Establishment of Hevea brasiliensis lines overexpressing genes involved in ethylene signalling pathway

Gas etilen merupakan hormon pada tumbuhan yang telah banyak digunakan dalam pengembangan pertanian dan hortikultura. Sebagian besar kerja beberapa gen yang responsif terhadap hormon etilen dikendalikan oleh faktor transkripsi yang disebut Ethylene Response Factors (ERF). Dua faktor transkripsi pada tanaman Hevea HbERF-IXc4 dan HbERFIXc5 merupakan gen yang ortholog dengan ERF1 pada Arabidopsis thaliana. ERF1 merupakan faktor yang mengatur integrasi sinyal antara etilen dan jasmonat. Kedua gen tersebut diduga berperan dalam pengaturan metabolisme sel lateks pada proses penyadapan dan stimulasi ethephon. Tanaman transgenik Hevea yang mengandung dua gen HbERF-IXc4 dan HbERFIXc5 di bawah kendali promoter 35S CaMV dan HEV2.1 telah berhasil dirakit menggunakan metode transformasi genetik melalui Agrobacterium tumefaciens. Transformasi genetik melalui perantara A. tumefaciens dikembangkan menggunakan material kalus remah dari klon PB260. Agregat kalus Hevea tersebut kemudian disubkultur ke dalam medium yang mengandung paromomisin. Kalus transgenik yang diperoleh menunjukkan hasil yang positif dalam uji aktivitas GFP. Sembilan galur transgenik dikonfirmasi telah terinsersi gen HbERF-IXc4 dan HbERFIXc5 dengan metode Southern blot, dan tujuh di antaranya hanya mengandung satu kopi T-DNA. Regenerasi dan karakterisasi dari tanaman transgenik perlu dilakukan untuk memahami fungsi HbERF-IXc4 dan HbERF-IXc5 yang diduga berperan dalam produksi lateks.


Abstract
The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture.Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes.Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1, a key regulator at the crosstalk of ethylene and jasmonate signalling pathways.These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation.In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted.Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation.The somatic embryogenesis process was affected by these modifications.Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260.Hevea callus was sub-cultured as small aggregates on paromomycin selection medium.Transgenic callus lines established from sub-aggregates showing full GFP activity.The insertion of HbERF-IXc4 and HbERF-IXc5 genes were confirmed by Southern blot hybridization in nine transgenic lines, and seven of these lines have only one T-DNA copy.Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex production.

Introduction
Hevea brasiliensis (Willd.ex A. Juss.)Müll Arg. is the source of natural rubber (NR), which is synthesized in latex cells.NR is an important industrial material for transportation, consumer, and medical.The demand for NR is increasing from year to year.Nowadays, about 93% of NR world supplies is produced in Asia, Africa (4%), and Latin America (3%).The largest NR producing countries are Thailand (38%), Indonesia (30%), and Vietnam (9%).NR is the main product from H. brasiliensis which is obtained from latex in laticifer after tapping the bark.
Ethylene is an important hormone to stimulate latex production in rubber tree (Clément-Demange et al., 2007;Putranto et al., 2015;Vaysse et al., 2012).The ethylene signalling pathway activates the Ethylene Response Factor (ERF) transcription factors.ERF genes are associated with response to abiotic and biotic stresses.ERF1 was suggested to be a key component for the defence responses through the integration of ethylene (ET) and jasmonic acid (JA) signalling pathways (Benavente & Alonso, 2006;Lorenzo et al., 2003;Wasternack & Hause, 2013).The crosstalk between ET and JA signalling pathways acts together synergistically during plant defence against herbivores, necrotrophic fungi infections, plant pathogens, and in responses of wounding (Lorenzo et al. 2003;Zhu & Lee, 2015).Crosstalk connection between pathways in plants could be interpreted as the interaction between specific signal transduction pathways which resulted in a rapid and efficient mechanism for optimizing non-cognitive behaviour in response to various combinations of stimuli (Weiss and Ori, 2007).
Several genes in H. brasiliensis were regulated independently in signalling pathways.Defence responses in Hevea were dramatically mediated by wounding, methyl jasmonate (MeJA), and ethylene (ET) (Duan et al., 2010).Two Hevea ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways (Duan et al., 2013;Piyatrakul et al., 2014;Putranto et al., 2015).Functional analysis of a candidate gene, HbCuZnSOD, was carried out in Hevea (Leclercq et al., 2012), with both somatic embryogenesis and A. tumefaciensmediated genetic transformation procedures (Blanc et al., 2006;Leclercq et al., 2010).One of commonly used promoters is the 35S RNA promoter sequence from the Cauliflower Mosaic Virus (CaMV) (Wu et al., 2014).In the other hand, promoter of the HEV2.1 gene was reported to drive expression in latex and leaves (Montoro et al., 2008).This paper aims to establish H. brasiliensis transgenic lines for the two recently identified transcription factors HbERF-IXc4 and HbERF-IXc5 under the control of 35S CaMV or latex-specific HEV2.1 promoter.

Materials and methods
The construct used for this transformation experiment consisted of a binary pCamway 2300 vector with the cassette harbouring GFP gene, NPTII gene, and one candidate gene (Leclercq et al., 2015).In this study, HbERF-IXc4 and HbERF-IXc5 genes were cloned under the control of 35S CaMV or HEV2.1 promoter (Duan et al., 2013;Leclercq et al., 2010;Montoro et al., 2008).The binary vectors were introduced into A. tumefaciens strain EHA105 by electroporation.Plant material used in this study was the friable callus line CI07060 from clone PB 260, which was established from integument calli (Lardet et al., 2009).This callus line was sub-cultured every two weeks on a maintenance culture medium (MM).After getting the transgenic cell, the process was continued by using the green fluorescent protein (GFP) selection as a visual marker (Leclercq et al., 2010).The GFP visualisation was performed under a fluorescent stereo microscope (MZ FLIII, Leica Microsystems, Wetzlar, Germany).The production of embryos and their conversion into plantlets were carried out as described by Lardet et al. (2007).The development of pro-embryos was then carried out in a temporary immersion system (RITA ® , CIRAD, Montpellier, France).For plant regeneration, well-shaped mature embryos were collected and transferred to DEV3 medium for germination (Lardet et al., 2007).
Southern-blot hybridization analysis were conducted to verify the insertion and overexpression of two candidate genes (HbERF-IXc4 and HbERF-IXc5) into Hevea transgenic plants genome.DNA extraction from leaves of transgenic lines was conducted as described in Leclercq et al. (2010).Samples of genomic DNA were fragmented with EcoR1 restriction enzyme and fractionated by electrophoresis in a 0.8% agarose gel in TAE buffer.The hybridization was performed as described in Sambrook et al. (1989) using random primed 32 P radio-labelled probes corresponding to NPTII gene (Amersham TM Megaprime DNA Labelling System, Buckinghamshire, UK).

Results and Discussion
Hevea callus was successfully sub-cultured as small aggregates on paromomycin selection medium (Figure 1A).Somatic embryogenesis was initiated for 4 weeks by sub-culturing 1 g of callus showing full GFP activity on semi-solid embryogenesis expression medium (EXP).All GFP-positive lines were obtained from DM medium then continued to transfer on EXP medium (Figure 1B and 1C).Pro-embryo development was carried out in a temporary immersion system (RITA ® , CIRAD, Montpellier) for 4 weeks with 1 min of immersion per day in the liquid development medium (DEV) (Figure 1D).Each RITA was considered as an experimental replication.Conversion of mature embryos was carried out according to Lardet et al. (1999).Well-shaped mature embryos were produced (Figure 1E).Plantlets were successfully derived from normal embryos (Figure 1F).

Construct/ Konstruk
The role of gene regulatory regions (promoters) is important for understanding the regulation of plant gene expression.Both of promoters have been demonstrated to drive a candidate gene in genetic transformation via A. tumefaciens (Blanc et al. 2006;Montoro et al. 2000;Rattana et al. 2001).The 35S CaMV promoter allows strong constitutive expression in all tissues, in the other hand HEV2.1 promoter allows targeted expression in laticifer cells and in leaves (Montoro et al. 2008).
Twenty-nine GFP-positive lines were established on paromomycin selection medium.Paromomycin is more efficient than kanamycin for the selection of transformed cells and can inhibit the growth of non-transformed cells more quickly (Montoro et al. 2003;Perez-Barranco et al. 2009).Nine lines were confirmed by Southern blot hybridization and seven of these lines have only one T-DNA copy.This revealed that the genetic modification methodology did not affect too much the Hevea genome compared to other procedures using particle bombardment or too virulent Agrobacterium strains.For that reason, most of transgenic lines can be compared to each other without any strong effect of the copy number.Each line with one copy of T-DNA had a higher gene expression of their corresponding transgene.
HbERF-IXc4 gene having lower callus proliferation was observed with the 35S CaMV promoter compared to HEV2.1.For instance, HbERF-IXc4 gene with 35S CaMV need more subcultures than with HEV2.1 to get a sufficient quantity of callus for further plant regeneration and callus cryopreservation.All lines could produce abundant yellow callus.At the beginning of culture, the callus appeared creamy in colour and then gradually became yellow or dark yellow.Finer (1988) has been classified the cotyledon callus based on the colour as green, yellow, white, brown, and red.Only yellow callus could produce embryonic culture (Finer 1988;Perera and Dahanayake 2015;Sen et al. 2014).Callus turned brown at the advance of embryo formation.Brown calli produced a large number of somatic embryos compared to yellow calli.The abnormal types of embryos cannot develop into plantlet.Some studies verified that the constitutive promoter 35S CaMV is a weak promoter for transgene expression in young olive somatic embryos.This promoter was more active in an organized tissue of mature alfalfa somatic embryos than in the less-organised tissues of young embryos (Perez-Barranco et al. 2009;Tian et al. 2000).
The number of total embryos was similar for the two promoters (35S CaMV and HEV2.1).The rate of conversion of embryos into plantlets was lower for 35S CaMV than HEV2.1.Finally, that led to produce a low quantity of plantlets for lines harbouring 35S::HbERF-IXc4 compared to lines harbouring HEV2.1::HbERF-IXc4.
Transgenic lines had lower plant regeneration efficiency than wild-type, and lines harbouring ERF genes under the control of the HEV2.1 promoter provided a larger number of plantlets/RITA compared with those under the control of 35S CaMV promoter especially for HbERF-IXc4 gene.This observation suggests that constitutive expression of ERF transgenes decreased regeneration efficiency.By contrast, the use of specific promoter had less effect on somatic embryogenesis process except a stronger callus browning during embryo induction.

Conclusions
This work described the successful establishment of transgenic lines carrying the two transcription factors HbERF-IXc4 and HbERF-IXc5 under the control of 35S CaMV or HEV2.1 promoters.Transgenic lines obtained may lead to better understanding the functional analysis of overexpression of HbERF-IXc4 and HbERF-IXc5 by further application of biotic and abiotic stresses and histology analysis.