BACKGROUND
teak   is the most important and highly valued commercial hardwood timber in the tropics due to its high durability, dimensional stability, heartwood-sapwood proportions, weightlessness and resistance to weathering.
also, it is used for carpentry, floors, shipbuilding and agroforestry, thus becoming a high-class furniture and a standard timber in end-use classification of other tropical timbers .
it is a deciduous species presenting natural populations in thailand, laos, myanmar, india and java islands.
teak grows properly within 25-38 °c, between  <dig>  and  <dig>  mm/year of rainfall, presenting the best yields under  <dig> m above sea level and produces better wood quality with long dry periods, from  <dig> to 5 month long .
this species is the major component of the forest economies of many tropical countries.
it is the only valuable hardwood that constitutes a globally emerging forest resource with a planted area of  <dig>  million ha  and natural forest of  <dig>  million ha  around the world, and brazil presents the largest teak reforestation in south america  <cit> .
due to its importance, many efforts have focused on the study of teak population variability .
however, there are no genetic studies nor next-generation sequencing regarding wood formation in teak.
wood comes from secondary growth, starting with the vascular cambium expansion and cell division in stems of young trees, followed by a differentiation of secondary xylem and several events such as xylem cells expansion, secondary cell wall deposition and programmed cell death .
in most tropical america, including brazil, wood harvesting occurs at 20 years, producing small-dimension logs, which are not in demand on the international market  <cit> .
teak is not a fast growing species but can produce a timber of optimum strength in relatively short rotations of 21 years  <cit>  depending of the sapwood-heartwood percentages.
the timber quality produced will be the overriding commercial factor for the near future  <cit> , and usually relates to the amount, color and durability of the heartwood  <cit> .
for that reason, techniques such as ests and microarrays have been used extensively to understand wood formation in trees such as pinus  <cit>  and populus  <cit> .
however, today, large-scale studies of biological phenomena are unthinkable without the use of next-generation sequencing technologies , such as rna sequencing , which encourages developmental and genomics research of woody growth in trees  <cit> , especially for species without a sequenced genome and no molecular information available  <cit>  as teak.
in tropical trees, the use of next-generation sequencing in order to find differentially expressed unigenes involved in secondary xylem is restricted to some species  <cit> .
availability of nondestructive wood analysis methods such as core sampling would provide a valuable way to study teak wood in different aspects and avoid depletion of both natural and plantation teak resources  <cit> .
heartwood and sapwood are complex tissues in which percentages are not easily assessed on standing trees, but they can be determined from a bore core  <cit> .
also, their study in the area of molecular biology is challenging because of their rigid woody tissues with high contents of polysaccharides, which hinders its maceration and extraction of genetic material.
the sapwood is a heterogeneous tissue with a mixture of earlywood and latewood and differing levels of lignification.
sapwood is composed of xylem and other dead as well as living cells, reserves of starch or sugar and lower extractives content  <cit> .
the same author explains that a larger proportion of sapwood is preferred in wood for pulp manufacture and preservative treatment, and heartwood is desirable in construction timber, high quality veneers and joinery because of its resistance to biotic attack and darker color.
in a cross-section of logs, sapwood is usually observed as a pale annulus surrounding concentric heartwood  <cit> .
in teak, it is certainly needed to identify genes such as those controlling secondary xylem, vessel formation, sapwood and heartwood differentiation, volume growth and abiotic stress.
those studies have been documented in populus tremula  <cit> , populus euphratica  <cit> , populus trichocarpa  <cit> , eucalyptus  <cit> , conifers  <cit> , and fraxinus spp.
<cit> , but it needs to be done in teak to help improving wood quality, growth speed and environmental adaptability  <cit> .
the expression of several genes has been related to the wood formation processes, including some families of transcription factors  <cit> .
the myb transcription factors have been related to the coordination of genes which drive the lignin biosynthesis, with a great range of regulation and operating at all points of the phenylpropanoid pathway  <cit> .
the r2r3-myb proteins  belong to a large family of transcription factors with over  <dig> members in angiosperms, also defined by an n-terminal dna- binding domain , a c-terminal modulator region with regulatory activity; also r2r3-myb proteins show a potential of binding ac elements , which belong to the most abundant type in plants with essential roles in vascular organization  <cit> .
therefore, genetic examination of the superior growth of a prized woody plant such as t. grandis would provide a collection of expressed genes from several tissues, as it has been done in another forestry species such as eucalyptus, where a digital expression profiling of xylogenic and non-xylogenic tissues was obtained via rna-seq  <cit> .
a better understanding of secondary xylem formation is essential not only as a fundamental part of plant biology , but also because it is crucial to obtain solutions for problems in forest conservation, improving the offerings of woody products  <cit> .
also, it is hoped that through genetic selection and plant transformation, the non-durable core could be reduced or eliminated, the growth could be increased and the epicormic branches could be controlled, making the so-called “juvenile wood” problem a thing of the past  <cit> .
sapwood/hardwood characteristics are reliable predictors of overall genetic improvement of timber strength  <cit> .
therefore, this is the first rna sequencing in this tropical woody plant.
firstly, the aim of this study was to unveil the transcriptome of teak at a large-scale to later compare the transition of young  to mature  trees in order to reveal differentially expressed transcripts since this transition gives wood strength, endurance, color differences, natural chemicals and biotic and abiotic resistance to older trees, important features in the teak market.
we detected  <dig>  transcripts in stem secondary xylem and found that more than  <dig> unigenes were differentially expressed in a temporal and tissue specific fashion.
we also supplied several heat-shock proteins and analyzed the expression of some myb-related transcription factors differentially expressed in teak secondary xylem, including sapwood tissue.
RESULTS
quality of the rna and the reads
based on the bioanalyzer results , all samples  showed appropriate rin factor.
the libraries had a size of 280 bp, approximately.
we generated almost  <dig> million paired-end reads, covering  <dig>  gigabases of sequence data with a sequence length of 100 bp .
the dataset of raw reads was deposited in ncbi sra database under sra study number srp <dig>  after cleaning the data with the “trimmed” procedure  <cit> , the “per base quality”, “per base sequence content”, “per sequence gc content”, “per sequence quality”, “duplication levels” and “sequence length distribution” were improved .
then,  <dig>  % of the reads , and between  <dig>  %  and  <dig>  %   were lost after cleaning.
more than  <dig> million sequence reads with a size of  <dig>  gigabases  were obtained.
consequently, with this quality it was possible to continue the subsequent analyses .fig.
<dig> teak tissue and organ sample set.
a
in vitro seedling.
b) in vitro leaf.
c) in vitro root.
d) flower.
e) stem secondary xylem.
f) branch secondary xylem.
g) use of pressler core barrel  at diameter breast high .
h) core sample containing “s”  and “h” .
all samples were immediately placed on aluminum foil and transported in liquid nitrogen for a subsequent rna extraction
de novo assembly
the assembly of the transcriptome from the leaf, root, seedling, flower, secondary xylem of teak branch and stem was performed using the trinity assembler  <cit> .
for lignified tissues such as branch secondary xylem of both tree ages , we used between  <dig> , <dig> and  <dig> , <dig> reads, and for stem secondary xylem of both tree ages  we used between  <dig> , <dig> and  <dig> , <dig> reads .
flower, leaf, root and seedling were  <dig> , <dig>   <dig> , <dig>   <dig> , <dig> and  <dig> , <dig> reads, respectively.
unpaired reads were from  <dig> , <dig>  to  <dig> , <dig>  in all samples.
using those reads as input for trinity  <cit> , we obtained  <dig> ,  <dig> ,  <dig>  and  <dig>  contigs for stem secondary xylem, branch secondary xylem, non-lignified tissues  and unpaired reads, respectively, , with a mean for n <dig> length of  <dig>  bp.
contigs coming from lignified samples were subsequently used for differential expression analyses.
unigenes differentially expressed in lignified tissues between 12- and 60-year-old trees
differentially expressed transcripts in all the comparison groups with deseq program were obtained with a false discovery rate of  <dig>  .
in the case of the branch secondary xylem transcripts differentially expressed from both 12- and 60-year-old teak trees with repetitions, the dispersion plot  showed the presence of significant genes differentially expressed between both ages, showing a normalized grouping tendency in most of the transcripts with the fitted curve.
also, in additional file 4b all the differentially expressed transcripts are exposed in red dots.
the dispersion plot  of stem secondary xylem transcripts differentially expressed from both 12- and 60-year-old teak trees  showed a normalized grouping tendency with a fitted curve.
several differentially expressed transcripts in stem secondary xylem were also obtained .
additionally, looking for differentially expressed genes between all branch and stem samples , the contrast between both tissues is clear.
as well, additional file  <dig> exhibited almost the same quantity of differentially expressed and shared genes between both tissues.
when plotting stem and branch against non-lignified tissues  , still stem exhibited more genes differentially expressed compared to branch.
finally, with deseq, we obtained  <dig>  and  <dig> differentially expressed genes for stem and branch secondary xylem, respectively, when comparing 12- and 60-year-old trees .
the dataset of differentially expressed genes was deposited in ncbi tsa database under tsa study number gdlt <dig>  also, differential expression between branch and stem secondary xylem, stem secondary xylem against non-lignified tissues  and branch secondary xylem against non-lignified tissues provided  <dig> ,  <dig>  and  <dig>  genes, respectively .fig.
<dig> venn diagram showing number of differentially expressed genes in the different tissues and ages.
for the diagram, we used leaf, flower, root, seedling, stem and branch secondary xylem, comparing young  and mature  trees for the last two tissues
functional annotations of unigenes differentially expressed in lignified tissues
from the  <dig>  and  <dig> differentially expressed transcripts for stem and branch secondary xylem, respectively , an annotation of  <dig>  and  <dig>  genes was achieved with a known function by blast2go, respectively .
among the  <dig> genes annotated for stem secondary xylem, 48 %  exhibited strong homology .
also, for the same tissue, the similarity distribution showed that 89 % of the genes have more than 60 % identity with other plants  and for the species distribution, t. grandis had the greatest number of matches with vitis vinifera, followed by glycine max, theobroma cacao and populus trichocarpa .
on the other hand, from the  <dig> genes annotated for branch secondary xylem, 33 %  revealed an homology with e-value smaller than 1e- <dig>  and in the identity comparison showed that 92 % of the genes have more than 60 % identity with other plants .
most of the differentially expressed genes had a size between  <dig>  and  <dig>  bp .
gene ontology  tool classified the unigenes in several sub-categories for biological process, cellular component and molecular function.
in stem secondary xylem , catabolic process , cellular protein modification process , response to stress  and carbohydrate metabolic process represented the most abundant sub-categories in the biological process category , indicating the expression of genes related to catabolic activities and stress, where several heat-shock proteins were found.
under the molecular function category, the top  <dig> sub-categories were nucleotide and protein binding  , where three r2r3-mybs and one cc-myb transcription factors were found and used for subsequent analysis.
in the cellular component category, plastid  and protein complex  were the most abundant .
in branch secondary xylem , all categories showed similar results to stem secondary xylem , except for the protein transport through plasma membrane function.
catabolic process and response to stress , nucleotide and protein binding , plastid and plasma membrane  are the main categories for both tissues .
further, three heat-shock proteins , one carboxylesterase  and one bax inhibitor  with significant up-regulation were found in stem secondary xylem , and subsequent expression analyzes of these genes were performed.fig.
<dig> homology analysis of t. grandis differentially expressed unigenes.
branch secondary xylem : a e-value distribution.
b) similarity distribution.
c) species distribution.
stem secondary xylem: d) e-value distribution.
e) similarity distribution.
f) species distribution
fig.
<dig> gene ontology  assignment for the unigenes differentially expressed of t. grandis stem secondary xylem.
go assignments  as predicted for a biological process,  molecular function and  cellular components.
the number of unigenes assigned to each go term is shown behind semicolon
metabolic pathways of unigenes
beyond finding transcription factors, heat-shock proteins and annotating genes from secondary xylem from teak, we searched for pathways related to those differentially expressed genes.
for branch secondary xylem between both ages,  <dig> paths were identified in the annotated genes , the most relevant of which, due to number of sequences, were starch and sucrose, amino sugar and purine metabolism.
in the case of stem secondary xylem between both ages,  <dig> metabolic pathways were identified for all annotated differentially expressed genes .
starch and sucrose, glycerol lipid and purine metabolism presented the highest number of sequences.
also, some relevant metabolisms were found , such as irinotecan  and azathioprine-mercaptopurine metabolisms , with the genes located inside the pathway.
the ali-esterase   has  <dig>  bp.
another relevant gene obtained from the gene ontologies and metabolic pathways is the beta-galactosidase 17-like involved in glycan degradation  .fig.
<dig>
a irinotecan metabolism with the teak ali-esterase enzyme in brown .
azathioprine-mercaptopurine metabolism with the teak phosphoribosyltransferase enzyme in blue
clustering analysis of the teak r2r3-myb gene family members
in order to find phylogenetic relationships between r2r3-myb members of different plant species and teak, we performed clustering analysis.
indeed, tgmyb <dig> protein showed a predicted coiled-coil  domain  , a subtype within the myb superfamily, as defined by  <cit> .
tgmyb <dig>  tgmyb <dig> and tgmyb <dig> were consistent with the consensus dna-binding domain sequences  defined for r2r3-myb family, finding r2r <dig> motifs similar to those found in arabidopsis, gymnosperm and angiosperm plants  <cit> .
tgmyb <dig>  tgmyb <dig> and tgmyb <dig> presented the wtx1eedx2lx3vx4gx6w and the rx4cx1lrwx3lx1p conserved motifs within the r <dig> region .
tgmyb <dig> and tgmyb <dig> presented the tx2eex2lix2hx3gnkw motif, tgmyb <dig> presented the bhlh protein-binding motif  and tgmyb <dig>  tgmyb <dig> and tgmyb <dig> presented the pgrx2nx1ikx2wn motif, all in the r <dig> region .
using the complete r2r3-myb family from arabidopsis, a dendrogram was obtained to elucidate functional grouping which could also be present in the teak myb family .
tgmyb <dig> is located in the epidermal cell fate group, and closely-related to the flavonol glycosides group and c <dig> repressor motif group, the members of which participate in bhlh interactions and promoter repression  <cit> .
tgmyb <dig> is inside the gamyb-like genes group, which are microrna-regulated genes that facilitate anther development  <cit> .
additionally, tgmyb <dig> seems to share a common ancestor with atmyb <dig>  which do not have related function yet.
however, it is unclear how both proteins are grouped, one being cc-myb  and r2r3-myb  type.
furthermore, using gymnosperm and angiosperm protein sequences to characterize teak mybs transcription factors, we schemed the three major groups  and subgroups  of r2r3-mybs as described by bedon et al.
.
therefore, tgmyb <dig> fell into group a, subgroup  <dig>  , which presents motifs involved in protein or dna interactions.
also, tgmyb <dig> is close to subgroup  <dig> , consistent with fig.
<dig>  indeed, tgmyb <dig> could be related with cell wall formation.
tgmyb <dig> is found in group b, subgroup  <dig>  , similar clustering when using all arabidopsis myb transcription factors .
group b was previously described as being present only in angiosperms  <cit> .
tgmyb <dig> is presented as a separate unit and located inside group c. group c is also composed by subgroups  <dig>   <dig>   <dig>   <dig>   <dig> and lignin biosynthesis sequences atmyb <dig>  atmyb <dig>  atmyb <dig>  pgmyb <dig>  pgmyb <dig>  and ptmyb <dig>  tgmyb <dig> is still apart from the r2r <dig> myb proteins, being clustered with atmyb <dig>  atmyb <dig> and atmyb <dig> , as found in the arabidopsis grouping , as expected.
altogether, although r2r <dig> motifs have several differences in t. grandis sequences, they grouped closely to secondary wall biosynthesis genes from other species.fig.
<dig> integrated dendrogram of the  <dig> arabidopsis r2r <dig> myb proteins with teak myb proteins.
consensus circular tree was conducted by neighbor-joining method and  <dig> bootstraps using mega <dig> software.
teak myb proteins are denoted with red dots.
each functional group is colored.
references for myb gene functions are defined by previous reports  <cit>
fig.
<dig> dendrogram of gymnosperm and angiosperm r2r3-myb proteins.
the neighbor-joining method was used using  <dig> bootstraps with several spruce, pine, arabidopsis and teak protein myb sequences.
teak myb proteins are denoted with a diamond.
the bar indicates the evolutionary distance of  <dig>  %.
arabidopsis proteins were chosen as landmarks indicating the three main groups  and subgroups  defined by  <cit>
gene expression of myb transcription factors in teak
quantitative real-time pcr analysis showed that four teak mybs are differentially expressed in lignified tissues, being tgmyb <dig>  tgmyb <dig>  tgmyb <dig> up-regulated and tgmyb <dig> down-regulated .
in leaves and roots, tgmyb <dig>  tgmyb <dig> and tgmyb <dig> showed almost no expression levels compared to lignified tissues.
tgmyb <dig> was expressed much higher in leaves than the other tissues, and stem secondary xylem of both ages is shown as down-regulated.
the up-regulated genes tgmyb <dig> and tgmyb <dig> showed comparatively higher expression in stem secondary xylem and sapwood   in mature  compared to young  trees.
inversely, tgmyb <dig> expression is 2-fold higher  and 60-fold higher  in stem secondary xylem and sapwood, respectively, of young teak trees.
the down-regulated gene tgmyb <dig> showed similar expression pattern in stem secondary xylem and sapwood of trees from both ages , although in the deseq expression level stem secondary xylem from 60-year-old trees showed almost 150-fold less expression compared to 12-year-old trees.
branch secondary xylem of 12-year-old trees seems to have considerable expression levels in tgmyb <dig> and tgmyb <dig> genes compared to leaves , but similar expression compared to stem secondary xylem at both ages, with a 95 % statistical confidence level.
these results confirm that the unigenes obtained from the transcriptome assembly were differentially expressed, with differences between both ages .
moreover, the real-time pcr is in agreement with deseq results  for tgmyb <dig>  tgmyb <dig> and tgmyb <dig>  although tgmyb <dig> displayed a down-regulated expression in both methods for all tissues when compared with leaf, this gene showed a discrepancy for secondary xylem down-regulated expression at both ages due to the differences of the methods.
overall, the rna-seq data was biologically validated by the quantitative real-time pcr analysis.fig.
<dig> expression patterns of four myb transcription factors with the deseq method.
we chose four myb transcription factors from the differentially expressed unigenes obtained when comparing stem secondary xylem from mature and young trees.
± means se of two biological replicate samples were included.
the fold changes of the genes were calculated as the log <dig> value
fig.
<dig> expression of teak myb genes with the qrt-pcr method.
relative quantification of expression was examined in different tissues .
the name of each gene is indicated at the top of each histogram.
tissues considered are shown at the bottom of the diagrams.
± means se of three biological replicate samples.
*p <  <dig>  according to f-test.
y-axis indicates the relative expression level of each gene compared to the control tissue .
ef1α was the endogenous control used according to  <cit>
fig.
<dig> relative expression levels of teak myb genes in sapwood with the qrt-pcr method.
the name of each gene is indicated at the top of each histogram.
tissues considered are shown at the bottom of the diagrams.
± means se of three biological replicate samples.
*p <  <dig>  according to f-test.
y-axis indicates the relative expression level of each gene compared to the control tissue .
ef1α was the endogenous control used according to  <cit>
gene expression of heat-shock proteins, carboxylesterase and bax inhibitor transcripts in teak
expression analysis  presented tghsp <dig>  tghsp <dig>  tghsp <dig>  tgbi and tgces as differentially expressed transcripts, being up-regulated in lignified tissues .
all five genes presented almost null expression in leaves and roots compared to secondary xylem of stem and branch, and all the genes presented more expression in stem compared to branch secondary xylem .
tghsp <dig>  tghsp <dig>  tghsp <dig> and tgbi showed higher expression in stem secondary xylem of 12-year-old trees compared to 60-year-old trees, with 2-fold, 2-fold, 4-fold and 3-fold more transcripts by deseq method, respectively , and 5-fold, 4-fold, 3-fold and 7-fold more expression by qrt-pcr method, respectively .
in contrast to these results, tgces exposed more gene expression in mature teaks  compared to young trees.
again, the quantitative real-time pcr results are similar to the deseq expression tendencies.fig.
<dig> expression patterns of three heat-shock proteins and two enzymatic genes with the deseq method.
we chose three heat-shock proteins , a carboxylesterase  and a bax inhibitor  from the differentially expressed unigenes obtained when comparing stem secondary xylem from mature and young trees.
± means se of two biological replicate samples were included.
the fold changes of the genes were calculated as the log <dig> value
fig.
<dig> expression of tghsp <dig>  tghsp <dig>  tghsp <dig>  tgces, tgbi genes with the qrt-pcr method.
relative quantification of expression was examined in different tissues .
the name of each gene, values and tissues considered are shown at the bottom of the diagrams.
± means se of three biological replicate samples.
y-axis indicates the relative expression level of each gene compared to the control tissue .
ef1α was the endogenous control used according to  <cit>
discussion
t. grandis transcriptome
the high sensitivity of sequencing technologies presents the rna-seq as the preferred choice for transcriptome studies  <cit> , widely replacing the microarray-based gene expression technology  <cit> , the sequencing of cdna libraries, the sage and supersage analysis  <cit> .
despite the forestry and economic importance of t. grandis around the world, it is very poorly characterized, with only  <dig> gene sequences deposited in genbank , most of them being alleles used for molecular markers .
also, previous genetic studies have focused on proteomic analysis and kinetics of t. grandis .
in this study, we have generated more than  <dig> million sequence reads  corresponding to  <dig>  gigabases of raw sequence data from several tissues .
t. grandis without a sequenced genome and a lack of a sequenced genome in the lamiales order makes analysis of the teak rnaseq dataset more difficult.
tectona grandis is a diploid species with 2n =  <dig> chromosomes  <cit> .
ohri & kumar   <cit>  estimated the size of its genome by cytogenetic studies, finding about  <dig> mbp , which is about the same and 2-fold larger than the genome of populus trichocarpa and arabidopsis thaliana, respectively.
a. thaliana has at least  <dig>  transcription factor genes   <cit> .
assuming a similar proportion of transcription for t. grandis, all the transcription factors could be estimated in  <dig>  mbp.
comparatively,  <dig> million reads were obtained from phaseolus vulgaris  <cit> ,  <dig> million reads were generated from stem-root of piper nigrum  <cit> ,  <dig> million reads were generated from vitis vinifera  <cit> ,  <dig> million reads were obtained in camellia sinensis  <cit>  and close to  <dig> million reads were obtained from petroselinum crispum  <cit>  and isatis indigotica  <cit> .
in eucalyptus, pyrosequencing gave  <dig>  million reads  <cit> .
in that sense, trinity appears as a good choice to assemble de novo full-length transcripts for species without reference genome  <cit>  because it corrects almost 99 % of the sequencing errors.
trinity is a strategy which assembles a set of unique sequences from reads aided by the creation of independent de bruijn graphs, each representing one group of sequences and assembles isoforms within the groups, running in parallel in a computational cluster  <cit> .
we obtained four different transcriptomes from all tissues using the trinity platform .
recent studies found  <dig>  unigenes in isatis indigotica  <cit> ,  <dig>  unigenes from phaseolus vulgaris representing 49 mb  <cit> ,  <dig>  unigenes from petroselinum crispum  <cit>  and  <dig>  unigenes in camellia sinensis  <cit> .
several trees have generated significantly higher numbers of genes, such as salix matsudana with  <dig>  unigenes  <cit> , populus trichocarpa with  <dig>  unigenes  <cit> , populus euphratica with  <dig>  unigenes  <cit>  and fraxinus spp.
with  <dig>  unigenes  <cit> .
rnaseq provided several useful unigenes differentially expressed in lignified tissues of t. grandis
from the transcriptome obtained, we were able to identify differentially expressed genes with deseq program, obtaining an invaluable gene dataset of lignified tissues of teak.
deseq method is a parametric approach which works with technical replicates, with the variance and mean linked by local regression, and uses the negative binomial distribution  to visualize the intensity-dependent ratio of expression data .
our analysis for differentially expressed genes is based in biological replicates, which allow a solid biological interpretation.
we found  <dig>  and  <dig> differentially expressed genes in stem and branch secondary xylem, respectively, between young and mature teak trees.
recent studies have shown substantial differences obtaining differentially expressed genes.
<cit>  obtained  <dig>  transcripts from stem-root of piper nigrum.
in stem, almost  <dig> ,  <dig>  and  <dig>  differentially expressed genes were obtained in populus trichocarpa  <cit> , alfalfa  <cit>  and brassica juncea  <cit> , respectively.
in eucalyptus,  <dig>  contigs were obtained  <cit>  and in salix matsudana  <dig> mirna stress-related differentially expressed genes  <cit> .
it is common to find in some treatments no more than  <dig>  differentially expressed genes, as the case of camellia sinensis  <cit> .
to compare between two general tissue types that are of interest for woody biomass production  <cit>  such as stem and branch, along with the comparison between young  and mature  trees, we properly performed the differential expression procedure with deseq program .
all the differentially expressed genes in both tissues presented high homology , matched with lignified plants and presented sizes between  <dig> - <dig>  bp .
after annotations, the catabolic processes, response to stress, carbohydrate metabolism, protein binding, transport and plastid localization were the most abundant sub-categories.
these annotations are consistent with biopolymers production, transport, storage and xylogenic-related genes as were found in the transcriptome of e. grandis × e. urophylla hybrid clone  <cit> , picea glauca  <cit>  and populus trichocarpa  <cit> .
several differentially expressed genes in the transition between young to mature trees in secondary xylem include glycan degradation cell wall carbohydrate  metabolic genes , diacylglycerol kinase, ali-esterase, pectin-related genes and galactosyl transferase  likely involved in cell wall synthesis and extension, plant defense, cellulose, hemicellulose, lignin and pectin formation were found.
in pinus taeda  <cit>  and in aspen  <cit> , several pectin esterases, carbohydrate genes and transcription factors highly expressed in woody tissues were found.
additionally, studies with drought have found differentially expressed genes from cell wall and carbohydrate biosynthetic processes which respond greatly to drought stress and enhance mechanical resistance of drought-exposed cells  <cit> .
also, several kind of stress in different plants have shown up- and down-regulation of metabolic pathways such as carbon metabolism, sucrose and starch synthesis in maize with drought stress  <cit> .
both, stem and branch secondary xylem indicated a high proportion of predicted genes localized in plastids and plasma membrane in t. grandis, as was found in p. nigrum stem  <cit> .
relevant biochemical pathways in secondary xylem in tectona grandis
starch and sucrose metabolism showed highest number of sequences for branch and stem secondary xylem .
traditionally, biomass production has been related with carbon partitioning and source-sink relationships within storage organs when generating sugars and increase atp for starch synthesis  <cit> .
understanding the aspects that control the assimilates distribution in plants is still a challenge, but the storage contribution of starch and sucrose from source  to sink tissues such as secondary xylem  <cit>  is essential for plant support and defense.
in the same way, galactosidases in glycan degradation were found in teak secondary xylem .
galactosidases catalyze carbohydrates, glycolipids and glycoproteins residues in plants, animals and microorganisms  <cit> .
particularly, beta-galactosidase gene has the ability to degrade cell wall fractions and act on small polysaccharide arrangements which hold galactose  <cit> .
additionally, stem secondary xylem presented irinotecan and azathioprine metabolisms , considered important plant derivatives in medical application.
irinotecan is a camptothecin-type metabolite, a plant alkaloid with antitumor properties in human gastrointestinal tract  <cit> .
azathioprine is an immunosuppressive drug used to treat autoimmune human diseases such as rheumatoid arthritis  <cit>  and to avoid organ rejection after transplant surgeries  <cit> .
stimulus response genes and heat-shock proteins
differentially expressed genes included several stimulus response genes, cell death-associated genes and phenylpropanoid biosynthetic genes .
consequently, three heat-shock proteins , a bax inhibitor  and a carboxylesterase  genes were found in stem secondary xylem with a noticeable expression by deseq .
then, quantitative real-time pcr confirmed the deseq analysis, indicating that tghsp <dig>  tghsp <dig>  tghsp <dig> and tgbi are expressed more in stem secondary xylem of 12-year-old trees compared to 60-year-old trees .
particularly, plant carboxylesterase gene has been related with fruit ripening  <cit> , but this gene could probably be related with several environmental stimulus in teak and other plants, being necessary to be more elucidated in future studies.
in addition, the bax inhibitor homologs exist in multiple eukaryotic species and translate a multi-membrane-spanning protein to provide cytoprotection against diverse stimuli and stresses, especially with h2o2− induced cell death downstream of reactive oxygen species  signaling  <cit> .
given that bax inhibitor gene in plants is related with enhanced stress tolerance and cell death suppression, it may be linked to cell death regulation in lignified tissues of tectona grandis.
in capsicum annum, bax inhibitor gene expression was induced by drought, aba, high salinity, flooding, heavy metal stresses and high or low temperatures  <cit> , which means a substantial role of tolerance to several types of environmental stresses.
also, transgenic cells overexpressing atbi- <dig> showed enhanced tolerance to cell death induced by various oxidative stress, such as h2o <dig>  salicylic acid and pathogen elicitor  <cit> .
similar to our results, during ecodormancy of quercus petraea several stress-related genes were found, including one heat shock protein , as one of the most expressed genes among all, which is regulated by aba  <cit> .
ecodormancy state occurs when temperatures rise from late winter to early spring to prevent bud burst, so heat shock proteins show chaperone activity in order to maintain the proteins in their functional conformation and prevent degradation and damage during heat stress  <cit> .
curiously, genes encoding enzymes related to heat stress and heat-shock proteins showed differential expression between climacteric treatments in pyrus ussuriensis fruits  <cit> .
also,  <cit>  compared regulatory networks between primary and secondary meristems, finding common regulatory mechanisms between both stages.
the same authors described several stress-related genes playing a role in protecting the secondary xylem under stress conditions.
occasionally, sucrose synthases and glycosylases show a connection with stress-related genes, playing a role in reconverting sugars with a further transport into the cambial zone  <cit> .
one heat-shock protein acting with cell-wall related genes were reported in pinus taeda  <cit> .
particularly, tghsp <dig>  tghsp <dig>  tghsp <dig> and tgbi showed in teak young secondary xylem more expression than mature ones .
this suggests elevated rates of protein turnover in younger stages of this tree, as might be expected for actively dividing cells compared to mature tissues .
myb transcription factors revealed clustering and distinct expression during maturity
differentially expressed transcription factors during vascular development and secondary growth are of high interest due to the wood’s economic value.
also, they play roles as regulators, controlling response networks and modifying wood and fiber qualities  <cit> .
myb transcription factor family plays a fundamental role in xylem development in different plant species and it is a critical regulator of phenylpropanoid pathway  <cit>  such as arabidopsis thaliana , maize  <cit> , wheat  <cit> , and trees such as picea glauca  <cit> , pinus taeda  <cit> , eucalyptus genera  <cit>  and populus genera  <cit> .
go process annotation in the differentially expressed genes from stem secondary xylem followed by an individual examination and verification of the transcription factors annotated, led to finding four tissue-specific myb transcription factors whose function is linked to teak maturation.
to classify and predict the biological role of the four differentially expressed myb transcription factors found in the stem secondary xylem, domain protein sequence was analyzed  and clustering distances were calculated comparatively with all myb transcription factors from arabidopis thaliana and other trees .
in that sense, tgmyb <dig> is part of the myb-cc family; tgmyb <dig>  tgmyb <dig> and tgmyb <dig> are part of the r2r3-myb family with tgmyb <dig> displaying the bhlh motif .
our data show that the dna-binding domains  of t. grandis are conserved.
however, tgmyb <dig> was found in the arabidopsis myb group which participates in bhlh interactions, promoter repression and lignin biosynthesis genes , while tgmyb <dig> is in the gammyb-like group and inside the group “b” which is only present in angiosperms .
also, tgmyb <dig> is close to secondary wall biosynthesis function and protein or dna interactions .
tgmyb <dig> is outside the groups and need to be more elucidated.
this diversity between t. grandis, arabidopsis and some trees might give different roles in the secondary xylem formation.
it has been identified in poplar  <dig> myb members  <cit>  and  <dig> r2r3-myb transcription factors in arabidopsis  <cit> .
but, with the transcript expression levels by deseq  and through qrt-pcr analysis of four of the myb transcription factors in t. grandis, it was found that tgmyb <dig> and tgmyb <dig> showed more expression in secondary xylem and sapwood of mature trees than young ones, tgmyb <dig> less expression levels in lignified tissues of mature than young trees and tgmyb <dig> a down-regulation in secondary xylem and sapwood at both ages.
high expression of the arabidopsis atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  atmyb <dig>  as a simplified example, has been associated with secondary wall thickening  <cit> .
in picea glauca, pgmyb <dig>  pgmyb <dig> and pgmyb <dig>  which are proteins inside group c by the clustering analysis , were expressed in stem and root  <cit> , curiously expressed preferentially in the secondary differentiating xylem of both juvenile and mature trees.
the same authors described that some myb genes were highly expressed in apical stem, such as pgmyb <dig> and pgmyb <dig>  being subgrouped with tgmyb <dig> with high statistical support of 99 % .
the species used for the cluster analysis obtained in fig.
<dig>  are grouped separately from teak due to a bias in the specimen sampling, using  <dig>  repetitions .
indeed, tgmyb <dig> remains as an orphan unity.
in terms of distances, groups a and b present high statistical supports .
in group c is present tgmyb <dig> with a boostrap value of 77 % and separates this teak protein with the rest of the cluster.
nevertheless, the lignin biosynthesis subgroup shows a bootstrap value of 62 % , which reflects an unproportional taxon sample density.
indeed, a unique protein group with different functions can be considered.
to conclude, the t. grandis myb family structure and expression is not all that divergent from the gymnosperm and small flowering plants, such as arabidopsis thaliana.
even though there is only a 5 % increase in wood density going from 50- to 51-year-old trees compared to trees going from 8- to 9-year-old trees ,  <cit>  speculated that much of the growth characteristics and biological changes related to wood traits  should be absent in later years when sapwood gives way to the comparatively stable heartwood.
in our results, tgmyb <dig> and tgmyb <dig> are differentially expressed in secondary xylem, and highly expressed in sapwood of 60-year-old trees compared to young ones, presumably because they are key in conferring some woody properties that 12-year-old sapwood does not have.
likely, tgmyb <dig> and tgmyb <dig> could explain the transition from sapwood  to heartwood and they could be clues in enhancing the heartwood content and natural resistance as a genetic character, something desirable for teak producers.
implications and perspectives of this study
these results, the first dataset of sequences of the lamiales order and tectona genus, will open new perspectives for studies of diversity, ecology, breeding and genomic programs aiming to understand deeply the biology of this species.
in tropical zones, woody plants go through seasonal cycles with two stages: a growing period when environmental conditions are favorable and a period of non-growth in winter, and these phenological cycles have been shown to be strongly affected by an increase in the temperature, which has an impact on the biological processes  <cit> .
heat-shock proteins have a crucial role in maintaining the proteins in their functional conformation when temperatures rise, preventing degradation and damage during heat stress, from late winter to early spring  <cit> .
indeed, heat-shock proteins aid defending t. grandis against those environmental changes in the region sampled and need to be studied more, and in different seasons.
similarly, the molecular mechanism underlying regulation of wood formation in tropical forest trees remains poorly understood.
our transcriptomic study reported changes in the accumulation of up-and down-regulated genes through the maturation of t. grandis.
among all these genes, nine were chosen, quantified and validated by qrt-pcr.
the up-regulation of tgmyb <dig>  tgmyb <dig> and tgmyb <dig> in teak secondary xylem  may also be triggered by other transcription factors, especially nac master regulators  <cit> , in response to cell wall thickening, regulation of phenylpropanoid genes, changing environmental conditions prevailing between winter and spring and as a possible response to other biotic and abiotic stimuli.
it is important to take into account how the maturation of teak can influence the expression of the tgmyb <dig> and tgmyb <dig> transcription factors and a decrease of tgmyb <dig>  once they are selectively expressed in mature sapwood.
the drastic differences in wood quality comparing young to mature trees are well known, and heartwood and sapwood are considered high heritability characters, so they seem to be important features to be included in breeding programs  <cit> , particularly when short rotations, such as the brazilian ones  are targeted.
also, the quality of the juvenile wood itself will be an important target for improvement, and this can be assessed at an earlier stage, along with seeking trees that keep up fast juvenile growth speed for more years reducing the rotation age and yielding higher percentage of heartwood  <cit> .
globally, the current study provides several novel observations:  it contributes an extensive transcriptome analysis for a tropical wood with respects to secondary growth;  we achieved transcription  disparity from a gradient of young to mature secondary xylem and sapwood, identifying several tissue- and developmental stage-specific genes;  the secondary growth has unique molecular biology processes, which includes dna interacting proteins, regulators of lignin pathway, multitude of stress-related proteins, peptide transporters, carbohydrate metabolic genes and pectin formation;  our results provide for the first time differentially expressed heat-shock proteins and myb transcription factors in teak , contributing to the understanding of the molecular mechanisms in tropical wood, incentives to conduct reverse genetics and plant transformation in t. grandis, and they will aid in understanding regulatory networks of wood formation.
CONCLUSIONS
the transcriptome of t. grandis was assembled using about  <dig> million reads without a reference genome.
more than  <dig>  differentially expressed genes, including highly expressed heat-shock proteins, carbohydrate metabolic genes and myb transcription factors were obtained, with two biological replicates of  <dig> and 60-year-old trees.
analyses using deseq revealed that there are transcriptome changes in maturation of teak secondary xylem from 12- to 60-year-old trees, while enriched go groups for branch and stem secondary xylem were found similar.
in addition, this is the first attempt to assemble transcripts and characterize myb transcription factors from secondary xylem of t. grandis.
four myb transcription factors were classified and characterized, finding three of them with high expression and one down-regulated in lignified tissues.
expression patterns of three heat-shock proteins, one carboxylesterase and a bax inhibitor were also obtained, with significant correlation between deseq and qrt-pcr expression analysis.
the understanding of gene function of woody tissues in forest tree species is highly challenging due to the lack of standard tree transformation, also, due to plant size, slow growth and long generation time, which make breeding programs a very long process.
in order to contribute to assist selection of highly productive trees, next-generation sequencing has become the closest technology to identify target genes among thousands of candidates.
in conclusion, the data obtained can be used in applied and basic science along with biotechnological approaches to improve tropical trees.

 teak is the most important and highly valued commercial hardwood timber in the tropics due to its high durability, dimensional stability, heartwood-sapwood proportions, weightlessness and resistance to weathering. also, it is used for carpentry, floors, shipbuilding and agroforestry, thus becoming a high-class furniture and a standard timber in end-use classification of other tropical timbers. it is a deciduous species presenting natural populations in thailand, laos, myanmar, india and java islands. teak grows properly within 25-38 °c, between and  mm/year of rainfall, presenting the best yields under m above sea level and produces better wood quality with long dry periods, from to 5 month long. this species is the major component of the forest economies of many tropical countries. it is the only valuable hardwood that constitutes a globally emerging forest resource with a planted area of million ha and natural forest of million ha around the world, and brazil presents the largest teak reforestation in south america. due to its importance, many efforts have focused on the study of teak population variability. however, there are no genetic studies nor next-generation sequencing regarding wood formation in teak. wood comes from secondary growth, starting with the vascular cambium expansion and cell division in stems of young trees, followed by a differentiation of secondary xylem and several events such as xylem cells expansion, secondary cell wall deposition and programmed cell death. in most tropical america, including brazil, wood harvesting occurs at 20 years, producing small-dimension logs, which are not in demand on the international market. teak is not a fast growing species but can produce a timber of optimum strength in relatively short rotations of 21 years depending of the sapwood-heartwood percentages. the timber quality produced will be the overriding commercial factor for the near future, and usually relates to the amount, color and durability of the heartwood. for that reason, techniques such as ests and microarrays have been used extensively to understand wood formation in trees such as pinus and populus. however, today, large-scale studies of biological phenomena are unthinkable without the use of next-generation sequencing technologies, such as rna sequencing, which encourages developmental and genomics research of woody growth in trees, especially for species without a sequenced genome and no molecular information available as teak. in tropical trees, the use of next-generation sequencing in order to find differentially expressed unigenes involved in secondary xylem is restricted to some species. availability of nondestructive wood analysis methods such as core sampling would provide a valuable way to study teak wood in different aspects and avoid depletion of both natural and plantation teak resources. heartwood and sapwood are complex tissues in which percentages are not easily assessed on standing trees, but they can be determined from a bore core. also, their study in the area of molecular biology is challenging because of their rigid woody tissues with high contents of polysaccharides, which hinders its maceration and extraction of genetic material. the sapwood is a heterogeneous tissue with a mixture of earlywood and latewood and differing levels of lignification. sapwood is composed of xylem and other dead as well as living cells, reserves of starch or sugar and lower extractives content. the same author explains that a larger proportion of sapwood is preferred in wood for pulp manufacture and preservative treatment, and heartwood is desirable in construction timber, high quality veneers and joinery because of its resistance to biotic attack and darker color. in a cross-section of logs, sapwood is usually observed as a pale annulus surrounding concentric heartwood. in teak, it is certainly needed to identify genes such as those controlling secondary xylem, vessel formation, sapwood and heartwood differentiation, volume growth and abiotic stress. those studies have been documented in populus tremula, populus euphratica, populus trichocarpa, eucalyptus, conifers, and fraxinus spp., but it needs to be done in teak to help improving wood quality, growth speed and environmental adaptability. the expression of several genes has been related to the wood formation processes, including some families of transcription factors. the myb transcription factors have been related to the coordination of genes which drive the lignin biosynthesis, with a great range of regulation and operating at all points of the phenylpropanoid pathway. the r2r3-myb proteins belong to a large family of transcription factors with over members in angiosperms, also defined by an n-terminal dna- binding domain, a c-terminal modulator region with regulatory activity; also r2r3-myb proteins show a potential of binding ac elements, which belong to the most abundant type in plants with essential roles in vascular organization. therefore, genetic examination of the superior growth of a prized woody plant such as t. grandis would provide a collection of expressed genes from several tissues, as it has been done in another forestry species such as eucalyptus, where a digital expression profiling of xylogenic and non-xylogenic tissues was obtained via rna-seq. a better understanding of secondary xylem formation is essential not only as a fundamental part of plant biology, but also because it is crucial to obtain solutions for problems in forest conservation, improving the offerings of woody products. also, it is hoped that through genetic selection and plant transformation, the non-durable core could be reduced or eliminated, the growth could be increased and the epicormic branches could be controlled, making the so-called “juvenile wood” problem a thing of the past. sapwood/hardwood characteristics are reliable predictors of overall genetic improvement of timber strength. therefore, this is the first rna sequencing in this tropical woody plant. firstly, the aim of this study was to unveil the transcriptome of teak at a large-scale to later compare the transition of young to mature trees in order to reveal differentially expressed transcripts since this transition gives wood strength, endurance, color differences, natural chemicals and biotic and abiotic resistance to older trees, important features in the teak market. we detected transcripts in stem secondary xylem and found that more than unigenes were differentially expressed in a temporal and tissue specific fashion. we also supplied several heat-shock proteins and analyzed the expression of some myb-related transcription factors differentially expressed in teak secondary xylem, including sapwood tissue.  quality of the rna and the reads based on the bioanalyzer results, all samples showed appropriate rin factor. the libraries had a size of 280 bp, approximately. we generated almost million paired-end reads, covering gigabases of sequence data with a sequence length of 100 bp. the dataset of raw reads was deposited in ncbi sra database under sra study number srp after cleaning the data with the “trimmed” procedure, the “per base quality”, “per base sequence content”, “per sequence gc content”, “per sequence quality”, “duplication levels” and “sequence length distribution” were improved. then,  % of the reads, and between  % and  % were lost after cleaning. more than million sequence reads with a size of gigabases were obtained. consequently, with this quality it was possible to continue the subsequent analyses.fig. teak tissue and organ sample set. a in vitro seedling. b) in vitro leaf. c) in vitro root. d) flower. e) stem secondary xylem. f) branch secondary xylem. g) use of pressler core barrel at diameter breast high. h) core sample containing “s” and “h”. all samples were immediately placed on aluminum foil and transported in liquid nitrogen for a subsequent rna extraction de novo assembly the assembly of the transcriptome from the leaf, root, seedling, flower, secondary xylem of teak branch and stem was performed using the trinity assembler. for lignified tissues such as branch secondary xylem of both tree ages, we used between, and, reads, and for stem secondary xylem of both tree ages we used between, and, reads. flower, leaf, root and seedling were,,, and, reads, respectively. unpaired reads were from, to, in all samples. using those reads as input for trinity, we obtained,, and contigs for stem secondary xylem, branch secondary xylem, non-lignified tissues and unpaired reads, respectively,, with a mean for n length of  bp. contigs coming from lignified samples were subsequently used for differential expression analyses. unigenes differentially expressed in lignified tissues between 12- and 60-year-old trees differentially expressed transcripts in all the comparison groups with deseq program were obtained with a false discovery rate of. in the case of the branch secondary xylem transcripts differentially expressed from both 12- and 60-year-old teak trees with repetitions, the dispersion plot showed the presence of significant genes differentially expressed between both ages, showing a normalized grouping tendency in most of the transcripts with the fitted curve. also, in additional file 4b all the differentially expressed transcripts are exposed in red dots. the dispersion plot of stem secondary xylem transcripts differentially expressed from both 12- and 60-year-old teak trees showed a normalized grouping tendency with a fitted curve. several differentially expressed transcripts in stem secondary xylem were also obtained. additionally, looking for differentially expressed genes between all branch and stem samples, the contrast between both tissues is clear. as well, additional file exhibited almost the same quantity of differentially expressed and shared genes between both tissues. when plotting stem and branch against non-lignified tissues, still stem exhibited more genes differentially expressed compared to branch. finally, with deseq, we obtained and differentially expressed genes for stem and branch secondary xylem, respectively, when comparing 12- and 60-year-old trees. the dataset of differentially expressed genes was deposited in ncbi tsa database under tsa study number gdlt also, differential expression between branch and stem secondary xylem, stem secondary xylem against non-lignified tissues and branch secondary xylem against non-lignified tissues provided, and genes, respectively.fig. venn diagram showing number of differentially expressed genes in the different tissues and ages. for the diagram, we used leaf, flower, root, seedling, stem and branch secondary xylem, comparing young and mature trees for the last two tissues functional annotations of unigenes differentially expressed in lignified tissues from the and differentially expressed transcripts for stem and branch secondary xylem, respectively, an annotation of and genes was achieved with a known function by blast2go, respectively. among the genes annotated for stem secondary xylem, 48 % exhibited strong homology. also, for the same tissue, the similarity distribution showed that 89 % of the genes have more than 60 % identity with other plants and for the species distribution, t. grandis had the greatest number of matches with vitis vinifera, followed by glycine max, theobroma cacao and populus trichocarpa. on the other hand, from the genes annotated for branch secondary xylem, 33 % revealed an homology with e-value smaller than 1e- and in the identity comparison showed that 92 % of the genes have more than 60 % identity with other plants. most of the differentially expressed genes had a size between and  bp. gene ontology tool classified the unigenes in several sub-categories for biological process, cellular component and molecular function. in stem secondary xylem, catabolic process, cellular protein modification process, response to stress and carbohydrate metabolic process represented the most abundant sub-categories in the biological process category, indicating the expression of genes related to catabolic activities and stress, where several heat-shock proteins were found. under the molecular function category, the top sub-categories were nucleotide and protein binding, where three r2r3-mybs and one cc-myb transcription factors were found and used for subsequent analysis. in the cellular component category, plastid and protein complex were the most abundant. in branch secondary xylem, all categories showed similar results to stem secondary xylem, except for the protein transport through plasma membrane function. catabolic process and response to stress, nucleotide and protein binding, plastid and plasma membrane are the main categories for both tissues. further, three heat-shock proteins, one carboxylesterase and one bax inhibitor with significant up-regulation were found in stem secondary xylem, and subsequent expression analyzes of these genes were performed.fig. homology analysis of t. grandis differentially expressed unigenes. branch secondary xylem: a e-value distribution. b) similarity distribution. c) species distribution. stem secondary xylem: d) e-value distribution. e) similarity distribution. f) species distribution fig. gene ontology assignment for the unigenes differentially expressed of t. grandis stem secondary xylem. go assignments as predicted for a biological process, molecular function and cellular components. the number of unigenes assigned to each go term is shown behind semicolon metabolic pathways of unigenes beyond finding transcription factors, heat-shock proteins and annotating genes from secondary xylem from teak, we searched for pathways related to those differentially expressed genes. for branch secondary xylem between both ages, paths were identified in the annotated genes, the most relevant of which, due to number of sequences, were starch and sucrose, amino sugar and purine metabolism. in the case of stem secondary xylem between both ages, metabolic pathways were identified for all annotated differentially expressed genes. starch and sucrose, glycerol lipid and purine metabolism presented the highest number of sequences. also, some relevant metabolisms were found, such as irinotecan and azathioprine-mercaptopurine metabolisms, with the genes located inside the pathway. the ali-esterase has  bp. another relevant gene obtained from the gene ontologies and metabolic pathways is the beta-galactosidase 17-like involved in glycan degradation.fig. a irinotecan metabolism with the teak ali-esterase enzyme in brown. azathioprine-mercaptopurine metabolism with the teak phosphoribosyltransferase enzyme in blue clustering analysis of the teak r2r3-myb gene family members in order to find phylogenetic relationships between r2r3-myb members of different plant species and teak, we performed clustering analysis. indeed, tgmyb protein showed a predicted coiled-coil domain, a subtype within the myb superfamily, as defined by. tgmyb tgmyb and tgmyb were consistent with the consensus dna-binding domain sequences defined for r2r3-myb family, finding r2r motifs similar to those found in arabidopsis, gymnosperm and angiosperm plants. tgmyb tgmyb and tgmyb presented the wtx1eedx2lx3vx4gx6w and the rx4cx1lrwx3lx1p conserved motifs within the r region. tgmyb and tgmyb presented the tx2eex2lix2hx3gnkw motif, tgmyb presented the bhlh protein-binding motif and tgmyb tgmyb and tgmyb presented the pgrx2nx1ikx2wn motif, all in the r region. using the complete r2r3-myb family from arabidopsis, a dendrogram was obtained to elucidate functional grouping which could also be present in the teak myb family. tgmyb is located in the epidermal cell fate group, and closely-related to the flavonol glycosides group and c repressor motif group, the members of which participate in bhlh interactions and promoter repression. tgmyb is inside the gamyb-like genes group, which are microrna-regulated genes that facilitate anther development. additionally, tgmyb seems to share a common ancestor with atmyb which do not have related function yet. however, it is unclear how both proteins are grouped, one being cc-myb and r2r3-myb type. furthermore, using gymnosperm and angiosperm protein sequences to characterize teak mybs transcription factors, we schemed the three major groups and subgroups of r2r3-mybs as described by bedon et al.. therefore, tgmyb fell into group a, subgroup, which presents motifs involved in protein or dna interactions. also, tgmyb is close to subgroup, consistent with fig.  indeed, tgmyb could be related with cell wall formation. tgmyb is found in group b, subgroup, similar clustering when using all arabidopsis myb transcription factors. group b was previously described as being present only in angiosperms. tgmyb is presented as a separate unit and located inside group c. group c is also composed by subgroups and lignin biosynthesis sequences atmyb atmyb atmyb pgmyb pgmyb and ptmyb tgmyb is still apart from the r2r myb proteins, being clustered with atmyb atmyb and atmyb, as found in the arabidopsis grouping, as expected. altogether, although r2r motifs have several differences in t. grandis sequences, they grouped closely to secondary wall biosynthesis genes from other species.fig. integrated dendrogram of the arabidopsis r2r myb proteins with teak myb proteins. consensus circular tree was conducted by neighbor-joining method and bootstraps using mega software. teak myb proteins are denoted with red dots. each functional group is colored. references for myb gene functions are defined by previous reports fig. dendrogram of gymnosperm and angiosperm r2r3-myb proteins. the neighbor-joining method was used using bootstraps with several spruce, pine, arabidopsis and teak protein myb sequences. teak myb proteins are denoted with a diamond. the bar indicates the evolutionary distance of  %. arabidopsis proteins were chosen as landmarks indicating the three main groups and subgroups defined by gene expression of myb transcription factors in teak quantitative real-time pcr analysis showed that four teak mybs are differentially expressed in lignified tissues, being tgmyb tgmyb tgmyb up-regulated and tgmyb down-regulated. in leaves and roots, tgmyb tgmyb and tgmyb showed almost no expression levels compared to lignified tissues. tgmyb was expressed much higher in leaves than the other tissues, and stem secondary xylem of both ages is shown as down-regulated. the up-regulated genes tgmyb and tgmyb showed comparatively higher expression in stem secondary xylem and sapwood in mature compared to young trees. inversely, tgmyb expression is 2-fold higher and 60-fold higher in stem secondary xylem and sapwood, respectively, of young teak trees. the down-regulated gene tgmyb showed similar expression pattern in stem secondary xylem and sapwood of trees from both ages, although in the deseq expression level stem secondary xylem from 60-year-old trees showed almost 150-fold less expression compared to 12-year-old trees. branch secondary xylem of 12-year-old trees seems to have considerable expression levels in tgmyb and tgmyb genes compared to leaves, but similar expression compared to stem secondary xylem at both ages, with a 95 % statistical confidence level. these results confirm that the unigenes obtained from the transcriptome assembly were differentially expressed, with differences between both ages. moreover, the real-time pcr is in agreement with deseq results for tgmyb tgmyb and tgmyb although tgmyb displayed a down-regulated expression in both methods for all tissues when compared with leaf, this gene showed a discrepancy for secondary xylem down-regulated expression at both ages due to the differences of the methods. overall, the rna-seq data was biologically validated by the quantitative real-time pcr analysis.fig. expression patterns of four myb transcription factors with the deseq method. we chose four myb transcription factors from the differentially expressed unigenes obtained when comparing stem secondary xylem from mature and young trees. ± means se of two biological replicate samples were included. the fold changes of the genes were calculated as the log value fig. expression of teak myb genes with the qrt-pcr method. relative quantification of expression was examined in different tissues. the name of each gene is indicated at the top of each histogram. tissues considered are shown at the bottom of the diagrams. ± means se of three biological replicate samples. *p <  according to f-test. y-axis indicates the relative expression level of each gene compared to the control tissue. ef1α was the endogenous control used according to fig. relative expression levels of teak myb genes in sapwood with the qrt-pcr method. the name of each gene is indicated at the top of each histogram. tissues considered are shown at the bottom of the diagrams. ± means se of three biological replicate samples. *p <  according to f-test. y-axis indicates the relative expression level of each gene compared to the control tissue. ef1α was the endogenous control used according to gene expression of heat-shock proteins, carboxylesterase and bax inhibitor transcripts in teak expression analysis presented tghsp tghsp tghsp tgbi and tgces as differentially expressed transcripts, being up-regulated in lignified tissues. all five genes presented almost null expression in leaves and roots compared to secondary xylem of stem and branch, and all the genes presented more expression in stem compared to branch secondary xylem. tghsp tghsp tghsp and tgbi showed higher expression in stem secondary xylem of 12-year-old trees compared to 60-year-old trees, with 2-fold, 2-fold, 4-fold and 3-fold more transcripts by deseq method, respectively, and 5-fold, 4-fold, 3-fold and 7-fold more expression by qrt-pcr method, respectively. in contrast to these results, tgces exposed more gene expression in mature teaks compared to young trees. again, the quantitative real-time pcr results are similar to the deseq expression tendencies.fig. expression patterns of three heat-shock proteins and two enzymatic genes with the deseq method. we chose three heat-shock proteins, a carboxylesterase and a bax inhibitor from the differentially expressed unigenes obtained when comparing stem secondary xylem from mature and young trees. ± means se of two biological replicate samples were included. the fold changes of the genes were calculated as the log value fig. expression of tghsp tghsp tghsp tgces, tgbi genes with the qrt-pcr method. relative quantification of expression was examined in different tissues. the name of each gene, values and tissues considered are shown at the bottom of the diagrams. ± means se of three biological replicate samples. y-axis indicates the relative expression level of each gene compared to the control tissue. ef1α was the endogenous control used according to discussion t. grandis transcriptome the high sensitivity of sequencing technologies presents the rna-seq as the preferred choice for transcriptome studies, widely replacing the microarray-based gene expression technology, the sequencing of cdna libraries, the sage and supersage analysis. despite the forestry and economic importance of t. grandis around the world, it is very poorly characterized, with only gene sequences deposited in genbank, most of them being alleles used for molecular markers. also, previous genetic studies have focused on proteomic analysis and kinetics of t. grandis. in this study, we have generated more than million sequence reads corresponding to gigabases of raw sequence data from several tissues. t. grandis without a sequenced genome and a lack of a sequenced genome in the lamiales order makes analysis of the teak rnaseq dataset more difficult. tectona grandis is a diploid species with 2n =  chromosomes. ohri & kumar   estimated the size of its genome by cytogenetic studies, finding about mbp, which is about the same and 2-fold larger than the genome of populus trichocarpa and arabidopsis thaliana, respectively. a. thaliana has at least transcription factor genes. assuming a similar proportion of transcription for t. grandis, all the transcription factors could be estimated in mbp. comparatively, million reads were obtained from phaseolus vulgaris, million reads were generated from stem-root of piper nigrum, million reads were generated from vitis vinifera, million reads were obtained in camellia sinensis and close to million reads were obtained from petroselinum crispum and isatis indigotica. in eucalyptus, pyrosequencing gave million reads. in that sense, trinity appears as a good choice to assemble de novo full-length transcripts for species without reference genome because it corrects almost 99 % of the sequencing errors. trinity is a strategy which assembles a set of unique sequences from reads aided by the creation of independent de bruijn graphs, each representing one group of sequences and assembles isoforms within the groups, running in parallel in a computational cluster. we obtained four different transcriptomes from all tissues using the trinity platform. recent studies found unigenes in isatis indigotica, unigenes from phaseolus vulgaris representing 49 mb, unigenes from petroselinum crispum and unigenes in camellia sinensis. several trees have generated significantly higher numbers of genes, such as salix matsudana with unigenes, populus trichocarpa with unigenes, populus euphratica with unigenes and fraxinus spp. with unigenes. rnaseq provided several useful unigenes differentially expressed in lignified tissues of t. grandis from the transcriptome obtained, we were able to identify differentially expressed genes with deseq program, obtaining an invaluable gene dataset of lignified tissues of teak. deseq method is a parametric approach which works with technical replicates, with the variance and mean linked by local regression, and uses the negative binomial distribution to visualize the intensity-dependent ratio of expression data. our analysis for differentially expressed genes is based in biological replicates, which allow a solid biological interpretation. we found and differentially expressed genes in stem and branch secondary xylem, respectively, between young and mature teak trees. recent studies have shown substantial differences obtaining differentially expressed genes. obtained transcripts from stem-root of piper nigrum. in stem, almost, and differentially expressed genes were obtained in populus trichocarpa, alfalfa and brassica juncea, respectively. in eucalyptus, contigs were obtained and in salix matsudana mirna stress-related differentially expressed genes. it is common to find in some treatments no more than differentially expressed genes, as the case of camellia sinensis. to compare between two general tissue types that are of interest for woody biomass production such as stem and branch, along with the comparison between young and mature trees, we properly performed the differential expression procedure with deseq program. all the differentially expressed genes in both tissues presented high homology, matched with lignified plants and presented sizes between -  bp. after annotations, the catabolic processes, response to stress, carbohydrate metabolism, protein binding, transport and plastid localization were the most abundant sub-categories. these annotations are consistent with biopolymers production, transport, storage and xylogenic-related genes as were found in the transcriptome of e. grandis × e. urophylla hybrid clone, picea glauca and populus trichocarpa. several differentially expressed genes in the transition between young to mature trees in secondary xylem include glycan degradation cell wall carbohydrate metabolic genes, diacylglycerol kinase, ali-esterase, pectin-related genes and galactosyl transferase likely involved in cell wall synthesis and extension, plant defense, cellulose, hemicellulose, lignin and pectin formation were found. in pinus taeda and in aspen, several pectin esterases, carbohydrate genes and transcription factors highly expressed in woody tissues were found. additionally, studies with drought have found differentially expressed genes from cell wall and carbohydrate biosynthetic processes which respond greatly to drought stress and enhance mechanical resistance of drought-exposed cells. also, several kind of stress in different plants have shown up- and down-regulation of metabolic pathways such as carbon metabolism, sucrose and starch synthesis in maize with drought stress. both, stem and branch secondary xylem indicated a high proportion of predicted genes localized in plastids and plasma membrane in t. grandis, as was found in p. nigrum stem. relevant biochemical pathways in secondary xylem in tectona grandis starch and sucrose metabolism showed highest number of sequences for branch and stem secondary xylem. traditionally, biomass production has been related with carbon partitioning and source-sink relationships within storage organs when generating sugars and increase atp for starch synthesis. understanding the aspects that control the assimilates distribution in plants is still a challenge, but the storage contribution of starch and sucrose from source to sink tissues such as secondary xylem is essential for plant support and defense. in the same way, galactosidases in glycan degradation were found in teak secondary xylem. galactosidases catalyze carbohydrates, glycolipids and glycoproteins residues in plants, animals and microorganisms. particularly, beta-galactosidase gene has the ability to degrade cell wall fractions and act on small polysaccharide arrangements which hold galactose. additionally, stem secondary xylem presented irinotecan and azathioprine metabolisms, considered important plant derivatives in medical application. irinotecan is a camptothecin-type metabolite, a plant alkaloid with antitumor properties in human gastrointestinal tract. azathioprine is an immunosuppressive drug used to treat autoimmune human diseases such as rheumatoid arthritis and to avoid organ rejection after transplant surgeries. stimulus response genes and heat-shock proteins differentially expressed genes included several stimulus response genes, cell death-associated genes and phenylpropanoid biosynthetic genes. consequently, three heat-shock proteins, a bax inhibitor and a carboxylesterase genes were found in stem secondary xylem with a noticeable expression by deseq. then, quantitative real-time pcr confirmed the deseq analysis, indicating that tghsp tghsp tghsp and tgbi are expressed more in stem secondary xylem of 12-year-old trees compared to 60-year-old trees. particularly, plant carboxylesterase gene has been related with fruit ripening, but this gene could probably be related with several environmental stimulus in teak and other plants, being necessary to be more elucidated in future studies. in addition, the bax inhibitor homologs exist in multiple eukaryotic species and translate a multi-membrane-spanning protein to provide cytoprotection against diverse stimuli and stresses, especially with h2o2− induced cell death downstream of reactive oxygen species signaling. given that bax inhibitor gene in plants is related with enhanced stress tolerance and cell death suppression, it may be linked to cell death regulation in lignified tissues of tectona grandis. in capsicum annum, bax inhibitor gene expression was induced by drought, aba, high salinity, flooding, heavy metal stresses and high or low temperatures, which means a substantial role of tolerance to several types of environmental stresses. also, transgenic cells overexpressing atbi- showed enhanced tolerance to cell death induced by various oxidative stress, such as h2o salicylic acid and pathogen elicitor. similar to our results, during ecodormancy of quercus petraea several stress-related genes were found, including one heat shock protein, as one of the most expressed genes among all, which is regulated by aba. ecodormancy state occurs when temperatures rise from late winter to early spring to prevent bud burst, so heat shock proteins show chaperone activity in order to maintain the proteins in their functional conformation and prevent degradation and damage during heat stress. curiously, genes encoding enzymes related to heat stress and heat-shock proteins showed differential expression between climacteric treatments in pyrus ussuriensis fruits. also, compared regulatory networks between primary and secondary meristems, finding common regulatory mechanisms between both stages. the same authors described several stress-related genes playing a role in protecting the secondary xylem under stress conditions. occasionally, sucrose synthases and glycosylases show a connection with stress-related genes, playing a role in reconverting sugars with a further transport into the cambial zone. one heat-shock protein acting with cell-wall related genes were reported in pinus taeda. particularly, tghsp tghsp tghsp and tgbi showed in teak young secondary xylem more expression than mature ones. this suggests elevated rates of protein turnover in younger stages of this tree, as might be expected for actively dividing cells compared to mature tissues. myb transcription factors revealed clustering and distinct expression during maturity differentially expressed transcription factors during vascular development and secondary growth are of high interest due to the wood’s economic value. also, they play roles as regulators, controlling response networks and modifying wood and fiber qualities. myb transcription factor family plays a fundamental role in xylem development in different plant species and it is a critical regulator of phenylpropanoid pathway such as arabidopsis thaliana, maize, wheat, and trees such as picea glauca, pinus taeda, eucalyptus genera and populus genera. go process annotation in the differentially expressed genes from stem secondary xylem followed by an individual examination and verification of the transcription factors annotated, led to finding four tissue-specific myb transcription factors whose function is linked to teak maturation. to classify and predict the biological role of the four differentially expressed myb transcription factors found in the stem secondary xylem, domain protein sequence was analyzed and clustering distances were calculated comparatively with all myb transcription factors from arabidopis thaliana and other trees. in that sense, tgmyb is part of the myb-cc family; tgmyb tgmyb and tgmyb are part of the r2r3-myb family with tgmyb displaying the bhlh motif. our data show that the dna-binding domains of t. grandis are conserved. however, tgmyb was found in the arabidopsis myb group which participates in bhlh interactions, promoter repression and lignin biosynthesis genes, while tgmyb is in the gammyb-like group and inside the group “b” which is only present in angiosperms. also, tgmyb is close to secondary wall biosynthesis function and protein or dna interactions. tgmyb is outside the groups and need to be more elucidated. this diversity between t. grandis, arabidopsis and some trees might give different roles in the secondary xylem formation. it has been identified in poplar myb members and r2r3-myb transcription factors in arabidopsis. but, with the transcript expression levels by deseq and through qrt-pcr analysis of four of the myb transcription factors in t. grandis, it was found that tgmyb and tgmyb showed more expression in secondary xylem and sapwood of mature trees than young ones, tgmyb less expression levels in lignified tissues of mature than young trees and tgmyb a down-regulation in secondary xylem and sapwood at both ages. high expression of the arabidopsis atmyb atmyb atmyb atmyb atmyb atmyb atmyb atmyb atmyb atmyb atmyb as a simplified example, has been associated with secondary wall thickening. in picea glauca, pgmyb pgmyb and pgmyb which are proteins inside group c by the clustering analysis, were expressed in stem and root, curiously expressed preferentially in the secondary differentiating xylem of both juvenile and mature trees. the same authors described that some myb genes were highly expressed in apical stem, such as pgmyb and pgmyb being subgrouped with tgmyb with high statistical support of 99 %. the species used for the cluster analysis obtained in fig.  are grouped separately from teak due to a bias in the specimen sampling, using repetitions. indeed, tgmyb remains as an orphan unity. in terms of distances, groups a and b present high statistical supports. in group c is present tgmyb with a boostrap value of 77 % and separates this teak protein with the rest of the cluster. nevertheless, the lignin biosynthesis subgroup shows a bootstrap value of 62 %, which reflects an unproportional taxon sample density. indeed, a unique protein group with different functions can be considered. to conclude, the t. grandis myb family structure and expression is not all that divergent from the gymnosperm and small flowering plants, such as arabidopsis thaliana. even though there is only a 5 % increase in wood density going from 50- to 51-year-old trees compared to trees going from 8- to 9-year-old trees, speculated that much of the growth characteristics and biological changes related to wood traits should be absent in later years when sapwood gives way to the comparatively stable heartwood. in our results, tgmyb and tgmyb are differentially expressed in secondary xylem, and highly expressed in sapwood of 60-year-old trees compared to young ones, presumably because they are key in conferring some woody properties that 12-year-old sapwood does not have. likely, tgmyb and tgmyb could explain the transition from sapwood to heartwood and they could be clues in enhancing the heartwood content and natural resistance as a genetic character, something desirable for teak producers. implications and perspectives of this study these results, the first dataset of sequences of the lamiales order and tectona genus, will open new perspectives for studies of diversity, ecology, breeding and genomic programs aiming to understand deeply the biology of this species. in tropical zones, woody plants go through seasonal cycles with two stages: a growing period when environmental conditions are favorable and a period of non-growth in winter, and these phenological cycles have been shown to be strongly affected by an increase in the temperature, which has an impact on the biological processes. heat-shock proteins have a crucial role in maintaining the proteins in their functional conformation when temperatures rise, preventing degradation and damage during heat stress, from late winter to early spring. indeed, heat-shock proteins aid defending t. grandis against those environmental changes in the region sampled and need to be studied more, and in different seasons. similarly, the molecular mechanism underlying regulation of wood formation in tropical forest trees remains poorly understood. our transcriptomic study reported changes in the accumulation of up-and down-regulated genes through the maturation of t. grandis. among all these genes, nine were chosen, quantified and validated by qrt-pcr. the up-regulation of tgmyb tgmyb and tgmyb in teak secondary xylem may also be triggered by other transcription factors, especially nac master regulators, in response to cell wall thickening, regulation of phenylpropanoid genes, changing environmental conditions prevailing between winter and spring and as a possible response to other biotic and abiotic stimuli. it is important to take into account how the maturation of teak can influence the expression of the tgmyb and tgmyb transcription factors and a decrease of tgmyb once they are selectively expressed in mature sapwood. the drastic differences in wood quality comparing young to mature trees are well known, and heartwood and sapwood are considered high heritability characters, so they seem to be important features to be included in breeding programs, particularly when short rotations, such as the brazilian ones are targeted. also, the quality of the juvenile wood itself will be an important target for improvement, and this can be assessed at an earlier stage, along with seeking trees that keep up fast juvenile growth speed for more years reducing the rotation age and yielding higher percentage of heartwood. globally, the current study provides several novel observations: it contributes an extensive transcriptome analysis for a tropical wood with respects to secondary growth; we achieved transcription disparity from a gradient of young to mature secondary xylem and sapwood, identifying several tissue- and developmental stage-specific genes; the secondary growth has unique molecular biology processes, which includes dna interacting proteins, regulators of lignin pathway, multitude of stress-related proteins, peptide transporters, carbohydrate metabolic genes and pectin formation; our results provide for the first time differentially expressed heat-shock proteins and myb transcription factors in teak, contributing to the understanding of the molecular mechanisms in tropical wood, incentives to conduct reverse genetics and plant transformation in t. grandis, and they will aid in understanding regulatory networks of wood formation.  the transcriptome of t. grandis was assembled using about million reads without a reference genome. more than differentially expressed genes, including highly expressed heat-shock proteins, carbohydrate metabolic genes and myb transcription factors were obtained, with two biological replicates of and 60-year-old trees. analyses using deseq revealed that there are transcriptome changes in maturation of teak secondary xylem from 12- to 60-year-old trees, while enriched go groups for branch and stem secondary xylem were found similar. in addition, this is the first attempt to assemble transcripts and characterize myb transcription factors from secondary xylem of t. grandis. four myb transcription factors were classified and characterized, finding three of them with high expression and one down-regulated in lignified tissues. expression patterns of three heat-shock proteins, one carboxylesterase and a bax inhibitor were also obtained, with significant correlation between deseq and qrt-pcr expression analysis. the understanding of gene function of woody tissues in forest tree species is highly challenging due to the lack of standard tree transformation, also, due to plant size, slow growth and long generation time, which make breeding programs a very long process. in order to contribute to assist selection of highly productive trees, next-generation sequencing has become the closest technology to identify target genes among thousands of candidates. in conclusion, the data obtained can be used in applied and basic science along with biotechnological approaches to improve tropical trees. 
analysis of stem and branch secondary xylem indicates substantial similarity in gene ontologies including carbohydrate enzymes, response to stress, protein binding, and allowed us to find transcription factors and heat-shock proteins differentially expressed. expression patterns of myb transcription factors and heat-shock proteins in lignified tissues are dissimilar when tree development was evaluated, obtaining more expression of tgmyb and tgmyb in lignified tissues of 60-year-old trees, and more expression in tghsp tghsp tghsp and tgbi in stem secondary xylem of 12-year-old trees. these results will open new perspectives for studies of diversity, ecology, breeding and genomic programs aiming to understand deeply the biology of this species. investigation of some of the key regulators of lignin biosynthesis in teak, however, could be a valuable step towards understanding how rigidity of teak wood and extractives content are different from most other woods. considering how important the secondary xylem and sapwood transition from young to mature trees is, little is known about the expression differences between those successional processes and which transcription factors could regulate lignin biosynthesis in this tropical tree. currently, tectona grandis is one of the most valuable trees in the world and no transcript dataset related to secondary xylem is available. we present a widely expressed gene catalog for t. grandis using illumina technology and the de novo assembly. tgmyb tgmyb and tgces presented higher expression in mature secondary xylem, in contrast with tgmyb tghsp tghsp tghsp and tgbi whose expression is higher in young lignified tissues. although myb transcription factors are one of the largest superfamilies in plants related to secondary metabolism, it has not yet been characterized in teak. the obtained transcriptome data represents new sequences of t. grandis deposited in public databases, representing an unprecedented opportunity to discover several related-genes associated with secondary xylem such as transcription factors and stress-related genes in a tropical tree. a total of transcripts were obtained, with and genes differentially expressed for stem and branch secondary xylem, respectively, during age transition. tgmyb is expressed at lower level in secondary xylem.
 currently, tectona grandis is one of the most valuable trees in the world and no transcript dataset related to secondary xylem is available. considering how important the secondary xylem and sapwood transition from young to mature trees is, little is known about the expression differences between those successional processes and which transcription factors could regulate lignin biosynthesis in this tropical tree. although myb transcription factors are one of the largest superfamilies in plants related to secondary metabolism, it has not yet been characterized in teak. these results will open new perspectives for studies of diversity, ecology, breeding and genomic programs aiming to understand deeply the biology of this species.  we present a widely expressed gene catalog for t. grandis using illumina technology and the de novo assembly. a total of transcripts were obtained, with and genes differentially expressed for stem and branch secondary xylem, respectively, during age transition. analysis of stem and branch secondary xylem indicates substantial similarity in gene ontologies including carbohydrate enzymes, response to stress, protein binding, and allowed us to find transcription factors and heat-shock proteins differentially expressed. tgmyb displays a myb domain and a predicted coiled-coil domain, while tgmyb tgmyb and tgmyb showed r2r3-myb domain and grouped with mybs from several gymnosperms and flowering plants. tgmyb tgmyb and tgces presented higher expression in mature secondary xylem, in contrast with tgmyb tghsp tghsp tghsp and tgbi whose expression is higher in young lignified tissues. tgmyb is expressed at lower level in secondary xylem.  expression patterns of myb transcription factors and heat-shock proteins in lignified tissues are dissimilar when tree development was evaluated, obtaining more expression of tgmyb and tgmyb in lignified tissues of 60-year-old trees, and more expression in tghsp tghsp tghsp and tgbi in stem secondary xylem of 12-year-old trees. we are opening a door for further functional characterization by reverse genetics and marker-assisted selection with those genes. investigation of some of the key regulators of lignin biosynthesis in teak, however, could be a valuable step towards understanding how rigidity of teak wood and extractives content are different from most other woods. the obtained transcriptome data represents new sequences of t. grandis deposited in public databases, representing an unprecedented opportunity to discover several related-genes associated with secondary xylem such as transcription factors and stress-related genes in a tropical tree. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 