BACKGROUND
rubber tree  müll.
arg.
), cultivated in many countries of tropical area, is the primary commercial source of natural rubber.
natural rubber  produced from rubber tree has many outstanding performance properties and is an important industrial material which cannot be reproduced by synthetic alternatives  <cit> .
natural rubber is synthesized through the mevalonic acid  pathway with isopentenyl pyrophosphate  as the substrate and building block .
but there were evidences that the possibility that 1-dexoxy-d-xylulose 5-phosphate/2-c-methyl-d-erythritol 4-phosphate  pathway is involved in rubber biosynthesis cannot be excluded .
whatever the isoprenoid biosynthesis pathway, sucrose is the only precursor of natural rubber  <cit> .
natural rubber biosynthesis takes place in latex and the latex is the cytoplasm of laticifer cells .
the latex harvesting is conducted by tapping or cutting the bark of rubber tree at regular intervals ranging from  <dig> to 5 days.
since ethephon  was known to enhance rubber production, stimulation with ethephon has become a common practice in worldwide rubber tree plantations  <cit> .
usually, a  <dig> - to 2-fold increase of latex yield can be achieved by the treatment with ethephon  <cit> .
much effort has been devoted to the elucidation of the mechanism of ethylene action.
stimulation with ethephon was shown to prolong the flow of latex after tapping  <cit> .
two ethylene-responsive aquaporin genes  had been characterized and their functions possibly favor the prolongation of latex flow through regulation of water exchanges between inner liber and latex cells  <cit> .
physiological and biochemical studies showed that ethylene activated the general metabolism for latex regeneration between tappings, with adenylic pool, polysomes and rrna contents accumulated in laticifers, and the activities of glutamine synthetase  and chitinase up-regulated  <cit> .
specifically, tupy  showed that bark application of auxins and ethylene enhanced the invertase activity and sucrose utilization in latex by increase of latex ph.
cytosolic alkalinization might be explained by the early activation of h+-translocating atpase by ethylene  <cit> .
in view of the fact that the laticifer is a strong sink for sucrose and sucrose importation into laticfer and quebrachitol absorption may be important for sustained sucrose demands for latex regeneration, dusotoit-coucaud et al.
and tang et al.
<cit>  characterized several sucrose transporters and a polyol transporter, and suggested that they might be involved in ethylene-induced stimulation of latex production.
liu et al.
<cit>  constructed and screened two ethephon-induced latex ssh cdna libraries, and found that the cdnas associated with sucrose metabolism, regulation of coagulation, stability of lutoids and signal transduction were up-regulated and might be related to the ethephon action.
due to the complexity of ethylene stimulation and the limitations of the methodologies, many researches have been undertaken on understanding the mechanism of ethylene action but it has still not resolved  <cit> .
in contrast with the conventional methods such as single gene cloning and dna microarrays which yield a limited amount of genetic information, rna sequencing is powerful tool for analyzing differential gene expression with high resolution on the whole genome level  <cit> .
particularly, transcriptome analysis can be employed to reveal relationships between plant gene expression and phenotypes .
transcriptome sequencing technology has been applied to investigate the biology of rubber tree for generation of tissue-specific transcriptomal data  <cit>  and genome sequence  <cit> , development of molecular markers , identification of novel micrornas  <cit> , specific genes and gene families .
in the present study, the first rna sequencing project for deciphering the molecular mechanism of ethylene stimulation of latex yield in rubber tree was performed.
our objective was to identify the relevant metabolic pathways or major ethylene-responsive genes.
it should be noted that, we treated the rubber tree bark with ethephon for  <dig> and 24 h, considering that it should be an effect of relatively long duration and the response to the stimulation should at last be transmitted to certain metabolic pathways.
RESULTS
sequencing and de novo assembly
three cdna libraries from bark tissue, c , e <dig>  and e <dig> , were sequenced by illumina deep-sequencing and a total of approximately  <dig>   <dig> and  <dig> million raw reads for c, e <dig> and e <dig> were generated, respectively.
after removal of low-quality reads, adaptor sequences and ambiguous reads, about  <dig>   <dig> and  <dig> million high-quality clean reads for c, e <dig> and e <dig> were obtained, respectively, with the q <dig>  over 98 % for the three samples .
assembly of all trimmed reads produced  <dig> - <dig>  bp contigs from the three libraries with the average length exceeding 350 bp.
the contigs were joined into unigenes based on the paired-end information, generating  <dig>  ,  <dig>   and  <dig>   unigenes, with an average length of  <dig>  bp and n <dig> of  <dig>  bp  .
all unigenes were longer than 200 bp, the length of  <dig>   unigenes ranged from  <dig> to 400 bp, and unigenes with length longer than 2000 bp accounted for  <dig>  %  of total unigenes.
the length distribution of the unigenes is shown in fig.
<dig> table  <dig> overview of the sequencing and assembly
fig.
<dig> the length distribution of the unigenes.
the length distribution of the unigenes of c sample , e <dig> sample , e <dig> sample  and the all-unigenes
functional annotation and classification
function annotation of the generated unigenes was performed by searching the reference sequences using blastx against nt , ncbi’s non-redundant protein databases , swissprot, kyoto encyclopedia of genes and genomes  and cog .
a total of  <dig>  significant blast hits  were returned.
among them,  <dig>  ,  <dig>  ,  <dig>  ,  <dig>  ,  <dig>  ,  <dig>   unigenes were found in nr, nt, swiss-prot, kegg, cog and go database, respectively.
summary of functional annotations of unigenes by aligning to the nr, nt, swiss-prot, cog, go and kegg databases is shown in additional file  <dig>
cog classification revealed that  <dig>  out of all the assembled unigenes were clustered into  <dig> functional categories .
the largest category was “general function prediction only” , followed by “transcription” , “replication, recombination and repair” , “signal transduction mechanisms”  and “posttranslational modification, protein turnover, chaperones” .
“nuclear structure”  was the smallest group and  <dig>  unigenes were annoted as “function unknown”.fig.
<dig> cog classification of the total assembled unigenes
go assignments were used to classify the predicted functions of the unigenes and total of  <dig>  unigenes were classified into three main categories: biological process , cellular components  and molecular function  .
in the “biological process” category, the top six largest categories were “cellular process” , “metabolic process” , “single-organism process” , “response to stimulus” , “biological regulation”  and “regulation of biological process” .
as for the molecular function, unigenes with binding , catalytic activity  formed the largest groups.fig.
<dig> go classification of the total assembled unigenes.
the left y-axis indicates the percentage of genes for each functions; the right-axis indicates the correspondent number of genes
analysis of kegg pathways and differentially expressed genes
kegg pathway-based analysis was conducted to obtain a better understanding of the biological functions of the unigenes.
the results showed that  <dig>  annotated transcripts were mapped to  <dig> kegg pathways.
of the  <dig>  unigenes,  <dig> ,  <dig> ,  <dig>  and  <dig>  were involved in the metabolic pathways, the biosynthesis of secondary metabolites, the plant-pathogen interaction and the plant hormone signal transduction, respectively.
fpkm  method was used to calculate the expression levels of the unigenes to identify differentially expressed genes .
a total of  <dig>  up-regulated unigenes and  <dig>  down-regulated unigenes were identified in e <dig> compared with c, and  <dig>  and  <dig>  unigenes were up-regulated and down-regulated in e <dig> compared with c. degs between c and e- <dig>  and c and e- <dig> are shown in additional file  <dig>  the top  <dig> most up-regulated and down regulated genes between c and e- <dig>  and c and e- <dig> are shown in additional file  <dig>
by performing the kegg pathway enrichment analysis,  <dig> and  <dig> significantly enriched metabolic pathways or signal transduction pathways of degs in e <dig> and e <dig> were identified, respectively .
to examine the molecular basis of the ethylene stimulation of latex yield in rubber tree, we concentrated on glycolysis, ipp biosynthesis pathway, c <dig> carbon fixation , plant hormone signal transduction and tfs in relation to rubber biosynthesis.
degs involved in glycolysis and ipp pathway
glycolysis pathway is shown in fig.
<dig>  in the glycolysis pathway, the genes of pfk , fba , pgm  and pk  were significantly up-regulated in both e <dig> and e <dig> compared to c, and the gene expression of gapdh  was up-regulated in e <dig> compared to c .
it is well known that the pfk reaction is the first irreversible step committed to glycolysis and pk is responsible for producing pyruvate as the end product of the pathway.
both pk and pfk reactions are crucial points in regulating the rate of glycolysis and in other words these steps are rate-determining steps  <cit> .
in the “bottom up” regulatory mode of plant glycolysis, primary and secondary regulation are exerted at the levels of pep and fructose 6-phosphate, respectively  <cit> .
in addition, transgenic study demonstrated that pgm also plays an important role in the control of glycolysis  <cit> .fig.
<dig> differential expression of unigenes involved in glycolysis in e <dig> and e <dig> compared to c samples of hevea brasiliensis.
glycolysis comprises  <dig> step reactions.
in step  <dig>  the enzyme hexokinase  phosphorylates glucose by transferring a phosphate group from atp to glucose forming glucose 6-phosphate.
in step  <dig>  glucose 6-phosphate is converted to its isomer fructose 6-phosphate by phosphoglucoisomerase .
in step  <dig>  the enzyme phosphofructokinase  catalyzes the conversion of fructose 6-phosphate into fructose  <dig> -bisphosphate using another atp to transfer a phosphate group to fructose 6-phosphate.
in step  <dig>  fructose  <dig> -bisphosphate aldolase  splits fructose  <dig> -bisphosphate into dihydroxyacetone phosphate  and glyceraldehyde 3-phosphate .
in step  <dig>  triose-phosphate isomerase  catalyzes the reversible interconversion of dhap and g3p.
in step  <dig>  g3p is converted to  <dig> -bisphosphoglycerate  by glyceraldehyde 3-phosphate dehydrogenase .
in step  <dig>  phosphoglycerate kinase  catalyzes the reversible transfer of the phosphate group from  <dig> -bpg to adp generating 3-phosphoglycerate  and atp.
in step  <dig>  the conversion of 3-pg to 2-phosphoglycerate  is catalyzed by phosphoglycerate mutase .
in step  <dig>  enolase  catalyzes the dehydration of 2-pg to form phosphoenolpyruvate .
in step  <dig>  pyruvate kinase  catalyzes the transfer of the phosphate group from pep to adp, yielding pyruvate and atp.
in an acetyl-coa metabolic pathway, acetyl-coa synthetase   catalyzes the interconversion between acetyl-coa and acetate , aldehyde dehydrogenase  reversibly catalyze the conversion of acetate into acetaldehyde  and alcohol dehydrogenase  facilitates the interconversion between aldehydes and alcohols .
cells with gray border lines in the upper rows represent differentially expressed unigenes in e <dig> compared to c and cells with green border lines in the lower rows represent differentially expressed unigenes in e <dig> compared to c. relative levels of expression are showed by a color gradient from low  to high
glycolysis pathway not only plays a crucial in energy generation for latex generation but also provides carbon building blocks for the biosynthesis of rubber and other organic constituents of latex  <cit> .
particularly, the breakdown of glucose by glycolysis produces the acetyl coenzyme a  as the direct precursor in the mvp pathway, as well as the g3p and pyruvate which are the precursors in dex/mep pathway  <cit> .
up-regulation of the key enzyme genes described above possibly leads to an elevated rate of flux throughout the entire glycolytic pathway.
thus, the ethephon treatment enhances the latex yield through the rapid acceleration of the glycolysis for replenishing the precursors consumed in the biosynthesis of ipp and natural rubber molecules.
moreover, we found that, in the metabolic pathway of acetyl-coa into alcohol, the acs  and aldh  were down-regulated in both e <dig> and e <dig> compared to c .
the down-regulation of the acs and aldh leads to the inhibition of the acetyl-coa metabolic pathway under ethephon treatment, which may favor for the acetyl-coa flowing toward the rubber synthesis.
in contrast, the ipp pathway was not found to be significantly enriched in this study and specifically, 3-hydroxy-3-metylglutaryl coenzyme a reductase , which plays only limiting role in plant isoprenoid biosynthesis of mva pathway  <cit> , was slightly down-regulated .
this implied that carbohydrate utilization by glycolysis and availability of the precursors for ipp biosynthesis may critically determine the rate of rubber production, and the ipp pathway accounts little for the effect of ethephon stimulation.
although biosynthesis of natural rubber begins with the ipp pathway but the result that ethylene has little direct effect on accelerating rubber biosynthesis has been reported  <cit> .
interestingly, in one report about the mechanisms underlying super productivity of rubber tree, many rubber-biosynthesis-pathway genes showed no differential expressions between the super-high-yielding clone and the control  <cit> .
degs involved in carbon fixation
the c <dig> carbon fixation  is demonstrated in fig.
<dig>  the expressions of rubisco, gadph, fba, aldolase and sbp were shown to be up-regulated in response to ethylene in both e <dig> and e <dig> samples .
in the calvin cycle, extensive studies demonstrated that rubisco  <cit> , sbp  and aldolase  <cit>  dominate control of photosynthetic carbon fixation and they represent the rate-limiting enzymes in the calvin cycle .
the up-regaulation of these enzymes in governing substrate flux through the calvin cycle in response to the application of ethephon speeds up carbon fixation and further enhances the sustainable rubber productivity.fig.
<dig> differential expression of unigenes involved in carbon fixation in e <dig> and e <dig> compared to c samples of hevea brasiliensis.
the c <dig> carbon fixation proceeds through  <dig> steps in three phases.
in carboxylation phase, ribulose- <dig> -bisphosphate carboxylase/oxygenase  adds carbon dioxide to ribulose- <dig> -bisphosphate  to generate 3-pg .
reduction phase include two reactions: pgk catalyses the phosphorylation of 3-pg by atp to form  <dig> -bpg  and adp ; gadph catalyses the reduction of  <dig> -bpg by nadph to produce g3p .
the regeneration phase of the cycle comprises  <dig> steps and is initiated by the enzyme tpi which catalyses the interconversion of g3p and dhap .
then fructose- <dig> -bisphosphate aldolase  reversibly catalyses the aldol condensation of g3p and dhap into fructose- <dig> -bisphosphate .
the resulting fructose- <dig> -bisphosphatase is converted into fructose 6-phosphate by fructose- <dig> -bisphosphatase  .
transketolase  catalyzes the transfer of a 2-carbon fragment from fructose 6-phosphate to g3p, affording erythrose-4-phosphate and xylulose-5-phosphate .
erythrose-4-phosphate and a dhap are converted into sedoheptulose- <dig> -bisphosphate by aldolase .
the cleavage of sedoheptulose- <dig> -bisphosphate into sedoheptulose-7-phosphate and an inorganic phosphate ion is catalyzed by sedoheptulose- <dig> -bisphosphatase  .
the reversible interconversion of sedoheptulose-7-phosphate and g3p into ribose 5-phosphate and xylulose 5-phosphate is catalyzed by tk .
ribose-5-phosphate isomerase  interconverts ribose-5-phosphate and ribulose-5-phosphate .
finally, phosphoribulokinase  phosphorylates ribulose-5-phosphate into rubp .
cells with gray border lines in the upper rows represent differentially expressed unigenes in e <dig> compared to c and cells with green border lines in the lower rows represent differentially expressed unigenes in e <dig> compared to c. relative levels of expression are showed by a color gradient from low  to high
in plants, carbon-containing compounds including sucrose, ipp and natural rubber, ultimately comes from photosynthesis or from stored photosynthetic products.
wititsuwannakul  <cit>  found that the hmgr activity in latex of rubber tree showed a diurnal variation pattern, and suggested that the regulation of the light-dark phase was probably due to physiological processes associated with photosynthesis and one or more of the three components of the rubber biosynthesis  become a limiting factor causing the decline in hmgr activity during the dark period.
therefore, to a certain extent, the photosynthetic carbon fixation may play an important role in ethylene-induced stimulation of latex production by constantly supplying carbohydrate for glycolytic transformation.
degs of hormone signaling components and transcription factors
the responses of plant metabolism, growth, development and defense to exogenous ethylene application can hardly do without the ethylene perception and signal transduction  <cit> .
generally, hormone responses are the output of multiple pathway interaction and crosstalk .
ethylene signaling may exert its functions via interaction with other major plant hormones such as brassinosteroid , gibberellin ,auxin, cytokinin, salicylic acid , jasmonate , abscisic acid .
the transcripts of signaling components such as ethylene response , ethylene response factor  <dig> , ethylene insensitive  <dig>  and ein <dig> binding f-box protein 1/ <dig>  for ethylene, brassinosteroid-insensitive  <dig> , brassinazole resistant 1/ <dig>  and tch <dig> for br, della for ga, indole acetic acid  for auxin, type-a response regulator  for cytokinin, tga for sa, and myc <dig> for ja were found to be remarkably accumulated after ethephon treatment .
although all these hormones have been reported for being linked to growth regulation in certain manner  <cit> , how the ethylene interacts and coordinates with other hormones in relation to the stimulation of latex production in rubber tree still remains to be elucidated.
transcription factors  play important roles in the control of many of the biological processes in a cell or organism by the regulation of gene expression  <cit> .
in addition, tfs also play crucial roles in the cross-talk between hormone signalling pathways  <cit> .
a total of  <dig> degs of putative tfs were identified in this study  and the top five up-regulated and down-regulated tfs in e <dig> and e <dig> compared to c were listed in additional file  <dig>  among them, the largest gene family was the the v-myb avian myeloblastosis viral oncogene homolog family  , followed by myb related , the basic helix-loop-helix family  , c2h <dig> family , and the ethylene-responsive element binding factor family  .
although their functions relevant to ethylene responses for the production enhancement of rubber remained to be unknown but the data will be a valuable resource for the discovery of candidate genes related to the complex regulatory networks involved in the response.
the results obtained by qrt-pcr analysis matched the expression found by transcriptome analysis .
the consistent expression patterns further increased the confidence of our findings and the transcriptome dataset reported here which will be a valuable supplement to the rubber tree genomic and transcriptome information.fig.
<dig> qrt-pcr validation of three degs involved in glycolysis and carbon fixation of hevea brasiliensis bark.
the axis indicates treatments; the y-axis indicates relative expression level.
a
pfk; b
rubisco; c
gadph
differential gene expression, especially the up-regulated expression of the key genes in the rubber-biosynthesis-related metabolic pathways in response to ethylene allowed us to find the rubber-yield limiting factors.
based on our finding, it is possibly to develop a molecular diagnostic for rubber yield and to improve the rubber tree for high yield through the genetic modification of those key genes  <cit> .
CONCLUSIONS
in the present study, the molecular mechanism behind the ethylene stimulation of rubber yield was revealed.
up-regulation of the key enzymes in the glycolysis pathway and the c <dig> carbon fixation may be responsible for enhancing the rubber production by ethephon treatment.
identification of the rubber-yield limiting factors possibly leads to development of a molecular approach to assay and predict the productivity of rubber tree, and obtainment of new high yielding cultivars through genetic engineering.
moreover, our transcriptome data provides an useful resources for gene mining for high production of rubber.

 rubber tree müll. arg.), cultivated in many countries of tropical area, is the primary commercial source of natural rubber. natural rubber produced from rubber tree has many outstanding performance properties and is an important industrial material which cannot be reproduced by synthetic alternatives. natural rubber is synthesized through the mevalonic acid pathway with isopentenyl pyrophosphate as the substrate and building block. but there were evidences that the possibility that 1-dexoxy-d-xylulose 5-phosphate/2-c-methyl-d-erythritol 4-phosphate pathway is involved in rubber biosynthesis cannot be excluded. whatever the isoprenoid biosynthesis pathway, sucrose is the only precursor of natural rubber. natural rubber biosynthesis takes place in latex and the latex is the cytoplasm of laticifer cells. the latex harvesting is conducted by tapping or cutting the bark of rubber tree at regular intervals ranging from to 5 days. since ethephon was known to enhance rubber production, stimulation with ethephon has become a common practice in worldwide rubber tree plantations. usually, a - to 2-fold increase of latex yield can be achieved by the treatment with ethephon. much effort has been devoted to the elucidation of the mechanism of ethylene action. stimulation with ethephon was shown to prolong the flow of latex after tapping. two ethylene-responsive aquaporin genes had been characterized and their functions possibly favor the prolongation of latex flow through regulation of water exchanges between inner liber and latex cells. physiological and biochemical studies showed that ethylene activated the general metabolism for latex regeneration between tappings, with adenylic pool, polysomes and rrna contents accumulated in laticifers, and the activities of glutamine synthetase and chitinase up-regulated. specifically, tupy showed that bark application of auxins and ethylene enhanced the invertase activity and sucrose utilization in latex by increase of latex ph. cytosolic alkalinization might be explained by the early activation of h+-translocating atpase by ethylene. in view of the fact that the laticifer is a strong sink for sucrose and sucrose importation into laticfer and quebrachitol absorption may be important for sustained sucrose demands for latex regeneration, dusotoit-coucaud et al. and tang et al. characterized several sucrose transporters and a polyol transporter, and suggested that they might be involved in ethylene-induced stimulation of latex production. liu et al. constructed and screened two ethephon-induced latex ssh cdna libraries, and found that the cdnas associated with sucrose metabolism, regulation of coagulation, stability of lutoids and signal transduction were up-regulated and might be related to the ethephon action. due to the complexity of ethylene stimulation and the limitations of the methodologies, many researches have been undertaken on understanding the mechanism of ethylene action but it has still not resolved. in contrast with the conventional methods such as single gene cloning and dna microarrays which yield a limited amount of genetic information, rna sequencing is powerful tool for analyzing differential gene expression with high resolution on the whole genome level. particularly, transcriptome analysis can be employed to reveal relationships between plant gene expression and phenotypes. transcriptome sequencing technology has been applied to investigate the biology of rubber tree for generation of tissue-specific transcriptomal data and genome sequence, development of molecular markers, identification of novel micrornas, specific genes and gene families. in the present study, the first rna sequencing project for deciphering the molecular mechanism of ethylene stimulation of latex yield in rubber tree was performed. our objective was to identify the relevant metabolic pathways or major ethylene-responsive genes. it should be noted that, we treated the rubber tree bark with ethephon for and 24 h, considering that it should be an effect of relatively long duration and the response to the stimulation should at last be transmitted to certain metabolic pathways.  sequencing and de novo assembly three cdna libraries from bark tissue, c, e and e, were sequenced by illumina deep-sequencing and a total of approximately and million raw reads for c, e and e were generated, respectively. after removal of low-quality reads, adaptor sequences and ambiguous reads, about and million high-quality clean reads for c, e and e were obtained, respectively, with the q over 98 % for the three samples. assembly of all trimmed reads produced -  bp contigs from the three libraries with the average length exceeding 350 bp. the contigs were joined into unigenes based on the paired-end information, generating, and unigenes, with an average length of  bp and n of  bp. all unigenes were longer than 200 bp, the length of unigenes ranged from to 400 bp, and unigenes with length longer than 2000 bp accounted for  % of total unigenes. the length distribution of the unigenes is shown in fig.  table overview of the sequencing and assembly fig. the length distribution of the unigenes. the length distribution of the unigenes of c sample, e sample, e sample and the all-unigenes functional annotation and classification function annotation of the generated unigenes was performed by searching the reference sequences using blastx against nt, ncbi’s non-redundant protein databases, swissprot, kyoto encyclopedia of genes and genomes and cog. a total of significant blast hits were returned. among them,,,,,, unigenes were found in nr, nt, swiss-prot, kegg, cog and go database, respectively. summary of functional annotations of unigenes by aligning to the nr, nt, swiss-prot, cog, go and kegg databases is shown in additional file cog classification revealed that out of all the assembled unigenes were clustered into functional categories. the largest category was “general function prediction only”, followed by “transcription”, “replication, recombination and repair”, “signal transduction mechanisms” and “posttranslational modification, protein turnover, chaperones”. “nuclear structure” was the smallest group and unigenes were annoted as “function unknown”.fig. cog classification of the total assembled unigenes go assignments were used to classify the predicted functions of the unigenes and total of unigenes were classified into three main categories: biological process, cellular components and molecular function. in the “biological process” category, the top six largest categories were “cellular process”, “metabolic process”, “single-organism process”, “response to stimulus”, “biological regulation” and “regulation of biological process”. as for the molecular function, unigenes with binding, catalytic activity formed the largest groups.fig. go classification of the total assembled unigenes. the left y-axis indicates the percentage of genes for each functions; the right-axis indicates the correspondent number of genes analysis of kegg pathways and differentially expressed genes kegg pathway-based analysis was conducted to obtain a better understanding of the biological functions of the unigenes. the results showed that annotated transcripts were mapped to kegg pathways. of the unigenes,,, and were involved in the metabolic pathways, the biosynthesis of secondary metabolites, the plant-pathogen interaction and the plant hormone signal transduction, respectively. fpkm method was used to calculate the expression levels of the unigenes to identify differentially expressed genes. a total of up-regulated unigenes and down-regulated unigenes were identified in e compared with c, and and unigenes were up-regulated and down-regulated in e compared with c. degs between c and e- and c and e- are shown in additional file the top most up-regulated and down regulated genes between c and e- and c and e- are shown in additional file by performing the kegg pathway enrichment analysis, and significantly enriched metabolic pathways or signal transduction pathways of degs in e and e were identified, respectively. to examine the molecular basis of the ethylene stimulation of latex yield in rubber tree, we concentrated on glycolysis, ipp biosynthesis pathway, c carbon fixation, plant hormone signal transduction and tfs in relation to rubber biosynthesis. degs involved in glycolysis and ipp pathway glycolysis pathway is shown in fig.  in the glycolysis pathway, the genes of pfk, fba, pgm and pk were significantly up-regulated in both e and e compared to c, and the gene expression of gapdh was up-regulated in e compared to c. it is well known that the pfk reaction is the first irreversible step committed to glycolysis and pk is responsible for producing pyruvate as the end product of the pathway. both pk and pfk reactions are crucial points in regulating the rate of glycolysis and in other words these steps are rate-determining steps. in the “bottom up” regulatory mode of plant glycolysis, primary and secondary regulation are exerted at the levels of pep and fructose 6-phosphate, respectively. in addition, transgenic study demonstrated that pgm also plays an important role in the control of glycolysis.fig. differential expression of unigenes involved in glycolysis in e and e compared to c samples of hevea brasiliensis. glycolysis comprises step reactions. in step the enzyme hexokinase phosphorylates glucose by transferring a phosphate group from atp to glucose forming glucose 6-phosphate. in step glucose 6-phosphate is converted to its isomer fructose 6-phosphate by phosphoglucoisomerase. in step the enzyme phosphofructokinase catalyzes the conversion of fructose 6-phosphate into fructose -bisphosphate using another atp to transfer a phosphate group to fructose 6-phosphate. in step fructose -bisphosphate aldolase splits fructose -bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. in step triose-phosphate isomerase catalyzes the reversible interconversion of dhap and g3p. in step g3p is converted to -bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase. in step phosphoglycerate kinase catalyzes the reversible transfer of the phosphate group from -bpg to adp generating 3-phosphoglycerate and atp. in step the conversion of 3-pg to 2-phosphoglycerate is catalyzed by phosphoglycerate mutase. in step enolase catalyzes the dehydration of 2-pg to form phosphoenolpyruvate. in step pyruvate kinase catalyzes the transfer of the phosphate group from pep to adp, yielding pyruvate and atp. in an acetyl-coa metabolic pathway, acetyl-coa synthetase catalyzes the interconversion between acetyl-coa and acetate, aldehyde dehydrogenase reversibly catalyze the conversion of acetate into acetaldehyde and alcohol dehydrogenase facilitates the interconversion between aldehydes and alcohols. cells with gray border lines in the upper rows represent differentially expressed unigenes in e compared to c and cells with green border lines in the lower rows represent differentially expressed unigenes in e compared to c. relative levels of expression are showed by a color gradient from low to high glycolysis pathway not only plays a crucial in energy generation for latex generation but also provides carbon building blocks for the biosynthesis of rubber and other organic constituents of latex. particularly, the breakdown of glucose by glycolysis produces the acetyl coenzyme a as the direct precursor in the mvp pathway, as well as the g3p and pyruvate which are the precursors in dex/mep pathway. up-regulation of the key enzyme genes described above possibly leads to an elevated rate of flux throughout the entire glycolytic pathway. thus, the ethephon treatment enhances the latex yield through the rapid acceleration of the glycolysis for replenishing the precursors consumed in the biosynthesis of ipp and natural rubber molecules. moreover, we found that, in the metabolic pathway of acetyl-coa into alcohol, the acs and aldh were down-regulated in both e and e compared to c. the down-regulation of the acs and aldh leads to the inhibition of the acetyl-coa metabolic pathway under ethephon treatment, which may favor for the acetyl-coa flowing toward the rubber synthesis. in contrast, the ipp pathway was not found to be significantly enriched in this study and specifically, 3-hydroxy-3-metylglutaryl coenzyme a reductase, which plays only limiting role in plant isoprenoid biosynthesis of mva pathway, was slightly down-regulated. this implied that carbohydrate utilization by glycolysis and availability of the precursors for ipp biosynthesis may critically determine the rate of rubber production, and the ipp pathway accounts little for the effect of ethephon stimulation. although biosynthesis of natural rubber begins with the ipp pathway but the result that ethylene has little direct effect on accelerating rubber biosynthesis has been reported. interestingly, in one report about the mechanisms underlying super productivity of rubber tree, many rubber-biosynthesis-pathway genes showed no differential expressions between the super-high-yielding clone and the control. degs involved in carbon fixation the c carbon fixation is demonstrated in fig.  the expressions of rubisco, gadph, fba, aldolase and sbp were shown to be up-regulated in response to ethylene in both e and e samples. in the calvin cycle, extensive studies demonstrated that rubisco, sbp and aldolase dominate control of photosynthetic carbon fixation and they represent the rate-limiting enzymes in the calvin cycle. the up-regaulation of these enzymes in governing substrate flux through the calvin cycle in response to the application of ethephon speeds up carbon fixation and further enhances the sustainable rubber productivity.fig. differential expression of unigenes involved in carbon fixation in e and e compared to c samples of hevea brasiliensis. the c carbon fixation proceeds through steps in three phases. in carboxylation phase, ribulose- -bisphosphate carboxylase/oxygenase adds carbon dioxide to ribulose- -bisphosphate to generate 3-pg. reduction phase include two reactions: pgk catalyses the phosphorylation of 3-pg by atp to form -bpg and adp; gadph catalyses the reduction of -bpg by nadph to produce g3p. the regeneration phase of the cycle comprises steps and is initiated by the enzyme tpi which catalyses the interconversion of g3p and dhap. then fructose- -bisphosphate aldolase reversibly catalyses the aldol condensation of g3p and dhap into fructose- -bisphosphate. the resulting fructose- -bisphosphatase is converted into fructose 6-phosphate by fructose- -bisphosphatase. transketolase catalyzes the transfer of a 2-carbon fragment from fructose 6-phosphate to g3p, affording erythrose-4-phosphate and xylulose-5-phosphate. erythrose-4-phosphate and a dhap are converted into sedoheptulose- -bisphosphate by aldolase. the cleavage of sedoheptulose- -bisphosphate into sedoheptulose-7-phosphate and an inorganic phosphate ion is catalyzed by sedoheptulose- -bisphosphatase. the reversible interconversion of sedoheptulose-7-phosphate and g3p into ribose 5-phosphate and xylulose 5-phosphate is catalyzed by tk. ribose-5-phosphate isomerase interconverts ribose-5-phosphate and ribulose-5-phosphate. finally, phosphoribulokinase phosphorylates ribulose-5-phosphate into rubp. cells with gray border lines in the upper rows represent differentially expressed unigenes in e compared to c and cells with green border lines in the lower rows represent differentially expressed unigenes in e compared to c. relative levels of expression are showed by a color gradient from low to high in plants, carbon-containing compounds including sucrose, ipp and natural rubber, ultimately comes from photosynthesis or from stored photosynthetic products. wititsuwannakul found that the hmgr activity in latex of rubber tree showed a diurnal variation pattern, and suggested that the regulation of the light-dark phase was probably due to physiological processes associated with photosynthesis and one or more of the three components of the rubber biosynthesis become a limiting factor causing the decline in hmgr activity during the dark period. therefore, to a certain extent, the photosynthetic carbon fixation may play an important role in ethylene-induced stimulation of latex production by constantly supplying carbohydrate for glycolytic transformation. degs of hormone signaling components and transcription factors the responses of plant metabolism, growth, development and defense to exogenous ethylene application can hardly do without the ethylene perception and signal transduction. generally, hormone responses are the output of multiple pathway interaction and crosstalk. ethylene signaling may exert its functions via interaction with other major plant hormones such as brassinosteroid, gibberellin,auxin, cytokinin, salicylic acid, jasmonate, abscisic acid. the transcripts of signaling components such as ethylene response, ethylene response factor, ethylene insensitive and ein binding f-box protein 1/ for ethylene, brassinosteroid-insensitive, brassinazole resistant 1/ and tch for br, della for ga, indole acetic acid for auxin, type-a response regulator for cytokinin, tga for sa, and myc for ja were found to be remarkably accumulated after ethephon treatment. although all these hormones have been reported for being linked to growth regulation in certain manner, how the ethylene interacts and coordinates with other hormones in relation to the stimulation of latex production in rubber tree still remains to be elucidated. transcription factors play important roles in the control of many of the biological processes in a cell or organism by the regulation of gene expression. in addition, tfs also play crucial roles in the cross-talk between hormone signalling pathways. a total of degs of putative tfs were identified in this study and the top five up-regulated and down-regulated tfs in e and e compared to c were listed in additional file among them, the largest gene family was the the v-myb avian myeloblastosis viral oncogene homolog family, followed by myb related, the basic helix-loop-helix family, c2h family, and the ethylene-responsive element binding factor family. although their functions relevant to ethylene responses for the production enhancement of rubber remained to be unknown but the data will be a valuable resource for the discovery of candidate genes related to the complex regulatory networks involved in the response. the results obtained by qrt-pcr analysis matched the expression found by transcriptome analysis. the consistent expression patterns further increased the confidence of our findings and the transcriptome dataset reported here which will be a valuable supplement to the rubber tree genomic and transcriptome information.fig. qrt-pcr validation of three degs involved in glycolysis and carbon fixation of hevea brasiliensis bark. the axis indicates treatments; the y-axis indicates relative expression level. a pfk; b rubisco; c gadph differential gene expression, especially the up-regulated expression of the key genes in the rubber-biosynthesis-related metabolic pathways in response to ethylene allowed us to find the rubber-yield limiting factors. based on our finding, it is possibly to develop a molecular diagnostic for rubber yield and to improve the rubber tree for high yield through the genetic modification of those key genes.  in the present study, the molecular mechanism behind the ethylene stimulation of rubber yield was revealed. up-regulation of the key enzymes in the glycolysis pathway and the c carbon fixation may be responsible for enhancing the rubber production by ethephon treatment. identification of the rubber-yield limiting factors possibly leads to development of a molecular approach to assay and predict the productivity of rubber tree, and obtainment of new high yielding cultivars through genetic engineering. moreover, our transcriptome data provides an useful resources for gene mining for high production of rubber. 
treatment of the bark of rubber trees with ehephon has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. the expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and degs were not significantly enriched in isopentenyl diphosphate biosynthesis pathway. the rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of ipp and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. de novo sequencing and assembly of the bark transciptomes of hevea brasiliensis induced with ethephon for 8 h and 24 h were performed. rubber treehevea brasiliensisethephon treatmentethylene stimulationrubber productionhainan province major science and technology projectzdzx2013023liu jin-ping western plan and subject key areas construction project of hainan universityzxbjh-xk001liu jin-ping issue-copyright-statement© the author 2016 rubber tree is an important industrial crop cultivated in tropical areas for natural rubber production. the elevated rate of flux throughout the calvin cycle may account for some durability of ethylene-induced stimulation. in addition, up-regulated genes of great regulatory importance in carbon fixation were identified. deciphering the enigma is of great importance for improvement of rubber tree for high yield. our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.,, and, high-quality clean reads from e e and c samples were assembled into, and unigenes respectively, with a total of unigenes and an average length of  bp generated.
 rubber tree is an important industrial crop cultivated in tropical areas for natural rubber production. treatment of the bark of rubber trees with ehephon has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. deciphering the enigma is of great importance for improvement of rubber tree for high yield.  de novo sequencing and assembly of the bark transciptomes of hevea brasiliensis induced with ethephon for 8 h and 24 h were performed.,, and, high-quality clean reads from e e and c samples were assembled into, and unigenes respectively, with a total of unigenes and an average length of  bp generated. and differentially expressed genes in e and e compared with c were respectively detected. the expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and degs were not significantly enriched in isopentenyl diphosphate biosynthesis pathway. in addition, up-regulated genes of great regulatory importance in carbon fixation were identified.  the rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of ipp and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. the elevated rate of flux throughout the calvin cycle may account for some durability of ethylene-induced stimulation. our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rubber treehevea brasiliensisethephon treatmentethylene stimulationrubber productionhainan province major science and technology projectzdzx2013023liu jin-ping western plan and subject key areas construction project of hainan universityzxbjh-xk001liu jin-ping issue-copyright-statement© the author 2016 