confirmed that members of HbERF-VII was an activator-type transcription factor (Piyatrakul et al., 2014).
In parallel with the work of Piyatrakul, the characterization of Hevea AP2/ERF superfamily was similarly addressed by studying their stress response as well as during the occurrence of tapping panel dryness (Table 1) (Putranto et al., 2015b, Putranto et al., 2015a).A polyclonal test was initiated in 2003 at the Sembawa Research Station, Palembang, under collaboration of the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) and the Indonesian Rubber Research Institute (IRRI).The first tapping of mature trees was carried out in 2010.Since, latex diagnostic have been regularly measured in order to monitor the physiological state of rubber tree under different harvesting system.Latex and bark samples under the effect of harvesting system, including tapping and ethephon stimulation, and tapping panel dryness were then collected.The HbERFs as gene expression markers (GEMs) was identified using high-debit qPCR analysis from the juvenile plant samples which were generated from the somatic embryogenesis prepared in CIRAD (Putranto et al., 2015a).

Identification of AP2/ERF gene expression markers related with potential function in Rubber tree
The identification of gene expression markers was focused on the strongly expressed genes during latex harvesting and during the occurrence of tapping panel dryness.In regard to known function and well-characterized AP2/ERF superfamily, the Arabidopsis database was selected as comparison to Hevea brasiliensis.Duan et al. (2013) was also used this database to construct the classification of Hevea AP2/ERF transcription factors.The bibliographic and phylogenetic analyses were able to reveal the potential functions of these GEMs (Putranto et al., 2015a, Piyatrakul et al., 2014).However, these GEMs may or may not have all important function in rubber tree.Putranto et al. (2015b) and Putranto et al. (2015a) have identified 36 HbAP2/ERF genes including 5 genes of AP2 family and 31 genes of ERF family as GEMs (Table 1).Eighteen GEMs were predicted as ortholog with 19 Arabidopsis AP2/ERF genes.Nevertheless, several GEMs without knownortholog could have a specific and important function in Hevea.For now, the HbERF-IXb2 gene is linked to the occurrence of tapping panel dryness.It was the sole gene induced by the disease in laticifers.
In general results, high relative abundance of transcripts were detected in several groups of HbAP2/ERFs ( Putranto et al., 2014;2015a;2015b).Although some members of these groups have not proven to be GEMs, high level of expression leads us to suggest their involvement in some regulation at the molecular level.The transcripts of the HbERF-I, HbERF-VII, HbERF-VIII and HbERF-IX were highly accumulated in various responses such as development process, latex harvesting and tapping panel dryness.Among members of ERF group I, WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and RELATED TO AP2.4 (RAP2.4)proteins have known functions in A. thaliana.The WIND1 gene was associated with the control of wounding-induced cellular dedifferentiation (Iwase et al., 2011a;2011b).This gene was rapidly induced in the wounded site promoting cell dedifferentiation leading to cell proliferation until a mass of pluripotent cells called callus were formed (Iwase et al., 2013).The RAP2.4 gene was highly expressed in stems and roots under abiotic stress such as cold, dehydration and osmotic stress (Lin et al., 2008).It has been shown that the abundance of RAP2.4 protein was regulated by a BR-C, ttk and bab/Pox virus Zinc finger (BTP/POZ-MATH) or briefly known as BPM protein family (Weber & Hellmann, 2009).In addition, it is suggested that this gene plays a role in the regulation of water homeostasis (Rae et al., 2011).The HbERF-Ib7 gene is a GEM in response to latex harvesting stress but it has no known ortholog in Arabidopsis.
The ERF-VII was characterized by a protein motif MCGGAI(I/L) at the N-terminus ( van Veen et al., 2014;Piyatrakul et al., 2014;Nakano et al., 2006).This N-end rule regulation was associated with the targeted protein degradation by the proteasome (Licausi et al., 2011).The elimination of the methionine at N-terminal by methionine aminopeptidase (MAP) left the cysteine residue to be exposed and destabilized.Under the normal condition of the oxygen rate called normoxia, this cysteine was oxidized by oxygen or nitric oxide (Gibbs et al., 2014).This oxidized cysteine induced the addition of argynil residue at the Nterminal end which promotes polyubiquitination leading to the degradation of ERF-VII (Licausi et al., 2013b).The ERF-VII in Arabidopsis has been reported as a group of hypoxia-or anoxiaresponsive genes such as HYPOXIA RESPONSIVE 1 and 2 (HRE1 and HRE2), RELATED TO AP2.2 (RAP2.2) and RELATED TO AP2.12 (RAP2.12)(Seok et al., 2014;Licausi et al., 2010b).The ethylene-induced overexpression of RAP2.2 gene improves the plant survival under hypoxia (Hinz et al., 2010).By contrast, the overexpression of HRE1 gene increased tolerance to anoxia (Licausi et al., 2010b).Its role as repressor of ethylene during hypoxia has been demonstrated (Yang et al., 2011).The constitutive regulation of RAP2.12 and RAP2.3 genes involving a membrane protein named Acyl-CoA-binding protein (ACBP) has been shown (Bailey-Serres et al., 2012;Licausi et al., 2011).In rubber tree, a hypoxic condition has been suggested in the laticifers since the fermentative metabolism of pyruvate is the main degradation pathway of sugar in the latex cytosol (Tupý, 1989).The intermediate product, pyruvate can be used by both aerobic and anaerobic pathways to generate acetyl-CoA (Jacob et al., 1989a).The transcripts of HbERF-VIIs were accumulated in laticifers, suggesting a potential role of regulation to hypoxia in Hevea brasiliensis.Based on a phylogenetic analysis, the HbERF-VIIa12 and HbERF-VIIa20 genes were orthologous with Arabidopsis RAP2.12 and RAP2.3, respectively (Piyatrakul et al., 2014).
The ERF-IX group probably contains the transcription factors whose involvement in the response to pathogens has been most extensively studied (Licausi et al., 2013a;Zarei et al., 2011;Moffat et al., 2012).Members of this group were characterized by a protein motif of activator-type EDLL in the C-terminal (Tiwari et al., 2012).The involvement of ERF-IX (ERF1, ORA59, ERF5, and ERF6 genes) in the ethylene and jasmonate signalling was clearly demonstrated in Arabidopsis (Cheng et al., 2013a;Moffat et al., 2012;Zarei et al., 2011).The ERF1 and ORA59 genes belong to the subgroup of ERF-IXc while ERF5 and ERF6 genes are within the subgroup of ERF-IXb.Recently, it was shown that ERF5 also called AAL MODULATOR OF CELL DEATH (MACD1) was upregulated in response to programmed cell death via the ethylene signalling during the attack of pathogens (Mase et al., 2013).In Hevea, the HbERF-IXa3, HbERF-IXb3, HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6 genes were orthologous to AtERF1, ERF5/MACD1, ERF1 and ORA59 genes in Arabidopsis, respectively (Piyatrakul et al., 2014).
Table 1.The AP2/ERF gene expression markers (GEMs) during latex harvesting and under the occurrence of tapping panel dryness (TPD) in Hevea brasiliensis.The upregulated GEMs were shown in red meanwhile the downregulated GEMs were in blue colour.LH = latex harvesting; T = tapping; Et-= no ethephon treatment; Et+ = ethephon treatment.The GEMs were extracted from Putranto et al. (2015aPutranto et al. ( , 2015b)).

GEMs
The ERF group VIII contains an ERF-associated amphiphilic repression (EAR)-motif known as the transcriptional repressors (Ohta et al., 2001).These ERFs of repressor-type were demonstrated to be able to induce a programmed cell death (Ogata et al., 2011;2013).The multifunctional co-repressors, TOPLESS (TPL) and TPL-related (TPR) interact directly with the EAR motif of ERF group VIII.The interaction of TPL/TPR and other transcription factors was co-opted several times to modulate the expression of genes in various biological processes, including hormonal signalling and stress responses (Causier et al., 2012).The Arabidopsis ERF3, ERF4, ERF7, ERF10, ERF11, ERF12 and ESR1/DRN genes belong to the ERF-VIII.However, these genes showed differential expression and distinct roles in plant growth and development as well as in response to environmental stress.For instance, the ERF4 and ERF7 genes play an important role in the response to ABA (Yang et al., 2005;Song et al., 2005).The overexpression of ERF7 increased the sensitivity to ABA in guard cells (Song et al., 2005).The suppression of ERF-VIII also requires interactions with other transcription factors.The interaction of ERF11 gene with the bZIP-type transcription factor, HY5 has modulated the ethylene biosynthesis via ABA signalling (Li et al., 2011).The ESR1/DRN genes were involved in the development of petiole, the structuration of embryo and the development of cotyledons (Chandler & Werr, 2011;Cole et al. 2013).The high abundance of HbERF-VIII transcripts in bark and latex under harvesting stress suggest a post-transcriptional regulation of other HbERFs by their role as repressor (Putranto et al., 2015a).
Two GEMs such as HbERF-IIIB1 and HbERF-IVa3 did not have orthologs in Arabidopsis.However, these genes could have an important role in osmotic stress response especially linked to the production of latex.In the classification of Sakuma (2002), the HbERF-III genes were included in the CBF/DREB1 subfamily.The CBF/DREB1 genes have been extensively characterized in plants linked to the cold stress (Kurepin et al., 2013;Rehman & Mahmood, 2015).In Hevea, a small number of Cold Binding Factor/Dehydration Responsive Element Binding 1 (CBF/DREB1) genes were identified (Duan et al., 2013).This could be explained by a minor adaptation of Hevea tree to the cold zone.Additionally, HbERF-IV genes correspond to the DREB2 subfamily involved in the response to dehydration.The expression of HbERF-IVa3 gene was induced by tapping, ethephon stimulation and tapping panel dryness (Putranto et al. 2015b;Putranto et al., 2015a).The expression of HbERF-IVa3 gene was consistent with the hypothesis since laticifers are subject to a recurrent osmotic stress during the exploitation of rubber tree.In the future, these ERF groups, which have a strong expression but no known functions, deserve a deep characterization to determine their involvement in cell processes, including latex production.

Potential post-transcriptional and posttranslational regulations of hevea AP2/ERF genes
The characterization of AP2/ERF superfamily in Hevea brasiliensis was focused on transcriptional regulation.Yet, previous research works in different plant species has demonstrated that the AP2/ERF genes can be regulated at the posttranscriptional level, including the regulation by alternative splicing, control of protein stability, acetylation and nitrosylation (Licausi et al., 2013a).A strong accumulation of transcripts was observed for HbERF-I, HbERF-VII, HbERF-VIII and HbERF-IX members under harvesting stress and tapping panel dryness in bark and latex tissues (Putranto et al., 2015a;2015b).The constitutive expression of several HbERFs led us to consider a post-transcriptional regulation controlling the activity of these proteins.Yet, it has been shown in grapevine, tomato and Arabidopsis as ERF groups (ERF-II, ERF-III, ERF-VII and ERF-IX) were strongly expressed in different tissues (Licausi et al., 2010a;Pirrello et al., 2012;Bailey-Serres et al., 2012).A high abundance transcript in non-stressed condition was potentially a default defence system being present before the stress condition (Cheng et al., 2013b).
Post-transcriptional regulation of Dehydration Responsive Element Binding Proteins 2 (DREB2) genes by alternative splicing has been reported in Arabidopsis, tomato and wheat (Rehman & Mahmood, 2015;Pirrello et al., 2012;Lata & Prasad, 2011;Lucas et al., 2011;Mizoi et al. 2012).In the case of alternative splicing, two types of transcripts may exist.The first is an inactive portion having a transcribed intron sequence which generates a stop codon before the DNA binding domain.The second is an active transcript encoding the full-length protein.This type of regulation was also observed in the ERF-VII in tomato and Arabidopsis (Pirrello et al., 2006, Licausi et al., 2010b).Furthermore, post-transcriptional regulation of AP2/ERF genes by microRNAs was also demonstrated.The JcDREB gene in Jatropha curcas has been targeted by miR156 (Galli et al., 2014) while a repression of expression of OitaAP2 gene in Orchis italica was controlled by miR172 (Salemme et al., 2013).In some cases, the action of miRs can be specific.In Arabidopsis, the miR319b targeted transcript isoform of RAP2.12 containing an intron in order to inactivate the gene (Sobkowiak et al., 2012).In Hevea, several microRNAs including the well-known miR172 were predicted to target AP2/ERF transcripts (Duan et al., 2013).Although the inhibition mode was being achieved mainly by cleavage of target RNA messenger, translation inhibitions has been predicted for two genes (HbERF-VI5 and HbERF-IXc5).
The AP2/ERF genes are likewise regulated at the post-translational level.For now, the DREB2 belonging to ERF-IVa contains a rich region of Ser/Thr is a potential site of phosphorylation.A deletion of a Ser/Thr motif led to a constitutively active form of DREB2A in Arabidopsis (Sakuma et al., 2006).This finding was validated by overexpression of DREB2A in Arabidopsis and Oryza that did not result to an induction of downstream genes in normal conditions.Posttranslational modification was necessary to activate this phosphorylation site (Dubouzet et al., 2003;Liu et al., 1998).Phosphorylation regulation via histidine kinases has also been suggested for the ERF-VI (Raines et al., 2016).Another ERF belonging to the subgroup of ERF-IXb, AtERF104 required an interaction with the mitogen-activated protein kinase 6 (MPK6) for its stability (Bethke et al., 2009).The stability of ERF proteins occasionally involves the 26S proteasome.Once more, the ERF-VII gives an example of ubiquitin ligase involvement such as PROTEOLYSIS6 (PRT6) in the degradation of ERF-VII proteins via the N-end rule pathway (Gibbs et al., 2011;Licausi et al., 2011).There are not yet any reports about the post-translational regulation of AP2/ERF genes in Hevea brasiliensis.However, the low expression of HbERFs during the onset of TPD has brought a hypothesis in which a post-translational regulation was taken place (Putranto et al., 2015b).

Potential important role of hevea AP2/ERF in reversible tapping panel dryness generated by oxidative stress
The occurrence of tapping panel dryness depends on various factors, such as plant material (genotype sensitivity and rootstock-scion interaction), environment aspects (harvesting system, biotic and abiotic stresses, and soil compaction) (Putranto et al., 2015b).These factors result in a complex signalling involving molecular responses at transcriptional and post-transcriptional levels.Previous studies in Hevea have identified gene expression markers for TPD such as HbMyb1, HbTOM20 and HbTCTP genes (Venkatachalam et al., 2009;Peng et al., 2011).These genes were negative regulators of genes that positively regulate the programmed cell death.The inhibition of these genes in TPD-affected trees thus causes the activation of programmed cell death.HbTOM20 gene encodes a translocate protein of the outer mitochondrial membrane regulating the transport of nuclear proteins within mitochondria (Venkatachalam et al., 2009).It is likely that inhibition of this protein results to the mitochondrial dysfunction leading to impaired respiration and a low level of ATP.HbTCTP gene encodes cytosolic calcium binding protein that exact function remains to be elucidated in rubber tree.The transcript of this gene was also 60% lower during the occurrence of TPD in comparison to normal condition (Venkatachalam et al., 2007).In their study, Gébelin et al. (2013) predicted that the microRNA, HbmiR159b could target the Myb-like transcription factor (HbMyb1) and glutathione peroxidase (HbGPX) in Hevea brasiliensis.These post-transcriptional regulations may result in a change in the activity of target genes.Glutathione peroxidase is one of the key enzymes in the regulation of thiols in plants which function is to reduce the hydro-lipid peroxides in a form of alcohol as well as to hydrogen peroxide into H2O (Noctor et al., 2012).
The characterization of Hevea AP2/ERFs has led to the possible involvement of these transcription factors during the occurrence of TPD.In transcriptional level, Putranto et al. (2015b) has observed the induction of two HbERF genes (HbERF-IIb2 and HbERF-IXb2) and the inhibition of HbERF-VIIIa14 gene.The participation of jasmonate biosynthetic pathway could be suggested since the HbERF-IIb2 gene (ortholog of Arabidopsis ORA47) was induced.The ORA47 gene was demonstrated to induce the expression of LOX3, one of key genes to jasmonate (JA) biosynthesis (Khurshid 2012).Recently, Chen et al. (2016) has demonstrated the dual function of ORA47 protein in both JA and ABA biosynthesis leading to a clear involvement of ORA47 under osmotic stress.By contrast, the inhibition of HbERF-VIIIa14, a member of ERF-VIII during the occurrence of TPD suggests a control of the cell death regulation.
These responses at the molecular level generate a biochemical change in response to TPD.It is known that the thiol concentration and the enzymatic activities of catalase (CAS) and superoxide dismutase (SOD) were reduced during the occurrence of TPD (d'Auzac et al., 1993).Some of these results have been confirmed in Hevea (Putranto et al., 2015b).In addition, the reduction of the CAS and SOD activities was followed by the increase of NAD(P)H oxidase activity (Chrestin, 1989).These parameters highlighted the appearance of oxidative burst that could also become a response signal of TPD.From this oxidative stress, Putranto et al. (2015a) hypothesized two events by known mechanisms which can be drawn.First, the peroxidation of lutoidic membranes releases coagulation factors leading to reactive oxygen species-type of TPD (ROS-TPD) (Jacob et al., 1989b).Secondly, oxidative stress may lead to lipid peroxidation in the plasma membrane, release of linamarase and linamarin resulting to cyanide production (Kadow et al., 2012).In a worse condition, a bark necrosis can occur resulting finally brown bast-type of TPD (BB-TPD).
Future perspective for functional analysis of AP2/ERF candidate genes HbAP2/ERF genes, which were responsive to multiple harvesting stress and TPD, have been identified and characterized in transcriptional level.Further characterization of several HbAP2/ERF candidate genes using transgenesis was suggested in order to fully understand their functions, especially in latex production of Hevea brasiliensis.Targeted candidate genes should be selected from HbERF-VII, HbERF-VIII, and HbERF-IX previously described as the most abundant and potentially important groups involved in the tolerance to harvesting stress and tapping panel dryness.
The metabolism activities of laticifers consume oxygen and generate reactive oxygen species.Interestingly, the GEMs of HbERF-VII are potential activators of downstream genes in response to hypoxia such as HbADH and HbSUS1 (Piyatrakul et al., 2014).Among the members of this group, HbERF-VIIa12 gene, a potential ortholog of RAP2.12, could have an important role in the regulation of homeostasis of oxygen in the laticifers.In Arabidopsis, the RAP2.12 protein was known to be able to interact with alcohol dehydrogenase (ADH) and regulate their activity throughout the fermentation route.Further characterization of the gene, including a study of co-expression with response to hypoxia genes such as HbADH, a study of regulation by microRNAs and an analysis of transgenic line overexpressing or dowregulating HbERF-VII should clarify its involvement during a hypoxic condition in laticifers.
The rubber biosynthetic pathway belongs to the secondary metabolism (d'Auzac et al., 1993).For this reason, the HbERF-IX group could play an important role controlling latex metabolism, e.g. during sucrose loading into laticifers.Among members of this group, two genes (HbERF-IXc4 and HbERF-IXc5) were dramatically induced by the combination of ethylene and methyl jasmonate treatments in Hevea (Putranto et al., 2015a).These genes are potential ortholog to ERF1 famously known to be highly regulated at transcriptional level during multiple abiotic stresses.A further characterization of these genes requires the identification of their regulons (downstream target genes) by Chromatin Immunoprecipitation Sequencing (ChIP-seq) method and the functional analysis in transgenic lines.Transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 genes have been regenerated (Lestari et al., 2014).These lines showed a promising phenotype of highly tolerant to abiotic stress which will confirm the major role of both genes in latex production.
Another full characterization should be carried out on the ERF-VIII having an important role in the induction of programmed cell death biotic and abiotic stresses.In relation to TPD, members of this group should be considered as important candidate genes.An ortholog of Arabidopsis ERF12, HbERF-VIIIa13 gene has been demonstrated to be repressor-type transcription factor (Putranto et al., 2015a).In addition, HbERF-VIIIa14 gene was a gene expression marker specifically induced under the occurrence of TPD.The protein-protein interaction analysis with known repressor network such as TOPLESS protein could be an important aspect to be elucidated.

Conclusion and Remarks
The ethylene biosynthesis and signalling pathway is one of the most studied aspects in Hevea brasiliensis.These studies have been driven by the practice of latex harvesting involving an ethylene releaser.Understanding the hormonal regulation of latex production in cellular and molecular levels is an important aspect in Hevea brasiliensis.However, the crosstalk of other phytohormones such as jasmonate and ABA with ethylene cannot be taken aside.Clear evidence has shown that their regulation could be also key factors in latex production.As a transcription factor, the Hevea brasiliensis AP2/ERF superfamily has shown its potency to be the master regulator of phytohormones related directly to latex production, regulator of tolerance to abiotic and biotic stress, and regulator of tolerance to physiological disease.In the near future, molecular markers could be developed from HbAP2/ERFs in order to select Hevea clones tolerant to abiotic and biotic factors.