BACKGROUND
aquaporins , also known as major intrinsic proteins , are a class of integral membrane proteins that facilitate the passive transport of water and other small solutes across biological membranes  <cit> .
since their first discovery in 1990s, aqps have been found in almost all living organisms  <cit> .
compared with animals and microbes, aqps are particularly abundant and diverse in land plants and more than  <dig> homologs have been identified from arabidopsis thaliana, zea mays, oryza sativa, vitis vinifera, populus trichocarpa, gossypium hirsutum, solanum tuberosum, solanum lycopersicum and glycine max .
according to the sequence similarity, the aqps in model plant arabidopsis are divided into four subfamilies, i.e.
the plasma membrane intrinsic protein  subfamily that contains two subgroups, the tonoplast intrinsic protein  that contains five subgroups, the nod26-like intrinsic protein  that contains seven subgroups and small basic intrinsic protein  that contains two subgroups.
in moss and some dicots including p. trichocarpa, one more subfamily named x intrinsic protein  subfamily that contains three subgroups is also found  <cit> .
aqps assemble in tetramers in the cell membrane, although each monomer can act as a water channel  <cit> .
even though the overall pairwise sequence similarity can be low, aqps share some structural features such as harboring six transmembrane helices  connected by five loops .
lb and le from opposite sides dip into the membrane and form two half helices , at the n-termini of which, two highly conserved npa  motifs form one selectivity region.
and another region that determines the substrate specificity is known as the aromatic/arginine  selectivity filter   <cit> .
the npa motifs create an electrostatic repulsion of protons and act as a size barrier, where the ar/r filter renders the pore constriction site diverse in both size and hydrophobicity  <cit> .
in addition to water-conducting aqps, certain aqps called aquaglyceroporins  transport glycerol instead.
statistical analysis indicated that glps feather five highly conserved amino acid residues : an aromatic residue at p <dig>  an acidic residue at p <dig>  a basic residue at p <dig>  a proline followed by a nonaromatic residue at p <dig> and p <dig>  as y108-d207-k211-p236-l <dig> observed in the escherichia coli glycerol facilitator glpf in contrast to a103-s190-a194-f208-w <dig> in the pure water channel aqpz  <cit> .
very recently, nine specificity-determining positions  for non-aqua substrates, i.e.
urea, boric acid, silicic acid, ammonia , carbon dioxide  and hydrogen peroxide  were also proposed for each group via a comprehensive analysis of functionally characterized aqps  <cit> .
the para rubber tree  is a perennial tropical plant species that belongs to the euphorbiaceae family.
although it is native to the amazon basin, the economic importance and increasing demand of natural rubber has prompted its wide-domestication to southeast asia.
to date, the rubber tree is still the only commercial source of natural rubber for its high production and quality of rubber.
natural rubber  is specifically synthesized in the highly differentiated vessels termed laticifers, which are located in the secondary phloem of the tree trunk and are periodically differentiated from the vascular cambium  <cit> .
the rubber-containing latex which represents the cytoplasmic content of laticifers is harvested by tapping the bark every 2– <dig> days, and the latex yield is determined by the initial flow rate, duration of latex flow and latex regeneration between two tappings  <cit> .
ethylene, a gas phytohormone, plays crucial roles in numerous aspects of growth, development, and response to biotic and abiotic stresses in plants  <cit> .
in arabidopsis, the ethylene signaling pathway has been well established and its involvement in certain agronomically important processes such as seed dormancy, fruit ripening, abscission and senescence has made ethylene a target for manipulation by chemical and biotechnological methodologies  <cit> .
ethephon , an ethylene releaser, was initiatively tested for rubber yield promotion as early as the 1970s and now is widely used in rubber production all over the world.
although the molecular mechanism on ethephon stimulation of latex yield is still unclear, researches showed that the treatment of rubber tree barks with ethephon could significantly decrease latex dry rubber content  or total solid content , extend the bark drainage area, and prolong latex flow duration .
these effects are mainly benefited from water influx toward laticifers and the resultant latex dilution.
since water accounts for 60–70 % of total latex upon each tapping, sufficient water supply is essential for both latex flow and latex regeneration  <cit> .
nevertheless, the mature laticiferous vessel rings are devoid of functional plasmodesmata connections  <cit> , and thus the water inflow into laticifers is mediated largely by aqps.
however, the molecular characterization of rubber tree aqps  is still in its infancy.
as of june  <dig>  only eight full-length hbaqp cdnas have been reported .
among them, the water transport activity of hbpip <dig>  hbpip <dig>  hbpip1; <dig>  hbpip2; <dig> hbpip2; <dig> and hbtip1; <dig> was characterized by tungngoen et al.
and our group using xenopus laevis oocytes: hbtip1; <dig>  hbpip2; <dig> and hbpip2; <dig> were shown to be highly efficient, whereas hbpip1;1; hbpip1; <dig> and hbpip2; <dig> were less efficient .
lately, the rubber tree genome was sequenced by rahman et al.
<cit>  and rubber research institute, chinese academy of tropical agricultural sciences.
in addition, more than  <dig>  ests  and a high number of rna sequencing reads derived from several tissues such as shoot apex, leaf, laticifer, bark, root and somatic embryogenesis are also available in ncbi sra .
these datasets provide a good chance to analyze the rubber tree aqp gene family from a global view.
in the present study, a genome-wide search was carried out to identify aqp genes encoded in the rubber tree genome.
further, functional prediction was performed based on the analysis of the ar/r filter, froger’s positions and spds, and deep transcriptome sequencing and qrt-pcr expression analysis were also adopted to identify the most important aqp members expressed in the laticifer.
RESULTS
identification and classification of rubber tree aquaporin genes
via a comprehensive homology analysis,  <dig> or  <dig> loci putatively encoding aqp-like genes were identified from the rubber tree genome of clone ry7-33- <dig> or rrim <dig>  respectively .
since all aqp-encoding loci identified in the rrim <dig> genome were found in the genome of ry7-33- <dig> and some genes from rrim <dig> are incomplete and/or the sequences have a high number of “n”s, the aqp genes identified from the ry7-33- <dig> genome were selected for further analyses.
after discarding loci encoding partial aqp-like sequences which are truncated and lacking any of the npa motifs,  <dig> full-length aqp genes were retained and the gene models are available in additional file  <dig>
to analyze the evolutionary relationship and their putative function, an unrooted phylogenetic tree was constructed from the deduced amino acid sequences of hbaqps together with that from arabidopsis  and poplar   .
the reasons for choosing these two species are mainly as follows: the complete set of aqp genes in arabidopsis was firstly identified and then well characterized; the well-studied wood plant poplar harbors one more subfamily  that is not found in arabidopsis.
according to the phylogenetic analysis,  <dig> hbaqps were grouped into five subfamilies, i.e.
pip , tip , nip , sip  and xip  .
following the nomenclature of arabidopsis and poplar  <cit> , the hbpip subfamily was further divided into two phylogenetic subgroups , the hbtip subfamily into five subgroups , the hbnip subfamily into seven subgroups , the hbsip subfamily into two subgroups  and the hbxip subfamily into three subgroups  .
although the closest homolog of hbnip2; <dig> and hbnip3; <dig> is not atnip2; <dig> or atnip3; <dig>  their counterparts in poplar were identified and thus the names were assigned.
as shown in fig.
<dig>  many hbaqps were grouped in pairs, i.e.
hbpip1;1/hbpip1; <dig>  hbpip1;3/hbpip1; <dig>  hbpip2;1/hbpip2; <dig>  hbpip2;3/hbpip2; <dig>  hbpip2;5/hbpip2; <dig>  hbpip2;7/hbpip2; <dig>  hbtip1;1/hbtip1; <dig>  hbtip1;3/hbtip1; <dig>  hbtip1;5/hbtip1; <dig>  hbtip1;7/hbtip1; <dig>  hbtip2;1/hbtip2; <dig>  hbtip2;3/hbtip2; <dig>  hbtip3;1/hbtip3; <dig>  hbtip5;1/hbtip5; <dig>  hbnip1;1/hbnip1; <dig>  hbsip1;2/hbsip1; <dig>  hbxip1;3/hbxip1; <dig>  which exhibit sequence identities of  <dig> – <dig>  % and  <dig> – <dig>  % at the nucleotide or amino acid level, respectively .table  <dig> list of  <dig> hbaqp genes identified in this study
hbpip1;1
hbpip1;2
hbpip1;3
hbpip1;4
hbpip1;5
hbpip2; 1
hbpip2;2
hbpip2;3
hbpip2;4
hbpip2;5
hbpip2;6
hbpip2; 7
hbpip2;8
hbpp2;9
hbpip2; 1
hbtip1;1
hbtip1;2
hbtp1;3
hbtip1;4
hbtp1;5
hbtip1;6
hbtip1;7
hbtip1;8
hbtp2;1
hbtip2;2
hbtip2;3
hbtip2;4
hbtip3;1
hbtip3;2
hbtip4;1
hbtip5;1
hbtip5;2
hbnip1;1
hbnp1;2
hbnip2;1
hbnp3;1
hbnip4;1
hbnip4;2
hbnip5;1
hbnip6;1
hbnip7;1
hbxip1;1
hbxip1;2
hbxip1;3
hbxip1;4
hbxip2;1
hbxip3; 1
hbsip1;1
hbsip1;2
hbsp1;3
hbsip2;1
abased on the  <dig> transcriptome data under the ncbi sra accession number of drx000223; b based on the  <dig> transcriptome data of srx <dig> and illumina transcriptome data of srx <dig>  srx <dig>  srx <dig>  srx <dig>  srx <dig>  srx <dig>  srx <dig> and srx278515; c based on the  <dig> transcriptome data of srx <dig> and illumina transcriptome data of srx <dig>  srx <dig>  srx <dig> and srx278514; d based on the  <dig> transcriptome data of srx <dig> and illumina transcriptome data of srx278513; e based on the  <dig> transcriptome data of srx451710; f based on the  <dig> transcriptome data of srx <dig>  read-mapping was carried out using bowtie <dig> with default parameters, and mapped read number of more than one was counted as “yes” representing detected genes
fig.
<dig> phylogenetic analysis of deduced amino acid sequences of the  <dig> hbaqps with arabidopsis and poplar homologs.
amino acid sequences were aligned using clustalx and the phylogenetic tree was constructed using bootstrap maximum likelihood tree  method and mega <dig> software.
the distance scale denotes the number of amino acid substitutions per site.
the name of each subfamily is indicated next to the corresponding group.
species and accession numbers are listed in table  <dig> and additional file 2
homology search showed that  <dig> out of  <dig> hbaqp genes have est hits in genbank , i.e.
hbpip1; <dig>  hbpip1; <dig>  hbpip1; <dig>  hbpip1; <dig>  hbpip2; <dig>  hbpip2; <dig>  hbpip2; <dig>  hbpip2; <dig>  hbpip2; <dig>  hbtip1; <dig> and hbtip1; <dig>  eight full-length hbaqp cdnas, including hbpip1; <dig> , hbpip1; <dig> , hbpip2; <dig> , hbpip2; <dig> , hbpip2; <dig> , hbpip2; <dig> , hbtip1; <dig>  and hbtip1; <dig>  have been reported .
read alignments against rna sequencing data of rubber tree shoot apex, leaf, laticifer, bark, root and somatic embryogenesis  indicated that the expression of  <dig> hbaqp genes was observed in at least one of the examined tissues .
whereas, seven genes coding for two tips , one nip , four xips  might be expressed exclusively in response to a specific stimulus or in a very specific part of the plant and thus are excluded in the available datasets.
in arabidopsis, the orthologs of hbtip1; <dig>  and hbnip7; <dig>  were also shown to be pollen or anther-specific, respectively  <cit> .
besides supported by ests and/or rna sequencing reads, the exon-intron structures of laticifer-expressed hbaqp genes  were also confirmed with cloned cdnas .
analysis of exon-intron structure
the exon-intron structures of the  <dig> hbaqp genes were analyzed based on the gene models.
although the orf  length of each gene is similar , the gene size  is distinct .
the introns of hbaqp genes harbor an average length of 404 bp, with the minimum of 71 bp in hbnip2; <dig> and the maximum of 13000 bp in hbsip2; <dig>  genes in different subfamilies harbor distinct exon-intron structures.
all members of the hbpip subfamily feature three introns .
except for hbtip1; <dig>  hbtip1; <dig>  hbtip1; <dig> and hbtip1; <dig> that contain only one intron, other hbtip genes contain two introns instead.
hbnip genes usually have four introns except for hbnip5; <dig> containing three introns.
most hbsip genes contain two introns except for hbsip1; <dig> without any intron.
subgroups of hbxip genes vary in the number of introns: one intron for subgroup one, two or zero for subgroups two and three, respectively .fig.
<dig> exon-intron structures of the  <dig> hbaqp genes.
shown is a graphic representation of the gene models of all  <dig> hbaqps identified in this study using gsds.
utrs are shown as gray boxes, exons are shown as white boxes and introns are shown as black lines
structural features of hbaqps
sequence analysis showed that the  <dig> deduced hbaqps consist of 227– <dig> amino acids, with a theoretical molecular weight of  <dig> – <dig>  kda and a pi value of  <dig> – <dig> .
homology analysis revealed a high sequence diversity existing within and between the five subfamilies.
the sequence similarities of  <dig> – <dig>  % were found within hbpips,  <dig> – <dig>  % within hbtips,  <dig> – <dig>  % within hbxips,  <dig> – <dig>  % within hbnips, and  <dig> – <dig>  % within hbsips.
hbpips share the highest sequence similarity of  <dig> – <dig>  % with hbtips,  <dig> – <dig>  % with hbxips,  <dig> – <dig>  % with hbnips, and the lowest of  <dig> – <dig>  % with hbsips.
hbtips show  <dig> – <dig>  %,  <dig> – <dig>  % and  <dig> – <dig>  % sequence similarities with hbnips, hbxips and hbsips, respectively.
hbnips share sequence similarities of  <dig> – <dig>  % and  <dig> – <dig>  % with hbxips and hbsips, whereas hbsips share the lowest similarity of  <dig> – <dig>  % with hbxips .
topological analysis showed that all hbaqps were predicted to harbor six transmembrane helical domains , which is consistent with the results from multiple alignments with structure proven aqps .
the subcellular localization of each hbaqp was also predicted .
hbpips with an average pi value of  <dig>  are localized to plasma membranes.
hbtips with an average pi value of  <dig>  are mainly localized to vacuoles , though several members were predicted to be localized to endoplasmic reticulum , chloroplast and cytosol.
hbnips with an average pi value of  <dig>  are mostly localized to plasma membranes, but hbnip2; <dig> and hbnip3; <dig> were predicted to be localized to the membrane of vacuole and chloroplast, respectively.
two members  of the sip subfamily  were predicted to be localized to plasma membranes, whereas hbsip1; <dig> and hbsip2; <dig> are localized to the membrane of vacuole and chloroplast, respectively.
although the xip subfamily harbors only six members , the predicted localizations are diverse, including the vacuole, chloroplast, plasma membrane and cytosol.
to learn more about the putative function of hbaqps, the conserved residues typical of dual npa motifs, the ar/r filter, five froger’s positions and nine sdps were also identified .table  <dig> structural and subcellular localization analysis of the hbaqps
a,b,crepresenting the numbers of transmembrane helices predicted by topcons, tmpred and tmhmm, respectively; dbest possible subcellular localization prediction by the wolf psort.
athe sdps  in rubber tree aquaporins differing from typical sdps determined in this study are highlighted in red
hbpip subfamily
all hbpips were identified to have similar sequence length, however, hbpip2s  can be distinguished from hbpip1s  by harboring relatively shorter n-terminal and longer c-terminal sequences .
the five hbpip1s have sequence similarities of  <dig> – <dig>  %, whereas the similarity percents of ten hbpip2s are  <dig> – <dig>  %.
between hbpip <dig> and hbpip <dig> members, sequence similarities of  <dig> – <dig>  % are observed .
the dual npa motifs, ar/r filter , and four out of five froger’s positions are highly conserved in hbpips .
in contrast, the p <dig> position is more variable with the appearance of an e, q or m residue .
in addition, two phosphorylation sites corresponding to s <dig> and s <dig> in sopip2; <dig>  <cit>  are invariable in hbpip2s, and the former one is even highly conserved in all hbpips, hbtips and hbxips except for the s → t substitution in several members , implying their regulation by phosphorylation.
hbtip subfamily
hbtips consist of 227– <dig> residues.
those belonging to hbtip1s  share  <dig> – <dig>  % sequence similarities, whereas hbtip2s  have sequence similarities of  <dig> – <dig>  % .
members of the hbtip <dig> subgroup exhibit sequence similarities of  <dig> – <dig>  %,  <dig> – <dig>  %,  <dig> – <dig>  % and  <dig> – <dig>  % with subgroups hbtip <dig>  hbtip <dig>  hbtip <dig> and hbtip <dig>  respectively.
hbtip2s share sequence similarities of  <dig> – <dig>  %,  <dig> – <dig>  % and  <dig> – <dig>  % with hbtip3s, hbtip <dig> and hbtip5s, respectively.
hbtip3s share sequence similarities of  <dig> – <dig>  % and  <dig> – <dig>  % with hbtip <dig> and hbtip5s, respectively .
and hbtip <dig> shares  <dig>  % and  <dig>  % sequence similarities with hbtip5; <dig> and hbtip5; <dig>  respectively.
dual npa motifs and p <dig>  p <dig> and p <dig> positions are highly conserved in hbtips .
residue substitution is observed at the p <dig> and p <dig> positions: t is replaced by a in hbtip2; <dig> or i in hbtip5s at the p <dig> position, and s is replaced by a in hbtip3s and hbtip5s .
of the ar/r filter, h at h <dig> and i at h <dig> positions are replaced by n and v in hbtip5s, respectively; a is found to be conserved in hbtip1s, hbtip3s, and hbtip <dig> and g in hbtip2s and hbtip5s at the le <dig> position, respectively; and residues at the le <dig> position are more variable, mainly v, r, s or c .
hbnip subfamily
hbnips consist of 267– <dig> residues .
with the exception of hbnip <dig> and hbnip <dig> subgroups that contain two members, each of the other five subgroups harbors a single member.
hbnip1; <dig> shares the highest sequence similarity of  <dig>  % with hbnip1; <dig>  whereas hbnip4; <dig> shares a similarity of  <dig>  % with hbnip4; <dig>  hbnip1s show sequence similarities of  <dig> – <dig>  %,  <dig> – <dig>  %,  <dig> – <dig>  %,  <dig>  %,  <dig> – <dig>  % and  <dig>  % within the subgroups of hbnip <dig>  hbnip <dig>  hbnip <dig>  hbnip <dig>  hbnip <dig> and hbnip <dig>  respectively.
hbnip <dig> shows  <dig>  %,  <dig> – <dig>  %,  <dig>  %,  <dig>  % and  <dig>  % sequence similarities with hbnip <dig>  hbnip4s, hbnip <dig>  hbnip <dig> and hbnip <dig>  respectively.
the hbnip <dig> shows  <dig> – <dig>  %,  <dig>  %,  <dig>  % and  <dig>  % sequence similarities with hbnip4s, hbnip <dig>  hbnip <dig> and hbnip <dig>  respectively.
hbnip4s show  <dig> – <dig>  %,  <dig> – <dig>  % and  <dig> – <dig>  % sequence similarities with hbnip <dig>  hbnip <dig> and hbnip <dig>  respectively.
hbnip <dig> shows  <dig>  % and  <dig>  % sequence similarities with hbnip <dig> and hbnip <dig>  respectively.
hbnip <dig> shows  <dig>  % sequence similarity with hbnip <dig>  the hbnips have typical dual npa motifs except for hbnip4; <dig>  hbnip5; <dig> and hbnip6; <dig> .
a is replaced by v in the second npa motif of hbnip4; <dig> and by s or v in the first or second npa motif of hbnip5; <dig> and hbnip6; <dig>  compared with other subfamilies, hbnips are highly variable in the ar/r filter and froger’s positions: w/g/a/t at the h <dig> position, f/l/v/i at the h <dig> position, a/g at the le <dig> position, f/l/y at the p <dig> position, s/t at the p <dig> position, f/y at the p <dig> position and l/i at the p <dig> position .
in addition, one cdpk phosphorylation site corresponding to s <dig> in gmnod <dig>  <cit>  was also found in the c-terminus of most hbnips .
hbsip subfamily
there are only four members in the hbsip subfamily.
three hbsip1s consist of  <dig> residues, while hbsip2; <dig> has  <dig> residues .
hbsip2; <dig> shares sequence similarities of  <dig> – <dig>  % with hbsip1s.
within the hbsip <dig> subgroup, hbsip1; <dig> shares the highest sequence similarity of  <dig>  % with hbsip1; <dig>  whereas hbsip1; <dig> shows the lowest similarity of  <dig>  % with hbsip1; <dig> .
the three hbsip1s harbor the same npt/npa motifs, ar/r filter  and froger’s positions , whereas hbsip2; <dig> exhibits npl/npa motifs, s-h-g-s ar/r filter and f-v-a-y-w froger’s positions .
hbxip subfamily
hbxips vary from  <dig> to  <dig> residues in length .
hbxip1s share sequence similarities of  <dig> – <dig>  % and  <dig> – <dig>  % with hbxip2; <dig> and hbxip3; <dig>  respectively, whereas hbxip2; <dig> shows  <dig>  % sequence similarity with hbxip3; <dig>  within the hbxip <dig> subgroup, hbxip1; <dig> shares the highest sequence similarity of  <dig>  % with hbxip1; <dig>  whereas hbxip1; <dig> shows the lowest similarity of  <dig>  % with hbxip1; <dig> .
in hbxip1s, the second npa motif and the le <dig>  p <dig>  p <dig> and p <dig> positions are highly conserved.
spv/spi/npt/npv/npl in the first npa motif, v/i at the h <dig> position, i/v at the h <dig> position, v/p/a at the le <dig> position, m/v at the p <dig> position and f/c at the p <dig> position were observed .
similar to most xips  <cit> , two highly conserved c residues in the lggc motif of lc and the nparc motif of le were also found in hbxips except for hbxip1; <dig>  in which the f residue is located at the corresponding position of le .
transcriptional profiles of hbaqp genes in the laticifer and their response to ethephon stimulation
the rubber tree laticifer is a single-cell-type tissue specifically for natural rubber biosynthesis.
to identify the aqp genes expressed in the laticifer and determine the most important members in the laticifer water balance, the latex representing the laticifer cytoplasm was collected and high-quality total rnas  were isolated from three biological replicates, respectively.
then, rnas were pooled and subjected to illumina rna sequencing.
approximate  <dig>  gigabase pairs of raw data  were generated.
after cleaning and quality checks, about  <dig>  million high-quality clean reads with an average length of 95 nt were retained and assembled into  <dig>  unigenes longer than 200 bp, with an average length of 775 bp and an n <dig> of 1260 bp .
expression profiling showed that  <dig> out of the  <dig> identified hbaqp genes were detected in the laticifer transcriptome, including genes coding for  <dig> pips ,  <dig> tips ,  <dig> nips  and  <dig> sips  .
based on the rpkm value, the total expression level of pip members was  <dig>   <dig>   <dig> folds more than the nip, tip or sip members, respectively, indicating a crucial role of the pip subfamily in the laticifer water balance.
among ten laticifer-expressed pip genes, hbpip2; <dig>  hbpip1; <dig>  hbpip2; <dig>  hbpip1; <dig> and hbpip2; <dig> were considerably more abundant, counting about 418-, 306-, 204-, 30- and 15-folds higher than the well-studied hbpip1; <dig>  whereas, hbsip2; <dig>  the sixth laticifer-abundant aqp gene, expressed relatively more than any other non-pip members .fig.
<dig> expression profiles of  <dig> hbaqp genes detected in the laticifer based on illumina sequencing
given the important role and wide application of ethephon stimulation on rubber yield promotion, the response of the above  <dig> laticifer-expressed hbaqp genes subjected to ethephon treatment was analyzed using qrt-pcr over a time course .
as described before, treating the rubber tree bark with ethephon was shown to induce a huge increase in latex yield, starting as early as 6 h after the treatment, and the yield increase was about  <dig> -,  <dig> -,  <dig> - and  <dig> -folds higher than the control at  <dig>   <dig>   <dig>  and 40 h after the treatment, respectively; the tsc was significantly decreased at the time points of  <dig> and 40 h; and the latex flow duration was significantly prolonged from the time point of 16 h  <cit> .
as shown in fig.
<dig>  except for hbnip6; <dig>  ethephon treatment had significant effects on all other tested hbaqp genes at one or more time points, implying their regulation by ethylene.
at the early stage of ethephon treatment, i.e.
6 h, the transcriptional levels of  <dig> hbaqp genes were significantly affected, including two up-regulated  and eleven down-regulated  genes.
at 16 h post treatment,  <dig> hbaqp genes were significantly regulated, including eight up-regulated  and eight down-regulated  genes.
at 24 h post treatment,  <dig> hbaqp genes were significantly regulated, including thirteen up-regulated  and four down-regulated  genes.
at 40 h post treatment,  <dig> hbaqp genes were significantly regulated, including seven up-regulated  and seven down-regulated  genes.
although the time points of  <dig> and 24 h post treatment harbored similar significantly regulated genes, the later had relatively more genes  that were up-regulated.
although the expression patterns of the regulated genes were diverse, they could be classified into seven groups: the cluster  <dig> that includes hbpip2; <dig> was gradually increased upon ethephon stimulation; the cluster  <dig> including hbpip2; <dig> and hbsip2; <dig> were firstly increased and then decreased, which is like a clock; the cluster  <dig> including hbtip1; <dig> was firstly increased, subsequently decreased and increased at the last time point tested; the cluster  <dig> including hbpip1; <dig> was firstly increased, subsequently decreased, then increased and finally decreased; the cluster  <dig> including hbnip1; <dig> and hbsip1; <dig> were firstly decreased and then increased; the cluster  <dig> that includes  <dig> genes  were firstly decreased, subsequently increased and finally decreased; the cluster  <dig> that includes hbpip1; <dig> and hbnip1; <dig> were firstly decreased, subsequently increased, then decreased and finally increased.
at 24 h post ethephon stimulation, eight genes  exhibited the highest expression levels, whereas the highest expression of six genes  occurred at 40 h. moreover, the transcript abundance of hbpip2; <dig> and hbnip1; <dig> were similar at the time points of 24 h and 40 h .
as describe above, hbpip2; <dig>  hbpip2; <dig> and hbpip1; <dig> were among the top  <dig> highly abundant aqp genes expressed in laticifers .
in addition, another highly abundant aqp genes  was expressed most at 16 h post ethephon stimulation .fig.
<dig> qrt-pcr analysis of  <dig> laticifer-expressed hbaqp genes upon ethephon stimulation.
data are mean ± sd .
different letters mean significant difference over the time course
discussion
high abundance and diversity of hbaqps
a total of  <dig> full-length aqp genes were identified from the rubber tree genome, which is comparable to  <dig> members reported in poplar   <cit> ; more than  <dig> in grapevine  <cit> ,  <dig> in rice  <cit> ,  <dig> in arabidopsis  <cit> ,  <dig> in maize  <cit> ,  <dig> in potato  <cit>  and  <dig> in tomato  <cit> ; less than  <dig> in soybean  <cit>  and  <dig> in cotton  <cit> .
since the aqp genes in arabidopsis and poplar were well characterized, their deduced proteins were added in the phylogenetic analysis of hbaqps, which assigned them to five subfamilies.
with the exception of the xip subfamily, the further classification of hbaqp subfamilies into subgroups is consistent with arabidopsis, i.e.
two pip subgroups, five tip subgroups, seven nip subgroups and two sip subgroups.
nevertheless, classing atnip2; <dig> and atnip3; <dig> into the nip <dig> subgroup was proposed.
as shown in fig.
<dig>  atnip2; <dig> and atnip3; <dig> were clustered with the nip <dig> subgroup, sharing the highest similarity with atnip1; <dig> in arabidopsis, hbnip1; <dig> or hbnip1; <dig> in rubber tree, ptnip1; <dig> or ptnip1; <dig> in poplar, respectively.
thereby, no nip2s and nip3s were retained in arabidopsis as seen in rubber tree and poplar .
since no xip homologs were found in the arabidopsis genome, the nomenclature for poplar proposed by lopez et al.
<cit>  was adopted to divide hbxips into three subgroups.
besides supported by high bootstrap values, xip1s are characterized by the ar/r filter of v-m-v/p/a-r, xip2s by i-i-v-r and xip3s by v-k-a-r.fig.
<dig> distribution of the  <dig> hbaqp genes and their arabidopsis and poplar homologs in subgroups
gene pairs were identified not only in rubber tree, but also in poplar and arabidopsis .
for example, five atpip1s were clustered together apart from pip1s of rubber tree and poplar; hbpip1; <dig> and hbpip1; <dig> were clustered with ptpip1; <dig> and ptpip1; <dig>  these results suggest the occurrence of more than one gene duplication events.
previous studies indicated that poplar underwent one whole-genome triplication event  and one doubling event, whereas arabidopsis underwent the same γ event and two independent doubling events, though the arabidopsis genome encodes relatively less aqp genes due to massive gene loss and chromosomal rearrangement after genome duplications .
the γ duplication occurred at approximate  <dig> million years ago, shortly before the origin of core eudicots  <cit> .
as a core eudicot plant, the rubber tree appears to share the γ duplication.
however, another one as the data suggested is likely to be a doubling event independent from both arabidopsis and poplar, probably occurred after the divergence of euphorbiaceae and salicaceae.
a genome-wide comparative analysis may provide more information.
functional inference of hbaqps
although plant aqps firstly raised considerable interest for their high water permeability, when heterologously expressed in xenopus oocytes or yeast cells, increasing evidence has shown that some of them are also participated in the transport of other small molecules such as glycerol, urea, boric acid, silicic acid, nh <dig>  co <dig> and h2o <dig>  <cit> .
based on atomic resolution structures and molecular dynamics stimulations of glpf, aqpz, aqp <dig> and other mips, several structural features determining their transport selectivity were identified, e.g.
the two opposite npa motifs, the ar/r filter and the amino acid residues at froger’s positions for discriminating between aqps and glps  <cit> .
as shown in table  <dig>  most hbaqps exhibit an aqpz-like froger’s positions to favor the permeability of water.
in contrast, hbsip2; <dig> and nip subfamily members possess mixed key residues of glpf for p <dig> and p <dig>  and aqpz for p2–p <dig>  the glycerol permeability of gmnod <dig> and arabidopsis nips was reported  <cit> , however, the potential glycerol transport ability of hbsip2;1-like sips have not be confirmed by experimental means yet.
in addition to high permeability to water, plant pips were reported to transport urea, boric acid, co <dig> and h2o <dig>  <cit> .
as shown in table  <dig>  all hbpips represent the f-h-t-r ar/r filter as observed in aqpz which harbors an extremely narrow and hydrophilic pore   <cit> , suggesting their high water permeability.
however, when expressed in xenopus, extremely low water permeability of hbpip <dig> members such as hbpip1; <dig> and hbpip1; <dig> was observed  <cit>  as seen in many other plant species  <cit> .
based on the sdp analysis proposed by hove and bhave  <cit> , all hbpips represent urea-type sdps ; hbpip1s represent boric acid-type sdps ; hbpip1; <dig> represents co2-type sdps ; hbpips except for hbpip2; <dig> represent h2o2-type sdps  , supporting their similar functionality.
although highly variable in the ar/r filter, plant tips were shown to transport water as efficiently as pips  <cit> .
additionally, they also allow urea, nh <dig> and h2o <dig> through  <cit> .
as shown in table  <dig>  all hbtips except for hbtip2; <dig> and hbtip4; <dig> represent urea-type sdps , whereas hbtip1; <dig>  hbtip1; <dig>  hbtip4; <dig>  hbtip5; <dig> and hbtip5; <dig> represent h2o2-type sdps , indicating similar functionality.
compared with typical nh3-sdps , hbtip2; <dig> seems to represent novel sdps with the substitution of s for a/r/t at sdp <dig>
besides glycerol and water, plant nips have been found to transport urea, boric acid, silicic acid, nh <dig> and h2o <dig> .
as shown in table  <dig> and additional file  <dig>  hbnip5; <dig> is promised to be a urea and boric acid transporter with nine sdps of h-p-i-a-l-p-g-s-n or t-i-h-p-e-l-l-a-p. hbnip2; <dig> represent typical urea sdps , and sdps of v-v-h-p-e-i-i-a-p with the substitution of v for i at sdp <dig> in comparison to typical boric acid sdps .
compared with typical urea and boric acid sdps, hbnip6; <dig> seems to represent novel sdps-types with the substitution of q for a/p at sdp <dig> or q for a/p/g at sdp <dig>  although characterized as an nip iii member, the silicic acid transport ability of hbnip2; <dig> needs to be experimentally validated since it seems to represent novel sdps  similar to that of gmnip2; <dig> and gmnip2; <dig>   <cit> .
although gmnod <dig> was reported to transport nh <dig>  <cit> , whether its close rubber tree homologs  represent novel sdps-types still needs to be tested.
hbnip3; <dig>  hbnip4; <dig> and hbnip5; <dig> represent h2o2-type sdps  slightly different from atnip1; <dig>   <cit> .
as a recently identified aqp subfamily, plant xips were shown to transport water, glycerol, urea, boric acid and h2o <dig>  <cit> .
according to phylogenetic relationships, xips are split into two independent clusters termed xip-a and xip-b, where xip-a includes only xip <dig> subgroup and xip-b contains at least four subgroups, i.e.
xip <dig>  xip <dig>  xip <dig>  and xip <dig>  <cit> .
consistent with poplar xips , six hbxips can be assigned to subgroups xip <dig> , xip <dig>  and xip <dig>  .
when expressed in xenopus oocytes, ptxip2; <dig> and ptxip3; <dig> transported water while other ptxips did not.
although the mechanism why ptxip1s, ptxip3; <dig> and ptxip3; <dig> do not transport water is still unclear, the close homologs of ptxip1s in nicotiana tabacum and potato were also reported to have undetectable water permeability.
in contrast, solanaceae xips showed high permeability to glycerol  <cit> .
therefore, although exhibiting an aqpz-like froger’s positions, all hbxips maybe transport glycerol.
meanwhile, hbxip2; <dig> and hbxip3; <dig> are probably capable of transporting water.
as shown in table  <dig>  hbxip1; <dig>  hbxip1; <dig>  hbxip2; <dig> and hbxip3; <dig> are promised to be urea transporters with nine sdps of h-p-f/l-a-l-g-g-g-n; hbxip1; <dig>  hbxip1; <dig> and hbxip2; <dig> may represent novel boric acid sdps-types with the substitution of q or t for a/g/k/p at sdp9; hbxip1; <dig> and hbxip1; <dig> harbor h2o2-type sdps  with a slight difference from some solanaceae xips   <cit> .
a crucial role of hbpips in the water balance of laticifers
as a unique site for rubber biosynthesis, the laticifers are present in a wide variety of rubber tree tissues, including shoots, roots, stems, leaves, flowers, fruits, cotyledons, inner seed coats, etc., and can be divided into primary and secondary laticifers according to their origin  <cit> .
compared with the procambium-derivation of primary laticifers, the secondary laticifers, mainly located in the soft inner bark of the rubber tree trunk, are periodically differentiated from the vascular cambium and serve as a sole source for the commercial latex  <cit> .
during the differentiation and maturation process, laticifer mother cells articulate with each other and further anastomose together into a successive vertical network  arranged as concentric sheaths in the secondary phloem  <cit> .
unlike other cells such as neighboring parenchyma cells, the mature laticiferous cells are totally devoid of plasmodesmata  <cit> , and thus its water exchanges with surrounding cells are mainly governed by aqps.
upon bark tapping, the laticifer cytoplasm is expelled in the form of latex due to the high turgor pressure inside  <cit> .
generally, latex flow can continue for several hours until coagulation processes lead to the plugging of severed laticifers  <cit> .
during the latex flow, a progressive decrease in drc was observed , indicating rapid water influx and latex dilution inside laticifers caused by the activity of hbaqps.
given that of hbpips and hbtips account for more than  <dig>  % of the total hbaqps and their aqpz-like froger’s positions favoring the high water permeability, we initially prospect that these two subfamilies may play important roles in the laticifer water balance: the plasma membrane-targeted hbpips facilitate the water transport from the extracellular space to the laticifer cytoplasm, whereas the lutoid-targeted hbtips play an essential role in maintaining the cell osmotic balance as observed in most plant cells  <cit> .
however, in contrast to the mature plant cells characterized by a large central vacuole which occupies 80 % or more of the intracellular space, the lutoids in laticifers are polydispersed microvacuoles occupying only 12 % of the total latex  <cit> , arguing the central role of hbtips in the laticifer water balance, though their potential role in the lutoid stability and latex vessel plugging should be noted.
to address this issue, the transcriptome of such a single-cell-type tissue was deeply sequenced.
results showed that pip members were the main aqp genes expressed in the laticifer , suggesting their crucial role, especially the highly abundant hbpip2; <dig>  hbpip1; <dig> and hbpip2; <dig>  in the laticifer water balance.
when expressed in xenopus oocytes, our previous study showed that hbpip2; <dig> could transport water as efficiently as hbpip2; <dig>  <cit> ; in contrast, hbpip1; <dig> and hbpip2; <dig> were shown to be less efficient  <cit> .
in addition, as a pip <dig> member, the poor efficiency of hbpip1; <dig> was also observed  <cit> .
therefore, the exact role of hbpip2; <dig>  hbpip1; <dig> and hbpip2; <dig> in the water balance of rubber tree laticifers needs further investigations.
to profile the aqp genes in response to ethylene stimulation in laticifers, the latex at different time points after ethephon treatment was collected from rubber tree clone pr <dig>  similar to pb <dig>  pr <dig> clone is characterized as a relatively late mature variety which has a high tsc, short latex flow duration and low latex metabolism, however, ethephon stimulation could significantly prolong its latex flow duration and enhance latex yield  <cit> .
our qrt-pcr analysis showed that the expression levels of most laticifer-expressed genes significantly changed at least one tested time point after ethephon application , indicating their involvements in the ethephon enhanced water influx into laticifers.
among these time points, the latex collected at  <dig> and 40 h  after ethephon treatment was shown to harbor the most abundant transcripts, which include four of the five highly abundant hbpip1; <dig>  hbpip2; <dig>  hbpip1; <dig> and hbpip2; <dig>  corresponding to the significantly decreased tsc, the longest latex flow duration and the highest latex yield as reported by wang et al.
who utilized the same materials  <cit> .
besides, similar effects of ethephon on latex yield and latex tsc of the pb <dig> clone were also observed by tungngoen et al., although they used mature virgin trees as materials  <cit> .
CONCLUSIONS
to our knowledge, this is the first genome-wide study of the rubber tree aqp gene family and using systematic nomenclature assigned  <dig> hbaqps into five subfamilies based on the sequence similarity and phylogenetic relationship with their arabidopsis and poplar counterparts.
furthermore, their structural and functional properties were investigated based on the analysis of the ar/r filter, froger’s positions and spds, which suggested the potentially key role of hbpips and hbtips in the laticifer water balance.
most importantly, the laticifer transcriptome was deeply sequenced to identify the most important aqps in such a single-cell-type tissue, and qrt-pcr analysis was also performed to investigate the expression profiles of laticifer-expressed hbaqp genes upon ethephon stimulation.
our results revealed that hbpips were the mainly aqp genes expressed in the laticifer.
among  <dig> hbaqp genes detected in the laticifer, most of them were significantly regulated by ethylene.
consistent with the significantly decreased tsc and increased latex yield, most laticifer-expressed pip genes were considerably induced at the time point of 24 h after ethephon application, supporting their crucial roles in the water balance of laticifers in the case of ethephon stimulation.
this study provides an important genetic resource of hbaqp genes, which will be useful to improve the water use efficiency and latex yield of hevea.

 aquaporins, also known as major intrinsic proteins, are a class of integral membrane proteins that facilitate the passive transport of water and other small solutes across biological membranes. since their first discovery in 1990s, aqps have been found in almost all living organisms. compared with animals and microbes, aqps are particularly abundant and diverse in land plants and more than homologs have been identified from arabidopsis thaliana, zea mays, oryza sativa, vitis vinifera, populus trichocarpa, gossypium hirsutum, solanum tuberosum, solanum lycopersicum and glycine max. according to the sequence similarity, the aqps in model plant arabidopsis are divided into four subfamilies, i.e. the plasma membrane intrinsic protein subfamily that contains two subgroups, the tonoplast intrinsic protein that contains five subgroups, the nod26-like intrinsic protein that contains seven subgroups and small basic intrinsic protein that contains two subgroups. in moss and some dicots including p. trichocarpa, one more subfamily named x intrinsic protein subfamily that contains three subgroups is also found. aqps assemble in tetramers in the cell membrane, although each monomer can act as a water channel. even though the overall pairwise sequence similarity can be low, aqps share some structural features such as harboring six transmembrane helices connected by five loops. lb and le from opposite sides dip into the membrane and form two half helices, at the n-termini of which, two highly conserved npa motifs form one selectivity region. and another region that determines the substrate specificity is known as the aromatic/arginine selectivity filter. the npa motifs create an electrostatic repulsion of protons and act as a size barrier, where the ar/r filter renders the pore constriction site diverse in both size and hydrophobicity. in addition to water-conducting aqps, certain aqps called aquaglyceroporins transport glycerol instead. statistical analysis indicated that glps feather five highly conserved amino acid residues: an aromatic residue at p an acidic residue at p a basic residue at p a proline followed by a nonaromatic residue at p and p as y108-d207-k211-p236-l observed in the escherichia coli glycerol facilitator glpf in contrast to a103-s190-a194-f208-w in the pure water channel aqpz. very recently, nine specificity-determining positions for non-aqua substrates, i.e. urea, boric acid, silicic acid, ammonia, carbon dioxide and hydrogen peroxide were also proposed for each group via a comprehensive analysis of functionally characterized aqps. the para rubber tree is a perennial tropical plant species that belongs to the euphorbiaceae family. although it is native to the amazon basin, the economic importance and increasing demand of natural rubber has prompted its wide-domestication to southeast asia. to date, the rubber tree is still the only commercial source of natural rubber for its high production and quality of rubber. natural rubber is specifically synthesized in the highly differentiated vessels termed laticifers, which are located in the secondary phloem of the tree trunk and are periodically differentiated from the vascular cambium. the rubber-containing latex which represents the cytoplasmic content of laticifers is harvested by tapping the bark every 2– days, and the latex yield is determined by the initial flow rate, duration of latex flow and latex regeneration between two tappings. ethylene, a gas phytohormone, plays crucial roles in numerous aspects of growth, development, and response to biotic and abiotic stresses in plants. in arabidopsis, the ethylene signaling pathway has been well established and its involvement in certain agronomically important processes such as seed dormancy, fruit ripening, abscission and senescence has made ethylene a target for manipulation by chemical and biotechnological methodologies. ethephon, an ethylene releaser, was initiatively tested for rubber yield promotion as early as the 1970s and now is widely used in rubber production all over the world. although the molecular mechanism on ethephon stimulation of latex yield is still unclear, researches showed that the treatment of rubber tree barks with ethephon could significantly decrease latex dry rubber content or total solid content, extend the bark drainage area, and prolong latex flow duration. these effects are mainly benefited from water influx toward laticifers and the resultant latex dilution. since water accounts for 60–70 % of total latex upon each tapping, sufficient water supply is essential for both latex flow and latex regeneration. nevertheless, the mature laticiferous vessel rings are devoid of functional plasmodesmata connections, and thus the water inflow into laticifers is mediated largely by aqps. however, the molecular characterization of rubber tree aqps is still in its infancy. as of june only eight full-length hbaqp cdnas have been reported. among them, the water transport activity of hbpip hbpip hbpip1; hbpip2; hbpip2; and hbtip1; was characterized by tungngoen et al. and our group using xenopus laevis oocytes: hbtip1; hbpip2; and hbpip2; were shown to be highly efficient, whereas hbpip1;1; hbpip1; and hbpip2; were less efficient. lately, the rubber tree genome was sequenced by rahman et al. and rubber research institute, chinese academy of tropical agricultural sciences. in addition, more than ests and a high number of rna sequencing reads derived from several tissues such as shoot apex, leaf, laticifer, bark, root and somatic embryogenesis are also available in ncbi sra. these datasets provide a good chance to analyze the rubber tree aqp gene family from a global view. in the present study, a genome-wide search was carried out to identify aqp genes encoded in the rubber tree genome. further, functional prediction was performed based on the analysis of the ar/r filter, froger’s positions and spds, and deep transcriptome sequencing and qrt-pcr expression analysis were also adopted to identify the most important aqp members expressed in the laticifer.  identification and classification of rubber tree aquaporin genes via a comprehensive homology analysis, or loci putatively encoding aqp-like genes were identified from the rubber tree genome of clone ry7-33- or rrim respectively. since all aqp-encoding loci identified in the rrim genome were found in the genome of ry7-33- and some genes from rrim are incomplete and/or the sequences have a high number of “n”s, the aqp genes identified from the ry7-33- genome were selected for further analyses. after discarding loci encoding partial aqp-like sequences which are truncated and lacking any of the npa motifs, full-length aqp genes were retained and the gene models are available in additional file to analyze the evolutionary relationship and their putative function, an unrooted phylogenetic tree was constructed from the deduced amino acid sequences of hbaqps together with that from arabidopsis and poplar. the reasons for choosing these two species are mainly as follows: the complete set of aqp genes in arabidopsis was firstly identified and then well characterized; the well-studied wood plant poplar harbors one more subfamily that is not found in arabidopsis. according to the phylogenetic analysis, hbaqps were grouped into five subfamilies, i.e. pip, tip, nip, sip and xip. following the nomenclature of arabidopsis and poplar, the hbpip subfamily was further divided into two phylogenetic subgroups, the hbtip subfamily into five subgroups, the hbnip subfamily into seven subgroups, the hbsip subfamily into two subgroups and the hbxip subfamily into three subgroups. although the closest homolog of hbnip2; and hbnip3; is not atnip2; or atnip3; their counterparts in poplar were identified and thus the names were assigned. as shown in fig.  many hbaqps were grouped in pairs, i.e. hbpip1;1/hbpip1; hbpip1;3/hbpip1; hbpip2;1/hbpip2; hbpip2;3/hbpip2; hbpip2;5/hbpip2; hbpip2;7/hbpip2; hbtip1;1/hbtip1; hbtip1;3/hbtip1; hbtip1;5/hbtip1; hbtip1;7/hbtip1; hbtip2;1/hbtip2; hbtip2;3/hbtip2; hbtip3;1/hbtip3; hbtip5;1/hbtip5; hbnip1;1/hbnip1; hbsip1;2/hbsip1; hbxip1;3/hbxip1; which exhibit sequence identities of –  % and –  % at the nucleotide or amino acid level, respectively.table list of hbaqp genes identified in this study hbpip1;1 hbpip1;2 hbpip1;3 hbpip1;4 hbpip1;5 hbpip2; 1 hbpip2;2 hbpip2;3 hbpip2;4 hbpip2;5 hbpip2;6 hbpip2; 7 hbpip2;8 hbpp2;9 hbpip2; 1 hbtip1;1 hbtip1;2 hbtp1;3 hbtip1;4 hbtp1;5 hbtip1;6 hbtip1;7 hbtip1;8 hbtp2;1 hbtip2;2 hbtip2;3 hbtip2;4 hbtip3;1 hbtip3;2 hbtip4;1 hbtip5;1 hbtip5;2 hbnip1;1 hbnp1;2 hbnip2;1 hbnp3;1 hbnip4;1 hbnip4;2 hbnip5;1 hbnip6;1 hbnip7;1 hbxip1;1 hbxip1;2 hbxip1;3 hbxip1;4 hbxip2;1 hbxip3; 1 hbsip1;1 hbsip1;2 hbsp1;3 hbsip2;1 abased on the transcriptome data under the ncbi sra accession number of drx000223; b based on the transcriptome data of srx and illumina transcriptome data of srx srx srx srx srx srx srx and srx278515; c based on the transcriptome data of srx and illumina transcriptome data of srx srx srx and srx278514; d based on the transcriptome data of srx and illumina transcriptome data of srx278513; e based on the transcriptome data of srx451710; f based on the transcriptome data of srx read-mapping was carried out using bowtie with default parameters, and mapped read number of more than one was counted as “yes” representing detected genes fig. phylogenetic analysis of deduced amino acid sequences of the hbaqps with arabidopsis and poplar homologs. amino acid sequences were aligned using clustalx and the phylogenetic tree was constructed using bootstrap maximum likelihood tree method and mega software. the distance scale denotes the number of amino acid substitutions per site. the name of each subfamily is indicated next to the corresponding group. species and accession numbers are listed in table  and additional file 2 homology search showed that out of hbaqp genes have est hits in genbank, i.e. hbpip1; hbpip1; hbpip1; hbpip1; hbpip2; hbpip2; hbpip2; hbpip2; hbpip2; hbtip1; and hbtip1; eight full-length hbaqp cdnas, including hbpip1;, hbpip1;, hbpip2;, hbpip2;, hbpip2;, hbpip2;, hbtip1; and hbtip1; have been reported. read alignments against rna sequencing data of rubber tree shoot apex, leaf, laticifer, bark, root and somatic embryogenesis indicated that the expression of hbaqp genes was observed in at least one of the examined tissues. whereas, seven genes coding for two tips, one nip, four xips might be expressed exclusively in response to a specific stimulus or in a very specific part of the plant and thus are excluded in the available datasets. in arabidopsis, the orthologs of hbtip1; and hbnip7; were also shown to be pollen or anther-specific, respectively. besides supported by ests and/or rna sequencing reads, the exon-intron structures of laticifer-expressed hbaqp genes were also confirmed with cloned cdnas. analysis of exon-intron structure the exon-intron structures of the hbaqp genes were analyzed based on the gene models. although the orf length of each gene is similar, the gene size is distinct. the introns of hbaqp genes harbor an average length of 404 bp, with the minimum of 71 bp in hbnip2; and the maximum of 13000 bp in hbsip2; genes in different subfamilies harbor distinct exon-intron structures. all members of the hbpip subfamily feature three introns. except for hbtip1; hbtip1; hbtip1; and hbtip1; that contain only one intron, other hbtip genes contain two introns instead. hbnip genes usually have four introns except for hbnip5; containing three introns. most hbsip genes contain two introns except for hbsip1; without any intron. subgroups of hbxip genes vary in the number of introns: one intron for subgroup one, two or zero for subgroups two and three, respectively.fig. exon-intron structures of the hbaqp genes. shown is a graphic representation of the gene models of all hbaqps identified in this study using gsds. utrs are shown as gray boxes, exons are shown as white boxes and introns are shown as black lines structural features of hbaqps sequence analysis showed that the deduced hbaqps consist of 227– amino acids, with a theoretical molecular weight of –  kda and a pi value of –. homology analysis revealed a high sequence diversity existing within and between the five subfamilies. the sequence similarities of –  % were found within hbpips, –  % within hbtips, –  % within hbxips, –  % within hbnips, and –  % within hbsips. hbpips share the highest sequence similarity of –  % with hbtips, –  % with hbxips, –  % with hbnips, and the lowest of –  % with hbsips. hbtips show –  %, –  % and –  % sequence similarities with hbnips, hbxips and hbsips, respectively. hbnips share sequence similarities of –  % and –  % with hbxips and hbsips, whereas hbsips share the lowest similarity of –  % with hbxips. topological analysis showed that all hbaqps were predicted to harbor six transmembrane helical domains, which is consistent with the results from multiple alignments with structure proven aqps. the subcellular localization of each hbaqp was also predicted. hbpips with an average pi value of are localized to plasma membranes. hbtips with an average pi value of are mainly localized to vacuoles, though several members were predicted to be localized to endoplasmic reticulum, chloroplast and cytosol. hbnips with an average pi value of are mostly localized to plasma membranes, but hbnip2; and hbnip3; were predicted to be localized to the membrane of vacuole and chloroplast, respectively. two members of the sip subfamily were predicted to be localized to plasma membranes, whereas hbsip1; and hbsip2; are localized to the membrane of vacuole and chloroplast, respectively. although the xip subfamily harbors only six members, the predicted localizations are diverse, including the vacuole, chloroplast, plasma membrane and cytosol. to learn more about the putative function of hbaqps, the conserved residues typical of dual npa motifs, the ar/r filter, five froger’s positions and nine sdps were also identified.table structural and subcellular localization analysis of the hbaqps a,b,crepresenting the numbers of transmembrane helices predicted by topcons, tmpred and tmhmm, respectively; dbest possible subcellular localization prediction by the wolf psort. athe sdps in rubber tree aquaporins differing from typical sdps determined in this study are highlighted in red hbpip subfamily all hbpips were identified to have similar sequence length, however, hbpip2s can be distinguished from hbpip1s by harboring relatively shorter n-terminal and longer c-terminal sequences. the five hbpip1s have sequence similarities of –  %, whereas the similarity percents of ten hbpip2s are –  %. between hbpip and hbpip members, sequence similarities of –  % are observed. the dual npa motifs, ar/r filter, and four out of five froger’s positions are highly conserved in hbpips. in contrast, the p position is more variable with the appearance of an e, q or m residue. in addition, two phosphorylation sites corresponding to s and s in sopip2; are invariable in hbpip2s, and the former one is even highly conserved in all hbpips, hbtips and hbxips except for the s → t substitution in several members, implying their regulation by phosphorylation. hbtip subfamily hbtips consist of 227– residues. those belonging to hbtip1s share –  % sequence similarities, whereas hbtip2s have sequence similarities of –  %. members of the hbtip subgroup exhibit sequence similarities of –  %, –  %, –  % and –  % with subgroups hbtip hbtip hbtip and hbtip respectively. hbtip2s share sequence similarities of –  %, –  % and –  % with hbtip3s, hbtip and hbtip5s, respectively. hbtip3s share sequence similarities of –  % and –  % with hbtip and hbtip5s, respectively. and hbtip shares  % and  % sequence similarities with hbtip5; and hbtip5; respectively. dual npa motifs and p p and p positions are highly conserved in hbtips. residue substitution is observed at the p and p positions: t is replaced by a in hbtip2; or i in hbtip5s at the p position, and s is replaced by a in hbtip3s and hbtip5s. of the ar/r filter, h at h and i at h positions are replaced by n and v in hbtip5s, respectively; a is found to be conserved in hbtip1s, hbtip3s, and hbtip and g in hbtip2s and hbtip5s at the le position, respectively; and residues at the le position are more variable, mainly v, r, s or c. hbnip subfamily hbnips consist of 267– residues. with the exception of hbnip and hbnip subgroups that contain two members, each of the other five subgroups harbors a single member. hbnip1; shares the highest sequence similarity of  % with hbnip1; whereas hbnip4; shares a similarity of  % with hbnip4; hbnip1s show sequence similarities of –  %, –  %, –  %,  %, –  % and  % within the subgroups of hbnip hbnip hbnip hbnip hbnip and hbnip respectively. hbnip shows  %, –  %,  %,  % and  % sequence similarities with hbnip hbnip4s, hbnip hbnip and hbnip respectively. the hbnip shows –  %,  %,  % and  % sequence similarities with hbnip4s, hbnip hbnip and hbnip respectively. hbnip4s show –  %, –  % and –  % sequence similarities with hbnip hbnip and hbnip respectively. hbnip shows  % and  % sequence similarities with hbnip and hbnip respectively. hbnip shows  % sequence similarity with hbnip the hbnips have typical dual npa motifs except for hbnip4; hbnip5; and hbnip6;. a is replaced by v in the second npa motif of hbnip4; and by s or v in the first or second npa motif of hbnip5; and hbnip6; compared with other subfamilies, hbnips are highly variable in the ar/r filter and froger’s positions: w/g/a/t at the h position, f/l/v/i at the h position, a/g at the le position, f/l/y at the p position, s/t at the p position, f/y at the p position and l/i at the p position. in addition, one cdpk phosphorylation site corresponding to s in gmnod was also found in the c-terminus of most hbnips. hbsip subfamily there are only four members in the hbsip subfamily. three hbsip1s consist of residues, while hbsip2; has residues. hbsip2; shares sequence similarities of –  % with hbsip1s. within the hbsip subgroup, hbsip1; shares the highest sequence similarity of  % with hbsip1; whereas hbsip1; shows the lowest similarity of  % with hbsip1;. the three hbsip1s harbor the same npt/npa motifs, ar/r filter and froger’s positions, whereas hbsip2; exhibits npl/npa motifs, s-h-g-s ar/r filter and f-v-a-y-w froger’s positions. hbxip subfamily hbxips vary from to residues in length. hbxip1s share sequence similarities of –  % and –  % with hbxip2; and hbxip3; respectively, whereas hbxip2; shows  % sequence similarity with hbxip3; within the hbxip subgroup, hbxip1; shares the highest sequence similarity of  % with hbxip1; whereas hbxip1; shows the lowest similarity of  % with hbxip1;. in hbxip1s, the second npa motif and the le p p and p positions are highly conserved. spv/spi/npt/npv/npl in the first npa motif, v/i at the h position, i/v at the h position, v/p/a at the le position, m/v at the p position and f/c at the p position were observed. similar to most xips, two highly conserved c residues in the lggc motif of lc and the nparc motif of le were also found in hbxips except for hbxip1; in which the f residue is located at the corresponding position of le. transcriptional profiles of hbaqp genes in the laticifer and their response to ethephon stimulation the rubber tree laticifer is a single-cell-type tissue specifically for natural rubber biosynthesis. to identify the aqp genes expressed in the laticifer and determine the most important members in the laticifer water balance, the latex representing the laticifer cytoplasm was collected and high-quality total rnas were isolated from three biological replicates, respectively. then, rnas were pooled and subjected to illumina rna sequencing. approximate gigabase pairs of raw data were generated. after cleaning and quality checks, about million high-quality clean reads with an average length of 95 nt were retained and assembled into unigenes longer than 200 bp, with an average length of 775 bp and an n of 1260 bp. expression profiling showed that out of the identified hbaqp genes were detected in the laticifer transcriptome, including genes coding for pips, tips, nips and sips. based on the rpkm value, the total expression level of pip members was folds more than the nip, tip or sip members, respectively, indicating a crucial role of the pip subfamily in the laticifer water balance. among ten laticifer-expressed pip genes, hbpip2; hbpip1; hbpip2; hbpip1; and hbpip2; were considerably more abundant, counting about 418-, 306-, 204-, 30- and 15-folds higher than the well-studied hbpip1; whereas, hbsip2; the sixth laticifer-abundant aqp gene, expressed relatively more than any other non-pip members.fig. expression profiles of hbaqp genes detected in the laticifer based on illumina sequencing given the important role and wide application of ethephon stimulation on rubber yield promotion, the response of the above laticifer-expressed hbaqp genes subjected to ethephon treatment was analyzed using qrt-pcr over a time course. as described before, treating the rubber tree bark with ethephon was shown to induce a huge increase in latex yield, starting as early as 6 h after the treatment, and the yield increase was about -, -, - and -folds higher than the control at and 40 h after the treatment, respectively; the tsc was significantly decreased at the time points of and 40 h; and the latex flow duration was significantly prolonged from the time point of 16 h. as shown in fig.  except for hbnip6; ethephon treatment had significant effects on all other tested hbaqp genes at one or more time points, implying their regulation by ethylene. at the early stage of ethephon treatment, i.e. 6 h, the transcriptional levels of hbaqp genes were significantly affected, including two up-regulated and eleven down-regulated genes. at 16 h post treatment, hbaqp genes were significantly regulated, including eight up-regulated and eight down-regulated genes. at 24 h post treatment, hbaqp genes were significantly regulated, including thirteen up-regulated and four down-regulated genes. at 40 h post treatment, hbaqp genes were significantly regulated, including seven up-regulated and seven down-regulated genes. although the time points of and 24 h post treatment harbored similar significantly regulated genes, the later had relatively more genes that were up-regulated. although the expression patterns of the regulated genes were diverse, they could be classified into seven groups: the cluster that includes hbpip2; was gradually increased upon ethephon stimulation; the cluster including hbpip2; and hbsip2; were firstly increased and then decreased, which is like a clock; the cluster including hbtip1; was firstly increased, subsequently decreased and increased at the last time point tested; the cluster including hbpip1; was firstly increased, subsequently decreased, then increased and finally decreased; the cluster including hbnip1; and hbsip1; were firstly decreased and then increased; the cluster that includes genes were firstly decreased, subsequently increased and finally decreased; the cluster that includes hbpip1; and hbnip1; were firstly decreased, subsequently increased, then decreased and finally increased. at 24 h post ethephon stimulation, eight genes exhibited the highest expression levels, whereas the highest expression of six genes occurred at 40 h. moreover, the transcript abundance of hbpip2; and hbnip1; were similar at the time points of 24 h and 40 h. as describe above, hbpip2; hbpip2; and hbpip1; were among the top highly abundant aqp genes expressed in laticifers. in addition, another highly abundant aqp genes was expressed most at 16 h post ethephon stimulation.fig. qrt-pcr analysis of laticifer-expressed hbaqp genes upon ethephon stimulation. data are mean ± sd. different letters mean significant difference over the time course discussion high abundance and diversity of hbaqps a total of full-length aqp genes were identified from the rubber tree genome, which is comparable to members reported in poplar; more than in grapevine, in rice, in arabidopsis, in maize, in potato and in tomato; less than in soybean and in cotton. since the aqp genes in arabidopsis and poplar were well characterized, their deduced proteins were added in the phylogenetic analysis of hbaqps, which assigned them to five subfamilies. with the exception of the xip subfamily, the further classification of hbaqp subfamilies into subgroups is consistent with arabidopsis, i.e. two pip subgroups, five tip subgroups, seven nip subgroups and two sip subgroups. nevertheless, classing atnip2; and atnip3; into the nip subgroup was proposed. as shown in fig.  atnip2; and atnip3; were clustered with the nip subgroup, sharing the highest similarity with atnip1; in arabidopsis, hbnip1; or hbnip1; in rubber tree, ptnip1; or ptnip1; in poplar, respectively. thereby, no nip2s and nip3s were retained in arabidopsis as seen in rubber tree and poplar. since no xip homologs were found in the arabidopsis genome, the nomenclature for poplar proposed by lopez et al. was adopted to divide hbxips into three subgroups. besides supported by high bootstrap values, xip1s are characterized by the ar/r filter of v-m-v/p/a-r, xip2s by i-i-v-r and xip3s by v-k-a-r.fig. distribution of the hbaqp genes and their arabidopsis and poplar homologs in subgroups gene pairs were identified not only in rubber tree, but also in poplar and arabidopsis. for example, five atpip1s were clustered together apart from pip1s of rubber tree and poplar; hbpip1; and hbpip1; were clustered with ptpip1; and ptpip1; these results suggest the occurrence of more than one gene duplication events. previous studies indicated that poplar underwent one whole-genome triplication event and one doubling event, whereas arabidopsis underwent the same γ event and two independent doubling events, though the arabidopsis genome encodes relatively less aqp genes due to massive gene loss and chromosomal rearrangement after genome duplications. the γ duplication occurred at approximate million years ago, shortly before the origin of core eudicots. as a core eudicot plant, the rubber tree appears to share the γ duplication. however, another one as the data suggested is likely to be a doubling event independent from both arabidopsis and poplar, probably occurred after the divergence of euphorbiaceae and salicaceae. a genome-wide comparative analysis may provide more information. functional inference of hbaqps although plant aqps firstly raised considerable interest for their high water permeability, when heterologously expressed in xenopus oocytes or yeast cells, increasing evidence has shown that some of them are also participated in the transport of other small molecules such as glycerol, urea, boric acid, silicic acid, nh co and h2o. based on atomic resolution structures and molecular dynamics stimulations of glpf, aqpz, aqp and other mips, several structural features determining their transport selectivity were identified, e.g. the two opposite npa motifs, the ar/r filter and the amino acid residues at froger’s positions for discriminating between aqps and glps. as shown in table  most hbaqps exhibit an aqpz-like froger’s positions to favor the permeability of water. in contrast, hbsip2; and nip subfamily members possess mixed key residues of glpf for p and p and aqpz for p2–p the glycerol permeability of gmnod and arabidopsis nips was reported, however, the potential glycerol transport ability of hbsip2;1-like sips have not be confirmed by experimental means yet. in addition to high permeability to water, plant pips were reported to transport urea, boric acid, co and h2o. as shown in table  all hbpips represent the f-h-t-r ar/r filter as observed in aqpz which harbors an extremely narrow and hydrophilic pore, suggesting their high water permeability. however, when expressed in xenopus, extremely low water permeability of hbpip members such as hbpip1; and hbpip1; was observed as seen in many other plant species. based on the sdp analysis proposed by hove and bhave, all hbpips represent urea-type sdps; hbpip1s represent boric acid-type sdps; hbpip1; represents co2-type sdps; hbpips except for hbpip2; represent h2o2-type sdps, supporting their similar functionality. although highly variable in the ar/r filter, plant tips were shown to transport water as efficiently as pips. additionally, they also allow urea, nh and h2o through. as shown in table  all hbtips except for hbtip2; and hbtip4; represent urea-type sdps, whereas hbtip1; hbtip1; hbtip4; hbtip5; and hbtip5; represent h2o2-type sdps, indicating similar functionality. compared with typical nh3-sdps, hbtip2; seems to represent novel sdps with the substitution of s for a/r/t at sdp besides glycerol and water, plant nips have been found to transport urea, boric acid, silicic acid, nh and h2o. as shown in table  and additional file hbnip5; is promised to be a urea and boric acid transporter with nine sdps of h-p-i-a-l-p-g-s-n or t-i-h-p-e-l-l-a-p. hbnip2; represent typical urea sdps, and sdps of v-v-h-p-e-i-i-a-p with the substitution of v for i at sdp in comparison to typical boric acid sdps. compared with typical urea and boric acid sdps, hbnip6; seems to represent novel sdps-types with the substitution of q for a/p at sdp or q for a/p/g at sdp although characterized as an nip iii member, the silicic acid transport ability of hbnip2; needs to be experimentally validated since it seems to represent novel sdps similar to that of gmnip2; and gmnip2;. although gmnod was reported to transport nh, whether its close rubber tree homologs represent novel sdps-types still needs to be tested. hbnip3; hbnip4; and hbnip5; represent h2o2-type sdps slightly different from atnip1;. as a recently identified aqp subfamily, plant xips were shown to transport water, glycerol, urea, boric acid and h2o. according to phylogenetic relationships, xips are split into two independent clusters termed xip-a and xip-b, where xip-a includes only xip subgroup and xip-b contains at least four subgroups, i.e. xip xip xip and xip. consistent with poplar xips, six hbxips can be assigned to subgroups xip, xip and xip. when expressed in xenopus oocytes, ptxip2; and ptxip3; transported water while other ptxips did not. although the mechanism why ptxip1s, ptxip3; and ptxip3; do not transport water is still unclear, the close homologs of ptxip1s in nicotiana tabacum and potato were also reported to have undetectable water permeability. in contrast, solanaceae xips showed high permeability to glycerol. therefore, although exhibiting an aqpz-like froger’s positions, all hbxips maybe transport glycerol. meanwhile, hbxip2; and hbxip3; are probably capable of transporting water. as shown in table  hbxip1; hbxip1; hbxip2; and hbxip3; are promised to be urea transporters with nine sdps of h-p-f/l-a-l-g-g-g-n; hbxip1; hbxip1; and hbxip2; may represent novel boric acid sdps-types with the substitution of q or t for a/g/k/p at sdp9; hbxip1; and hbxip1; harbor h2o2-type sdps with a slight difference from some solanaceae xips. a crucial role of hbpips in the water balance of laticifers as a unique site for rubber biosynthesis, the laticifers are present in a wide variety of rubber tree tissues, including shoots, roots, stems, leaves, flowers, fruits, cotyledons, inner seed coats, etc., and can be divided into primary and secondary laticifers according to their origin. compared with the procambium-derivation of primary laticifers, the secondary laticifers, mainly located in the soft inner bark of the rubber tree trunk, are periodically differentiated from the vascular cambium and serve as a sole source for the commercial latex. during the differentiation and maturation process, laticifer mother cells articulate with each other and further anastomose together into a successive vertical network arranged as concentric sheaths in the secondary phloem. unlike other cells such as neighboring parenchyma cells, the mature laticiferous cells are totally devoid of plasmodesmata, and thus its water exchanges with surrounding cells are mainly governed by aqps. upon bark tapping, the laticifer cytoplasm is expelled in the form of latex due to the high turgor pressure inside. generally, latex flow can continue for several hours until coagulation processes lead to the plugging of severed laticifers. during the latex flow, a progressive decrease in drc was observed, indicating rapid water influx and latex dilution inside laticifers caused by the activity of hbaqps. given that of hbpips and hbtips account for more than  % of the total hbaqps and their aqpz-like froger’s positions favoring the high water permeability, we initially prospect that these two subfamilies may play important roles in the laticifer water balance: the plasma membrane-targeted hbpips facilitate the water transport from the extracellular space to the laticifer cytoplasm, whereas the lutoid-targeted hbtips play an essential role in maintaining the cell osmotic balance as observed in most plant cells. however, in contrast to the mature plant cells characterized by a large central vacuole which occupies 80 % or more of the intracellular space, the lutoids in laticifers are polydispersed microvacuoles occupying only 12 % of the total latex, arguing the central role of hbtips in the laticifer water balance, though their potential role in the lutoid stability and latex vessel plugging should be noted. to address this issue, the transcriptome of such a single-cell-type tissue was deeply sequenced. results showed that pip members were the main aqp genes expressed in the laticifer, suggesting their crucial role, especially the highly abundant hbpip2; hbpip1; and hbpip2; in the laticifer water balance. when expressed in xenopus oocytes, our previous study showed that hbpip2; could transport water as efficiently as hbpip2;; in contrast, hbpip1; and hbpip2; were shown to be less efficient. in addition, as a pip member, the poor efficiency of hbpip1; was also observed. therefore, the exact role of hbpip2; hbpip1; and hbpip2; in the water balance of rubber tree laticifers needs further investigations. to profile the aqp genes in response to ethylene stimulation in laticifers, the latex at different time points after ethephon treatment was collected from rubber tree clone pr similar to pb pr clone is characterized as a relatively late mature variety which has a high tsc, short latex flow duration and low latex metabolism, however, ethephon stimulation could significantly prolong its latex flow duration and enhance latex yield. our qrt-pcr analysis showed that the expression levels of most laticifer-expressed genes significantly changed at least one tested time point after ethephon application, indicating their involvements in the ethephon enhanced water influx into laticifers. among these time points, the latex collected at and 40 h after ethephon treatment was shown to harbor the most abundant transcripts, which include four of the five highly abundant hbpip1; hbpip2; hbpip1; and hbpip2; corresponding to the significantly decreased tsc, the longest latex flow duration and the highest latex yield as reported by wang et al. who utilized the same materials. besides, similar effects of ethephon on latex yield and latex tsc of the pb clone were also observed by tungngoen et al., although they used mature virgin trees as materials.  to our knowledge, this is the first genome-wide study of the rubber tree aqp gene family and using systematic nomenclature assigned hbaqps into five subfamilies based on the sequence similarity and phylogenetic relationship with their arabidopsis and poplar counterparts. furthermore, their structural and functional properties were investigated based on the analysis of the ar/r filter, froger’s positions and spds, which suggested the potentially key role of hbpips and hbtips in the laticifer water balance. most importantly, the laticifer transcriptome was deeply sequenced to identify the most important aqps in such a single-cell-type tissue, and qrt-pcr analysis was also performed to investigate the expression profiles of laticifer-expressed hbaqp genes upon ethephon stimulation. our results revealed that hbpips were the mainly aqp genes expressed in the laticifer. among hbaqp genes detected in the laticifer, most of them were significantly regulated by ethylene. consistent with the significantly decreased tsc and increased latex yield, most laticifer-expressed pip genes were considerably induced at the time point of 24 h after ethephon application, supporting their crucial roles in the water balance of laticifers in the case of ethephon stimulation. this study provides an important genetic resource of hbaqp genes, which will be useful to improve the water use efficiency and latex yield of hevea. 
homology analysis supported the expression of hbaqp genes in at least one of the examined tissues. furthermore, deep sequencing of the laticifer transcriptome in the form of latex revealed a key role of several pip subfamily members in the laticifer water balance, and qrt-pcr analysis showed diverse expression patterns of laticifer-expressed hbaqp genes upon ethephon treatment, a widely-used practice for the stimulation of latex yield. this study provides an important genetic resource of hbaqp genes, which will be useful to improve the water use efficiency and latex yield of hevea. functional prediction based on the analysis of the aromatic/arginine selectivity filter, froger’s positions and specificity-determining positions showed a remarkable difference in substrate specificity among subfamilies. natural rubber, an important industrial raw material, is specifically synthesized in laticifers located inside the rubber tree trunk. the phylogenetic analysis assigned these aqps to five subfamilies, including plasma membrane intrinsic proteins, tonoplast intrinsic proteins, nod26-like intrinsic proteins, small basic intrinsic proteins and x intrinsic proteins. in this study, full-length aqp genes were identified from the rubber tree genome. due to the absence of plasmodesmata, the laticifer water balance is mediated by aquaporins.
 natural rubber, an important industrial raw material, is specifically synthesized in laticifers located inside the rubber tree trunk. due to the absence of plasmodesmata, the laticifer water balance is mediated by aquaporins. however, to date, the characterization of h. brasiliensis aqps is still in its infancy.  in this study, full-length aqp genes were identified from the rubber tree genome. the phylogenetic analysis assigned these aqps to five subfamilies, including plasma membrane intrinsic proteins, tonoplast intrinsic proteins, nod26-like intrinsic proteins, small basic intrinsic proteins and x intrinsic proteins. functional prediction based on the analysis of the aromatic/arginine selectivity filter, froger’s positions and specificity-determining positions showed a remarkable difference in substrate specificity among subfamilies. homology analysis supported the expression of hbaqp genes in at least one of the examined tissues. furthermore, deep sequencing of the laticifer transcriptome in the form of latex revealed a key role of several pip subfamily members in the laticifer water balance, and qrt-pcr analysis showed diverse expression patterns of laticifer-expressed hbaqp genes upon ethephon treatment, a widely-used practice for the stimulation of latex yield.  this study provides an important genetic resource of hbaqp genes, which will be useful to improve the water use efficiency and latex yield of hevea. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rubber tree laticifertranscriptomeaquaporinplasma membrane intrinsic proteinwater balanceissue-copyright-statement© the author 2015 