Involvement of ethylene-responsive microRNAs and their targets in increased latex yield in the rubber tree in response to ethylene treatment

The rubber tree is an economically important plant that produces natural rubber for various industrial uses. The application of ethylene contributes to increased latex production in rubber trees; however, the molecular biology behind the effects of ethylene on latex yield remains to be elucidated. Recently, the intersection between microRNA (miRNA) regulation and phytohormone responses has been revealed. Insight into the regulation of miRNAs and their target genes should help to determine the functional importance of miRNAs as well as the role of miRNAs in signaling under ethylene stimulation in the rubber tree. In this study, hbr-miR159 and hbr-miR166 were down-regulated in bark under ethylene treatment. The ethylene also down-regulated ATHB15-like (Class III Homeodomain Leucine Zipper, HD-ZIP III) which have been extensively implicated in the regulation of primary and secondary vascular tissue pattern formation. The strong negative-regulation of ARF6/ARF8 caused by hbr-miR167 involved in an attenuation of vascular development and may gradually lead to bark dryness syndrome in the long term ethylene treatment. The negative correlation of hbr-miR172 and its target REF3 in the inner soft bark under ethylene treatment results in dramatic increases in latex yield in the ethylene-sensitive clone of the rubber tree. The overall results suggested that the differential expression of HD-ZIP III, miR167/ARF6, ARF8, and miR172/REF3 and related genes may play possible roles in the response to ethylene treatment, resulting in longer latex flow and increased latex yield

Differential expression of microRNAs and their targets reveals a possible dual role in physiological bark disorder in rubber tree

Trunk phloem necrosis (TPN), a physiological bark disorder of the rubber tree (Hevea brasiliensis), is a serious problem that affects the yield of natural rubber. The resultant bark dryness occurs in up to half of a plantation's trees in almost every rubber tree plantation region, causing a great annual loss of dry rubber for natural rubber production. Different types of injury and physical damage caused by mechanical activation as well as environmental stresses cause physiological bark disorder in tree. Due to the essential role of miR166, miR393 and miR167 in vascular development and abiotic stress response in diverse plant species, it was interesting to investigate the role of these miRNAs in rubber trees, particularly during development of a physiological bark disorder. In this study, the expression pattern of miR166, miR393 and miR167; and their target genes, HD-ZIP III; TIR1 and ARF8, respectively; was demonstrated in healthy tree and different TPN trees. Their existence and function in vivo was validated using RNA ligase-mediated 5' rapid amplification of cDNA ends. Taken together, the results suggest a possible dual role of these three miRNAs in maintaining normal bark regeneration in healthy trees, coping with overtapping by affecting the wound healing system leading to abnormal bark regeneration in overtapped-TPN trees, and act as additional forces that enhance the attenuation of vascular development resulting in bark necrosis and cell death in the natural-TPN tree. This is the first study to address the molecular events of miRNAs involved in the physiological bark disorder TPN in rubber tree. Further study will open the possibility to better understanding of physiological and molecular perspectives during TPN development, and lead to improvement of monitoring the exploitation of rubber tree plantations

Development and mapping of functional expressed sequence tag-derived simple sequence repeat markers in a rubber tree RRIM600 x PB217 population

Sets of polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high-and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 x PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to fiber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease-in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbT1P1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2:1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbT1P1:1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1:1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbT1P1:1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1:1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2:1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow

Genome-wide analysis of microRNAs in rubber tree (Hevea brasiliensis L.) using high-throughput sequencing

MicroRNAs (miRNAs) are short RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high-throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 115 miRNAs belonging to 56 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly and predicted 20 novel miRNAs. Further, computational analysis uncovered potential targets of the known and novel miRNAs. Predicted target genes included not only transcription factors but also genes involved in various biological processes including stress responses, primary and secondary metabolism, and signal transduction. In particular, genes with roles in rubber biosynthesis are predicted targets of miRNAs. This study provides a basic catalog of miRNAs and their targets in rubber tree to facilitate future improvement and exploitation of rubber tree

Cloning and characterization of a new polyol transporter (HbPLT2) in Hevea brasiliensis

Quebrachitol is a cyclic polyol and, along with sucrose, is one of the main sugars in Hevea latex. However, in contrast to sucrose, the mechanism and regulation of quebrachitol absorption is still unknown. Screening a latex-derived cDNA library using polyol transporter-specific probes, two full-length cDNAs were isolated, and named HbPLT1 and HbPLT2 (for Hevea brasiliensis polyol transporter 1 and 2, respectively). Their respective sequences exhibited close similarity with the previously cloned acyclic sugar polyol transporters, and shared the main features of the major facilitative superfamily. The functional activity of one of the cDNAs was determined by using an HbPLT2-complemented yeast strain. These strains displayed a marginal absorption of cyclic (inositol) and acyclic (mannitol and sorbitol) polyol but no absorption of sucrose, hexose and glycerol. Active absorption for xylitol was detected, and was competitively inhibited by quebrachitol. HbPLT1 and HbPLT2 expression patterns varied in response to different stimuli. Bark treatment with ethylene resulted in an early and significant up-regulation of HbPLT2 transcripts in laticifers as well as in inner bark cells, when compared with HbPLT1. Other treatments, especially mechanical wounding, strongly induced HbPLT2 transcripts. These data were consistent with the presence of ethylene and a wound-responsive regulatory cis-element on the sequence of the HbPLT2 promoter. All these findings together with those recently obtained for sucrose transporters and aquaporins are discussed in relation to the different roles for quebrachitol in Hevea brasiliensi

Ethylene stimulation of latex yield depends on the expression of a sucrose transporter (HbSUT1B) in rubber tree (Hevea brasiliensis)

International audienceHevea brasiliensis is an important industrial crop for natural rubber production. Latex biosynthesis occurs in the cytoplasm of highly specialized latex cells and requires sucrose as the unique precursor. Ethylene stimulation of latex production results in high sugar flow from the surrounding cells of inner bark towards the latex cells. The aim of this work was to understand the role of seven sucrose transporters (HbSUTs) and one hexose transporter (HbHXT1) in this process. Two Hevea clones were used: PB217 and PB260, respectively described as high and low yielding clones. The expression pattern of these sugar transporters (HbSUTs and HbHXT1) was monitored under different physiological conditions and found to be maximal in latex cells. HbSUT1, one of the most abundant isoforms, displayed the greatest response to ethylene treatment. In clone PB217, ethylene treatment led to a higher accumulation of HbSUT1B in latex cells than in the inner bark tissues. Conversely, stronger expression of HbSUT1B was observed in inner bark tissues than in latex cells of PB260. A positive correlation with HbSUT1B transcript accumulation and increased latex production was further supported by its lower expression in latex cells of the virgin clone PB217.</p