Evaluation of eleven reference genes for Reverse Transcriptase Quantitative PCR of rubber tree under water deficit

Reverse Transcriptase Quantitative PCR (RTqPCR) is a powerful technique in order to detect low abundance of mRNA in the plant cell. The measurement of transcript abundance is relative to the control of expression such as housekeeping genes. Therefore, the reliability of RT-qPCR depends essentially to the choice of these internal controls also called reference genes. That is the reason why a prior validation of reference genes is suggested for every set of cDNA samples used in a new RT-qPCR experiment. This study aimed to analyze the stability of eleven selected housekeeping genes in three Hevea brasiliensis tissues (leaf, bark and root) under15 days of moderate water deficit. Total RNA was isolated from 18 samples consisting of control and stressed-plants collected at day-0 (D0), day-5 (D5) and day-15 (D15).The quality of cDNA synthesized was examined by PCR using HbActin primer. The eleventh primers encoding Hevea housekeeping genes (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S and HbUBI) were validated using PCR amplification. The Crossing-point (Cp) values were measured using a second derivative method after RT-qPCR analysis revealing a significantly higher Cp mean values for 11 housekeeping genes at D5 compared to D0 and D15 sampling points. This study suggests that a simple coefficient of variation (CV) method can be used to rank Hevea reference genes based on its stable expression. Five housekeeping genes (HbRH2b, HbRH8, HbUBC4, HbαTUB and HbActin) can be used for RT-qPCR analysis in Hevea brasiliensis under moderate water deficit. The HbRH2b gene was the most stable among others. (Résumé d'auteur

Isolation and characterization of three members of the multigenic family encoding ACC oxidase from H. brasiliensis during plant development : [Draft]

Applying Ethephon, an ethylene releaser, to increase rubber production in H. brasiliensis has been practised for a long time. The effect of ethylene on latex production has been amply described, notably for lengthening latex flow and its regeneration. Nevertheless, little is known about the expression of genes involved in ethylene biosynthesis and response to ethylene. In this paper, we isolated and characterized genes encoding ACC oxidase, a key enzyme in ethylene biosynthesis in the plant. We then studied the effect of ethylene stimulation on the expression of ACO genes in various tissues during plant development. Three members of the ACO multigenic family were isolated from a bark cDNA library with RACE technology: HbACO-H4, HbACO-H5, and HbACO-O48. Full length cDNA sequences encoded for peptides of 318, 315, and 318 amino acids respectively for these 3 members, which had 79 to 92% protein identity and 75 to 86% nucleotide homology between them. Two genomic sequences were isolated: HbACO-H4, which was 1504 bp long and consisted of 2 introns and 3 exons, while Hb-ACO-H5 was 1456 bp long and consisted of 3 introns and 4 exons. These three genes were differentially expressed in different plant organs in response to ethylene stimulation. (Résumé d'auteur

Production of mutants affected in hormone signalling to dissect defence mechanisms in Hevea brasiliensis: the case of ethylene

Production of mutants by genetic transformation is one alternative to dissect the response to hormonal treatment. Ethylene is an important plant hormone involved in latex production. Transgenic Hevea brasiliensis plants overexpressing an ethylene mutant receptor etrl -1 from Arabidopsis thaliana were regenerated. These plants did not show any morphological response the ethephon stimulation. This plant material is a source of information to understand the role of ethylene in Hevea brasiliensis. (Résumé d'auteur

Comparison of GUS activity in self-rooting and budded primary trans/ormant plants in Hevea brasiliensis

Successful both somatic embryogenesis and Agrobacterium tumefaciens-mediated genetic transformation systems have been developed in PB260 clone. Several transgenic callus lines and plants expressing two reporter genes (gusA and/or GFP) and CuZnSOD gene driven by the CAMV 35S promoter are respectively maintained by cryopreservation and in greenhouse. Functional genomic analysis requires homogenous population of plants for further characterization. However, the primary transformant plants are considered unstable leading to a variation in transgene expression. With regards to this epigenetic control, the variability of the beta-Dglucuronidase activity has been assessed by fluorimetric assays in primary transformant plants both self-rooting and budded state. Our preliminary results show a relative homogeneity of GUS activity in the population of self-rooting primary transformant plants obtained directly by somatic embryogenesis. After green budding, a decrease in GUS activity is recorded leading to a higher variability. The effects of budding, rootstock, rootstock/scion interaction, quality and position of the scion are discussed. (Résumé d'auteur

Gene expression analysis for identifying candidate genes for controlling oxidative stress in rubber tree

Hevea brasiliensis is the main source of natural rubber which is biosynthesized in latex cells. The high metabolic productivity required for latex regeneration after each tapping can be enhanced by ethylene application, which optimizes the production potential in rubber tree. However, excessive metabolism activation can lead to Tapping Panel Dryness (TPD). Expression analyses of several genes involved in the reactive oxygen species (ROS) scavenging systems have been studied in healthy trees, TPD trees and also in young budded plants. Here, we presented the expression patterns obtained by semi-quantitative and real time RT-PCR of genes involved in ascorbate-glutathione cycle. Three genotypes (PB260, PB217 and RRIM600) with contrasting metabolism have been selected at the immature stage for this study. Their expression patterns have been monitored along the day, and in response to both ethylene stimulation and wounding treatments. (Résumé d'auteur

Biotechnologies in rubber tree (Hevea brasiliensis) : [Draft]

Rubber tree breeding and the dissemination of planting material for plantations are closely linked to propagation methods. Since the progress made by switching from multiplication by seed to propagation by budding, the development of new techniques, such as micropropagation, has been awaited. An analysis of genetic diversity sets out to identify the agronomic traits to be incorporated into the best clones. More widely, genetic modification is a tool that will enable the introduction of new agronomic traits that are not available in the genetic diversity being assessed, and also to optimize the metabolism of the best cultivated clones in a targeted manner. In the next twenty years, a whole raft of innovations is set to contribute to better quality planting material through more efficient rubber tree breeding and propagation processes. Among those innovations, the establishment of a new generation of so-called juvenile budwood gardens is a possibility within the next five years. That transfer will be decisive for assessing the degree to which new technologies are taken on board in modern rubber growing. The involvement of growers and agro-industrialists upstream of the innovation process is decisive for the success of such an undertaking, as for the progress made last century. In vitro culture research has led to three types of micropropagation techniques and genetic modification. Microcuttings. This technique was developed from juvenile seedling material and rejuvenated clonal material by reiterated grafting on young seedling or somatic embryogenesis (Carron et al., 2003). The capacity of that technique therefore depends on the juvenility of the material treated in vitro. Although this procedure is labour consuming, it offers a strong advantage for true-to-type multiplication. Short-term somatic embryogenesis. This technique is now available for about 18 clones worldwide. Although the quality of the emblings is good, the multiplication rate is limited with this method. Long-term maintained somatic embryogenesis. This was the only technique by which mass propagation can be envisaged (Carron et al., 1995b). Although recent work shows this avenue to be highly promising, it is necessary to assess the emblings produced, since the risks of somaclonal variations can be detrimental to the quality of the plant material produced. Genetic modification. This technique combines gene transfer and plant regeneration techniques by micropropagation. In Hevea, gene transfer is possible by particle bombardment, but Agrobacterium tumefaciens is more commonly used, combined with regeneration by somatic embryogenesis (Blanc et al., 2006; Montoro et al., 2000; Montoro et al., 2003). Several cloning strategies can be considered, based on those different micropropagation techniques (Carron et al., 2005). - One-part-tree or self-rooted clones. Clones propagate directly by long-term maintained somatic embryogenesis, or indirectly by microcuttings from emblings produced by short-term embryogenesis (Carron et al., 1995a). (Texte intégral

Toward the Genome-wide analysis of Transcriptional and post-Transcriptionnal regulations in hevea

In model plant species, the involvement of small non-coding RNAs in transcriptional and post-transcriptional regulation of gene expressions under environmental constraints is well documented. In Hevea, microtranscriptomic studies were carried out on several Hevea clones, tissues with regard to various abiotic stress. Tapping Panel Dryness (TPD) syndrome is a major physiological disease, caused by an excessive ROS production that leads to loss in natural rubber production. Previous studies also revealed that small RNA distributions can change in response to rubber harvesting stress in a TPD susceptible clone PB 260. A majority of 21-nt sRNA was observed in latex of TPD-affected trees, whereas 24-nt sRNAs were more abundant in latex from healthy trees. The shift from transcriptional to post-transcriptional regulations in response to stress could be a biological function shared between rubber clones, and needs to be further explored. Deeper analyses are under way to annotate MIR genes as well of Repeats Elements and Transposable Elements in the Hevea genomic sequences available. Partial conservation of miRNA/target justifies research programs for Hevea as cleavage sites of some predicted target genes by sRNA can be partially conserved in Hevea compared to Arabidopsis. Discovery of miRNA/target couples through “degradome” analysis, TE-derived siRNAs and gene-related siRNAs can lead to a full comprehensive picture of post- transcriptional and transcriptional regulations of genome expression by sRNA. (Résumé d'auteur