BACKGROUND
coffee is the single most important tropical commodity traded worldwide and is a source of income for many developing countries in tropics  <cit> .
in the coffee genus, coffea arabica accounts for approximately 70 % of total production worldwide, estimated at  <dig>  million tons in  <dig>  <cit> .
coffee production is subject to regular fluctuations mainly due to the natural biennial cycle but also caused by adverse climatic effects.
among them, drought is a widespread limiting factor and affects flowering and bean development, hence coffee yield  <cit> .
marked variations in rainfall also increase bean defects and modify the biochemical composition of beans, hence the final quality of the beverage  <cit> .
periods of drought may become more pronounced as a consequence of global climate change and geographical coffee growing regions may shift considerably, leading to environmental, economic and social problems  <cit> .
in such a context, the creation of drought-tolerant coffee varieties has now become a priority for coffee research.
genetic variability for drought tolerance exits in the coffee genus, particularly in coffea canephora  <cit>  but also in c. arabica  <cit> .
although molecular mechanisms of drought tolerance have been widely studied in model plants  <cit> , they are less well understood in coffee sp.
in a previous study analysing the effects of drought on gene expression, we recently identified a set of  <dig> genes differentially expressed in the leaves of drought-tolerant and drought-susceptible clones of c. canephora grown in the greenhouse under control  and drought conditions  <cit> .
in that case, the expression of genes encoding glycine-rich proteins, heat shock proteins, dehydrins, ascorbate peroxidase, as well as trans-acting factors , for example, increased under drought conditions.
in coffea sp., est resources have been developed for various species and tissues including roots, leaves, and fruits .
however, no genomic resources are available for shoot apices, which are considered as key organs for plant development by integrating several signals, such as environmental stimuli as well as hormones  and transcription  <cit> .
on the other hand, next-generation sequencing  provides new opportunities to study transcriptomic responses and to combine high-throughput sequencing with the functional annotation capacity of generated ests  <cit> .
in order to identify candidate genes involved in drought tolerance in coffee plants, we collected the shoot apices from drought-tolerant iapar <dig> and drought-susceptible rubi cultivars of c. arabica under control and drought conditions to generate libraries that were sequenced using the gs-flx titanium strategy.
a reference full transcriptome was annotated and compared to pre-identify genes differentially expressed between cultivars and drought conditions.
the transcription profiles of these genes were further analysed by qpcr in the plagiotropic buds of these plants.
methods
plant material
we compared two cultivars of coffea arabica, the drought-susceptible  rubi mg <dig>  and the drought-tolerant  iapar <dig> .
rubi did not undergo recent introgression with c. canephora genomic dna, while iapar <dig> is the result of a cross between the timor hybrid ht832/ <dig> and the villa sarchi cultivar  <cit> .
field experiment
seeds of these two commercial cultivars came from fruits harvested in may  <dig> in the coffee experimental fields of the institute for research and rural assistance  and germinated  in greenhouse of this institute.
five-month-old plantlets of the rubi and iapar <dig> were then planted  in a field experiment  at the cerrado agricultural research center  under full-sunlight conditions in two blocks of  <dig> plants for each cultivar.
under the conditions of the cerrado climate  <cit> , the rainfall pattern is divided into a dry season  followed by a wet season  that concentrates more than 80 % of annual precipitations.
for each cultivar, one control  block was irrigated while the drought  block was not irrigated during the dry seasons.
for the control condition, irrigation was supplied by sprinklers  set up in the field in such a way that irrigation was uniform.
soil water content was monitored using pr <dig> profile probes , and irrigation was applied regularly so as to maintain a moisture content above  <dig>  cm <dig> h2o.cm- <dig>
sampling
for both cultivars and experiments, leaf predawn water potentials  were measured once a week during the  <dig> dry season  of  and only once in  <dig>   using a scholander-type pressure chamber  in fully expanded leaves  from the third pair from the apex of plagiotropic branches located in the upper third of the plant canopy.
for  <dig> sequencing, between  <dig> and  <dig> shoot apices were collected  from three different plants at the end of the dry season from rubi and iapar <dig> under the control and drought conditions, and further dissected to isolate the shoot apex .
for microscopic analyses, leaves identical to those used for Ψpd measurements were also collected from the same plants.
at the end of the  <dig> dry season, Ψpd were measured once for rubi and iapar <dig> plants under control and drought treatments, and shoot apices were collected  for gene expression analyses .fig.
<dig> tissue dissection of plagiotropic buds.
a the plagiotropic buds  were collected from plants during the  <dig> dry season and used to extract rna for qpcr expression analysis.
b meristem and leaf primordium dissected from plagiotropic buds harvested during the  <dig> dry season and used to extract rna for pyrosequencing.
the dotted circles show the position of meristem and leaf primordium.
the same scale  is used for both documents
rna isolation, dna synthesis and 454-sequencing
the plagiotropic buds were incubated for 5 min in the washing buffer   <cit>  and further incubated twice for 30 min under a vacuum in the fixation buffer  then cooled to 4 °c.
samples were stored in 75 % rnase-free ethanol.
for the control and drought conditions, shoot apices  of three different plants were separated from plagiotropic buds under a binocular microscope by dissection and then ground to powder in liquid nitrogen using a pestle and mortar.
total rna was extracted using the nucleospin rna plant kit , including a dnase-i treatment.
the quality and quantity of rna were checked with a bioanalyzer .
the 1st strand cdna synthesis was performed using 1 μg total rna and the smarter™ pcr cdna synthesis kit .
double-stranded dna was then produced for each library .
for each sample, dna  was nebulized to a mean fragment size of 650 bp, ligated to an adapter using standard procedures  <cit>  and then sequenced by performing two runs  using gs-flx titanium  which generated one million reads corresponding to more than 255 mb.
transcriptome assembly and automatic annotation
all 454-sequencing reads were inspected for low quality reads and  <dig> adapters that were identified by ssaha <dig> software  <cit> .
a reference full transcriptome was then built using c. arabica reads originating from the present project and from the brazilian coffee genome project  available in the genbank public database  <cit> .
the sanger and  <dig> reads were submitted for a trimming pipeline using bdtrimmer software  <cit>  that was used to exclude ribosomal, vector, low quality  and short sequences .
all sequences  were assembled using mira software  <cit> .
the contigs formed by only sanger reads were discarded from the full transcriptome assembly.
the reference full transcriptome was annotated by blast2go software version  <dig>   <cit>  using non-redundant protein , interpro and gene ontology  databases.
the same program was also used to group datasets in go according to the biological process.
further details on the automatic annotation of all contigs are provided in additional file 1: table s <dig>  the complete bioinformatic pipeline used for this work is described in additional file 2: figure s <dig>
digital gene expression analysis
the reference full transcriptome was also used to count all  <dig> reads/libraries individually by parsing the ace file generated by mira software.
the number of sequences anchored in each contig  was subjected to differential expression analysis between the libraries using deseq  <cit>  and edger  <cit>  software in the r/bioconductor package.
a unigene was considered as differentially expressed when it was identified in at least one software considering fold-change ≥  <dig>  and p-value ≤  <dig> .
the libraries were compared based on  differentially expressed genes in iapar <dig> between c  and d  conditions ,  differentially expressed genes in rubi between c and d conditions ,  differentially expressed genes in the control library between rubi and iapar <dig>  and  differentially expressed genes in the drought library between rubi and iapar <dig> .
further information about differentially expressed genes in all the libraries is given in additional file 3: table s <dig>
functional annotation of differentially expressed genes
the lists of differentially expressed genes in each analysis were separated into up and down regulated and subjected to go enrichment analysis to identify significantly enriched go slim terms  using blast2go software and a p-value ≤  <dig> .
selection of candidate genes
cagas2
a
cagrp2
a
cah2a
a
cahsp3
a
caips1
a
cajamt1
a
camas1
a
capp2
a
capsbb
a
casamt1
a
casdc1
a
caslp1
a
castk1
a
caunk1
c
caunk2
b
caunk3
b
caunk4
b
caunk5
b
caunk6
b
caunk7
b
caunk8
b
caunk9
b
caunk10
c
caunk11
b
caunk12
b
caunk13
b
caunk14
b
caunk15
b
caunk16
b
caunk17
b
caltp1
d
caltp2
d
caltp3
d
ltp
d
gene names were assigned based on the best blast hit obtained by comparing the coffee ests with public databases.
c. canephora means coffee sequences that aligned with the candidate genes using blastx searches against nr/ncbi and filtration .
genbank , atp  and sgn  accession numbers of coffee ests are also given, as well as the length of base pairs  of amplicons.
nf: no-hits found .
the size of amplicons is based on the unigene.
: candidate genes  identified during this study.
: orphan genes  previously described  <cit>  and analysed in this study.
: orphan genes  with expression already been studied in leaves of dt and ds clones of c. canephora conilon  <cit> .
: ltp-encoding genes were previously described  <cit>
real-time quantitative pcr assays
for qpcr experiments, plagiotropic buds containing shoot apices and small leaves  were immediately frozen in liquid nitrogen after collection, and stored at -80 °c before being extracted and converted into single-strand cdna as previously described  <cit> .
real-time qpcr assays were carried out using the protocol recommended for the use of  <dig> fast real-time pcr systems .
dna preparations were diluted  and tested by qpcr using cg primer pairs .
rt-qpcr was performed with 1 μl of diluted ss-dna and  <dig>  μm  of each primer in a final volume of 10 μl with sybr green fluorochrome .
the reaction mixture was incubated for 2 min at 50 °c , then for 5 min at 95 °c , followed by  <dig> amplification cycles of 3 sec at 95 °c and finally for 30 sec at 60 °c.
data were analysed using sds  <dig>  software  to determine cycle threshold  values.
the specificity of the pcr products generated for each set of primers was verified by analysing the tm  of amplified products.
pcr efficiency  was estimated using absolute fluorescence data captured during the exponential phase of amplification of each reaction with the equation e  =  -1) x  <dig>  <cit> .
efficiency values were taken into account in all subsequent calculations.
gene expression levels were normalized to expression levels of caubq <dig> as a constitutive reference.
relative expression was quantified by applying the formula −ΔΔct where Δct target = ct target gene – ct reference gene and ΔΔct = Δct target – Δct internal calibrator, with the internal reference always being the rubi-control  sample with relative expression equal to  <dig>
leaf histological analysis of cuticle
mature leaves of the iapar <dig> and rubi genotypes were fixed for 48 h in 100 mm phosphate buffer at ph  <dig> , supplemented with 1 %  glutaraldehyde, 2 %  paraformaldehyde, and 1 %  caffeine, at room temperature  <cit> .
the samples were dehydrated and embedded in technovit  <dig> resin  according to the manufacturer’s recommendations.
three-micrometer semi-thin sections were cut with glass knives on a leica rm <dig> microtome.
the resulting sections were double stained according to buffard-morel et al.
<cit> .
briefly, polysaccharides were stained dark pink with periodic acid schiff  and soluble proteins were stained blue with naphthol blue-black   <cit> .
sections were then mounted in mowiol.
the slides were observed with a leica dm <dig> microscope  under bright field or epifluorescent light .
pictures were taken with a retiga 2000r camera  and the images were processed with volocity  <dig> .
<dig> .
cuticle thickness was measured with the freeware image j software .
experiments were conducted on the “plate-forme d’histocytologie et imagerie cellulaire végétale ”  using microscopes belonging to the montpellier rio imaging platform .
the results are expressed as means  of  <dig> measured values.
the data were statistically processed using  an analysis of variance computer program , and  the student-newman-keuls  mean comparison test  <cit>  when the effect of the factor tested was found to be statistically significant.
a probability level of p ≤  <dig>  was considered significant for all the statistical analyses.
RESULTS
monitoring drought under field conditions
in  <dig>  leaf predawn water potential  values were similar in the leaves of irrigated rubi and iapar <dig> plants, ranging from - <dig>  to - <dig>  mpa .
this confirmed the unstressed status of these plants which were considered as the control in our experiment.
at the same time, the Ψpd values decreased gradually during the dry season in the leaves of rubi and iapar <dig> under drought conditions reaching the lowest values at the end of the dry season .
at that time, the less negative Ψpd values in iapar <dig> indicated that it had better access to soil water.
the first rains then occurred and the Ψpd values of drought-stressed plants increased almost to those measured in irrigated plants, illustrating the complete recovery of stressed plants.
in  <dig>  Ψpd was measured at the peak of the drought .
under drought conditions, both rubi and iapar <dig> had similar Ψpd values that were more negative than those measured in  <dig>  indicating more severe drought stress in  <dig> .fig.
<dig> predawn leaf water potentials  measured in plants of c. arabica.
rubi  and iapar <dig>  cultivars were grown under control  and drought  conditions.
Ψ
pd values  were measured once a week during the  <dig> dry season  .
the time scale is in days and months .
vertical bars are standard deviations  and the dashed vertical line  represents the harvest point of plagiotropic buds for rna extraction for  <dig> sequencing and leaves for microscopic analyses.
b
Ψ
pd of rubi and iapar <dig> plants  measured at the end of the 2011dry season.
in this case, Ψ
pd values ranged from - <dig>  to - <dig>  mpa for the control conditions, but were below  the range of use of a scholander-type pressure chamber for drought conditions
sequencing, assembly and annotation of the coffee shoot apex transcriptome
the final reference assembly generated a total of  <dig> ,872 bp  with coverage of  <dig> x and  <dig>  clusters, corresponding to  <dig>  contigs and  <dig>  singletons.
these data are composed of:   <dig>  clusters  from  <dig> sequences, exclusively; and   <dig>  hybrid clusters that contain  <dig> reads, and at least one contig from sanger sequencing .
the contigs formed by only sanger reads were discarded from the full transcriptome assembly.
on average,  <dig>  % and  <dig>  % of the total raw data were discarded from sanger and  <dig>  respectively, due to low quality.
after removing the adapters, these reads had a size of  <dig>  bp .
the statistical data for the sanger and  <dig> reads are listed in table  <dig> table  <dig> characteristics of reads used in this work
statistics of all reads used in this work: public sanger reads and  <dig> sequenced reads from two cultivars under two conditions.
cultivars  of c. arabica and treatments  are indicated.
the number of total reads, trimmed reads and average read length  are indicated
transcriptome annotation by blast2go using non-redundant protein  and interpro databases resulted in  <dig>  transcriptome clusters  with a known protein function,  <dig>  conserved proteins of unknown function ,  <dig>  proteins identified by interpro only  and  <dig>  unidentified proteins .
the results of the digital gene expression analysis  showed more differentially expressed genes  in the cultivars rubi  and iapar <dig>  cultivars under drought  conditions , totalling  <dig> clusters , with  <dig> clusters classified as up-regulated.
under the control  conditions, a few deg were found , corresponding to  <dig> clusters .
the comparison between control and drought conditions showed a prevalence of up-regulated genes  and a total of  <dig> deg in iapar <dig>  with  <dig>  % of total clusters, and  <dig> clusters in rubi  with  <dig>  % of total clusters.table  <dig> reads showing differential expression between cultivars and/or treatments
differentially expressed genes  were obtained with the r/bioconductor packages deseq and edger.
total deg values mean the union of deseq and edger results.
the calculation of percentage was based on total of clusters .
cultivars  of c. arabica and treatments  are indicated
the results of the gene ontology  enrichment analysis are shown in fig.
<dig> and all go enrichment data are listed in additional file 1: tables s <dig> and additional file 3: table s <dig>  for iapar <dig>  the comparison of drought and control conditions  identified over-represented go terms characterized by up-regulated genes involved in expression  and translation  processes and in the generation of precursor metabolites and energy .
for rubi, a comparison of the rub-d/rub-c libraries revealed an over-representation of the following go terms which were up-regulated: protein metabolic process , response to stress  and response to abiotic stimulus .
when comparing both cultivars under drought conditions , go terms were identified related to increased enrichment of tropism for up-regulated genes  and photosynthesis for down-regulated genes .
under the control conditions , proteins of translational machinery were identified for up-regulated genes  and photosynthesis for down-regulated genes .fig.
<dig> gene ontology  enrichment analysis on a list of differentially expressed genes up- and down-regulated under four conditions.
the calculation of fold change was based on the ratio of:  i59-d/i59-c;  rub-d/rub-c;  rub-c/i59-c; and  rub-d/i59-d. the y axis indicates the number of genes normalized by the total number of genes used in each comparison from each library.
cultivars  of c. arabica and treatments  are indicated
expression profiles of candidate genes
among the candidate genes  identified in silico as presenting up- and down-regulation, expression profiles from  <dig> of them were analysed by qpcr together with the expression of  <dig> orphan genes  and ltp genes  <cit> .
for all these genes, expression profiles were analysed in plagiotropic buds of rubi and iapar <dig> under control and drought conditions.
these results are presented in separate sections below, according to the observed expression patterns.
genes with induced expression under drought conditions
twenty-five genes showing up-regulated expression profiles under drought conditions, mainly in iapar <dig> and to a lesser extent in rubi, were identified .
this was observed for castk <dig> which encodes a putative oxidative stress response serine/threonine protein kinase with 87 % identity with a predicted protein of populus trichocarpa .
in that case, expression of this gene was highly induced by drought in the dt cultivar iapar <dig>  similar profiles were also observed for the casamt <dig> gene encoding a putative s-adenosyl-l-methionine-dependent methyltransferase and the orphan genes caunk <dig> and caunk <dig>  the latter gene had no open reading frame but presented high identity  with the sgn-u <dig> contig and also with various coffee ests mainly found in c. canephora cherries at early developmental stages .fig.
<dig> expression profiles of genes up-regulated under drought conditions.
gene expression was analysed in plagiotropic buds of rubi  and iapar <dig>  cultivars of c. arabica grown under control  and drought  conditions.
the gene names are indicated in the histograms.
transcript abundances were normalized using the expression of the caubq <dig> gene as the endogenous control.
results are expressed using rub-c as the reference sample .
values of three technical replications are presented as mean ± sd
expression of the caslp <dig> gene encoding a putative protein homologous  to a protein of nicotiana benthamiana containing a peptidase s8/subtilisin-related domain, was also higher in iapar <dig> than in rubi under drought conditions.
a similar situation was observed for the camas1gene encoding a protein of  <dig> amino acid residues sharing similarities  with momilactone a synthase-like protein from vitis vinifera  that contains a secoisolariciresinol dehydrogenase conserved domain.
similar expression profiles, characterized by high up-regulation under drought conditions particularly in iapar <dig>  were observed for the orphan genes caunk <dig>  caunk <dig>  caunk <dig>  caunk <dig>  and for capsbb  and casdc <dig> encoding a putative protein related  to the adenosylmethionine decarboxylase proenzyme of catharanthus roseus).
expression of the caunk <dig> gene was also induced under drought conditions but without significant difference in expression between the two cultivars.
interestingly, the expression profiles of orphan genes caunk <dig>  caunk <dig>  caunk <dig>  caunk <dig>  caunk <dig> and caunk <dig> were similar to that of hsp-encoding gene cahsp <dig> in the sense that gene expression was highly up-regulated under drought conditions in both cultivars.
in the case of caunk <dig>  it is worth noting that expression increased 145- and 88-fold under drought conditions in rubi and iapar <dig>  respectively.
under drought conditions, expression of the cagas <dig> gene encoding a putative protein homologous  to the arbutin synthase from rauvolfia serpentina , was slightly increased in iapar <dig> but reduced in rubi.
the cacab <dig>  cachi <dig> and caelip <dig> genes encoding a photosystem ii light harvesting chlorophyll a/b binding protein of gardenia jasminoides , a class iii chitinase of c. arabica  and an early light-induced protein  of glycine max , respectively, showed similar profiles but with lower expression in rubi than in iapar <dig>  under control and drought conditions.
lastly, expression of the capp <dig> gene encoding a putative phloem protein  <dig>  of vitis vinifera  increased under drought conditions in rubi but was quite stable in iapar <dig> under both conditions.
expression of type ii nsltp genes
the expression of type ii nsltp-encoding genes was also monitored using the primer pairs ltp-ft/ltp-r <dig> , ltp-ft/ltp-r <dig>  and ltp-f100/ltp-r <dig> recognizing all homologous genes  <cit> .
no expression of nsltp genes was detected under the control conditions in both cultivars .
however, expression of nsltp genes was highly up-regulated in iapar <dig> but not in rubi under drought conditions.
it is worth noting that the caltp1-caltp <dig> and caltp <dig> genes were co-expressed in iapar <dig>  and that the expression of cacc genes was slightly higher than that of cace genes.fig.
<dig> expression of nsltp genes.
expression of caltp1-caltp <dig> , caltp <dig>  and all  genes was analysed by qpcr in plagiotropic buds of rubi  and iapar <dig>  cultivars of c. arabica grown under control  and drought  conditions, using the ltp-ft/ltp-r <dig>  ltp-ft/ltp-r <dig> and ltp-f100/ltp-r <dig> primer pairs, respectively  <cit> .
expression levels are expressed in arbitrary units  of nsltp genes using the expression of the caubq <dig> gene as the endogenous control and rub-c  as the reference sample .
values of three technical replications are presented as mean ± sd
drought influences leaf cuticle thickness
leaf anatomical analyses were also performed, revealing that the abaxial epidermis of iapar <dig> had a thicker cuticle than rubi under drought conditions .
there was also a strong interaction between genotype and drought conditions .
for example, in the dt cultivar iapar <dig>  the abaxial epidermis cuticle thickness greatly increased under drought conditions compared with the control treatment .
however, no significant variation in abaxial epidermis cuticle thickness could be observed between the control and drought treatments for rubi leaves.fig.
<dig> comparative analysis of leaf histological cross sections of iapar <dig>  and rubi  cultivars of c. arabica under control  and drought  conditions.
samples were double stained with schiff and nbb and observed under wide field  and fluorescent microscopy .
le = lower  epidermis.
the white arrows indicate the fluorescent cuticle.
values of leaf cuticle thickness are given in table  <dig>  bars = 20 μm
leaves of iapar <dig> and rubi cultivars of c. arabica grown under control  and drought conditions were analysed to measure the cuticle thickness of the abaxial faces.
values  correspond to the average calculated from  <dig> independent measurements.
those marked with different letters are significantly different
genes with reduced expression under drought conditions
the qpcr experiments led to the identification of several genes whose expression was reduced under drought conditions .
in both cultivars, expression of the orphan genes caunk <dig> and caunk <dig>  and of the cadlp <dig> gene encoding a putative protein containing a dirigent-like protein domain homologous to the hypothetical protein  of vitis vinifera, was greatly reduced under drought conditions.
expression of the cachi <dig> gene encoding a protein homologous to the putative chitinase of catharanthus roseus , was 5-fold higher in iapar <dig> than in rubi under the control conditions but decreased under drought conditions.
however, the expression level of the cachi <dig> gene was similar in iapar <dig> and rubi under drought conditions.
for the genes cachi <dig> , caunk <dig> and cajamt <dig> , similar expression profiles were found.
in these cases, drought reduced gene expression in both cultivars but expression levels were always higher in iapar <dig> than in rubi, particularly for cajamt <dig> fig.
<dig> expression profiles of genes down-regulated under drought conditions.
gene expression was analysed in plagiotropic buds of rubi  and iapar <dig>  cultivars of c. arabica grown under control  and drought  conditions.
the gene names are indicated in the histograms.
transcript abundances were normalized using the expression of the caubq <dig> gene as the endogenous control.
results are expressed using rub-c as the reference sample .
values of three technical replications are presented as mean ± sd
gene expression levels of the cah2a , cagrp <dig>  and caunk <dig> genes, were similar in rubi and iapar <dig>  for the caaep <dig>  and caips <dig>  genes, gene expression remained high in iapar <dig> under both control and drought conditions, but decreased drastically in rubi under drought conditions.
discussion
in this study, we obtained  <dig>  mbp  of sequences with longer reads  from plagiotropic shoot apices enriched in meristems and primordium leaves of two cultivars of c. arabica under control  and drought conditions.
these sequences were assembled giving  <dig>  clusters  with a mean size ≥ 300 bp each.
these rnaseq data, which complement those already available in public databases for coffee ests , can be considered as innovative and relevant in the sense that they were produced from c. arabica tissues  that have never previously been studied  <cit> .
the transcriptome annotation by blast2go provided information based on the nomenclature and organism of origin of genes in the ncbi/nr database, the enzyme family, a functional analysis of proteins from the interpro database, and metabolic functions, biological processes and cellular location from gene ontology.
our results showed that a large percentage of transcriptome alignment had  <dig>  hits with known function ,  <dig>  genes with unknown function  both in the ncbi/nr database, and only  <dig>  hits in the interpro database , thereby enabling the identification of most genes.
with this analysis, we identified  <dig>  genes related to coffea sp.
.
we also found  <dig>  genes from solanum sp.,  <dig> genes from populus trichocarpa,  <dig> genes from vitis vinifera and  <dig> genes from arabidopsis sp.
thus, the transcriptome was aligned with several genes from different plant species and these genes may be conserved among these species, including coffea sp.
on the other hand, our results also included  <dig>  “no-hit” genes , perhaps indicating the presence of unannotated or new genes.
the comparisons of dna libraries undertaken during this work led to the identification of  <dig>  genes  with differential expression profiles in silico between the drought-susceptible  and drought-tolerant  cultivars of c. arabica with drought conditions.
the expression profiles of these genes, as well as those of other previously identified genes , were analysed by qpcr in plagiotropic buds  taken from control and drought-stressed plants of rubi and iapar <dig>  for most of the cgs identified during this work, in vivo gene expression profiles confirmed those deduced from in silico comparisons of dna libraries.
for example, this was the case for the cahsp <dig>  gene whose up-regulated expression under drought conditions can be considered as a “molecular control” of stress applied to the plants during this study and confirmed by leaf water potential  measurements.
many ests encoding putative hsps were also found in leaf cdna libraries of c. arabica  and c. canephora  plants grown under drought conditions  <cit> , heat stress  <cit> , leaf infection by hemileia vastatrix  <cit>  and also during bean development  <cit> .
our results also identified several genes differentially expressed in plagiotropic buds of iapar <dig> and rubi, as for the castk <dig> gene encoding a putative serine/threonine protein kinase containing a conserved domain  of mitogen-activated protein kinases .
these kinases are known to have a central role in the transduction of extra- and intracellular signals in plants, including cell division and differentiation, as well as in responses to various types of stress  <cit> .
in pisum sativum, there is evidence that the mapk cascade is involved in aba-regulated stomatal activity as well as aba-induced gene expression in the epidermal peels  <cit> .
in a recent study, shen et al.
<cit>  showed that the phosphorylation of oswrky <dig> protein by mapks is a key step in conferring drought tolerance in transgenic rice.
according to our results, higher castk <dig> expression under drought conditions in iapar <dig> than in rubi could enhance the mapk cascade and therefore be involved in the drought tolerance of iapar <dig>  in this cultivar, the over-expression of casamt <dig> under drought conditions is also particularly interesting because this sequence encodes a putative s-adenosyl-l-methionine-dependent methyltransferase related to the tumorous shoot development <dig>  gene.
in arabidopsis thaliana, tsd <dig> is a pleiotropic mutation that affects leaf, root and shoot meristem development  <cit> .
expression of a tsd2:: gus reporter gene has mainly been detected in meristems where this gene is essential for cell adhesion and coordinated plant development.
the weaker expression of casamt <dig> in rubi than in iapar <dig> under drought conditions, points to the existence of major developmental differences between these two cultivars.
the differential expression in rubi and iapar <dig> of the caslp <dig> gene encoding a putative subtilisin-like protein is also worth noting.
in arabidopsis, the subtilisin-like serine-protease sdd <dig>  gene was shown to be strongly expressed in stomatal precursor cells   <cit> .
in addition, sdd <dig> mutation increased leaf stomatal density  while sdd <dig> over-expression led to the opposite phenotype with decreased sd.
in c. arabica, maximum and minimum average stomatal densities were observed in full sunlight and shaded conditions respectively, providing evidence for the existence of plasticity for this characteristic in this coffee species  <cit> .
even though no sd were observed between rubi and iapar <dig> under moderate drought conditions  <cit> , the caslp <dig> expression profiles presented here do not preclude the involvement of this gene in the genetic determinism of drought tolerance in coffee.
another interesting response concerned the differential expression of the camas <dig> gene encoding a putative protein containing the conserved domain .
this domain is also found in secoisolariciresinol dehydrogenase-like proteins catalyzing the nad-dependent conversion of -secoisolariciresinol to -matairesinol, like the arabidopsis aba <dig> protein considered to be one of the key regulators of aba biosynthesis  <cit> .
based on the camas <dig> expression profiles presented here, it is possible that aba synthesis was enhanced by drought in plagiotropic buds of iapar <dig> but not  in those of rubi.
this hypothesis is also reinforced by the fact that higher cajamt <dig> expression was observed in iapar <dig> than in rubi buds.
indeed, in addition to well-known functions of jasmonates in plant defence mechanisms in response to biotic stress  <cit> , recent studies also demonstrated that methyl jasmonate stimulates aba biosynthesis under drought conditions in panicles of oryza sativa  <cit> .
higher expression of casdc <dig>  under drought conditions in iapar <dig> than in rubi is also worth noting because this enzyme catalyzes the synthesis of polyamines  involved in stress tolerance in higher plants  <cit> .
in theobroma cacao, aba and drought induced the expression of tcsamdc increasing spermine and spermidine leaf contents correlated with changes in stomatal conductance  <cit> .
more recently, samdc over-expression in transgenic rice was also shown to facilitate drought tolerance  <cit> .
investigation of polyamine levels in plagiotropic buds and leaves of iapar <dig> and rubi would be of particular interest to see if these compounds are involved in drought tolerance in coffee.
in mature plants, nuclear-encoded early-light inducible proteins  accumulate in response to various stress conditions including aba or desiccation  <cit> .
these proteins are presumed to protect the chloroplast apparatus from photo-oxidation occurring after stomatal limitation of photosynthesis  <cit> .
in a recent study, transgenic plants of medicago truncatula over-expressing the dsp <dig> gene from craterostigma plantagineum were shown to be able to recover from water deprivation better than wild type plants, thereby reinforcing the idea of using elip-encoding genes to improve abiotic stress resistance in crops  <cit> .
our results clearly highlight the increased expression of the caelip <dig> , capsbb  and cacab <dig>  genes, respectively, under drought conditions.
interestingly, the expression levels of all these genes were always higher in iapar <dig> than in rubi.
these results are also in accordance with electronic northern experiments which showed high accumulation of elip and cab-encoding ests in cdna libraries of c. arabica and c. canephora subjected to drought  <cit> .
another surprising result concerned the capsbb gene that was reverse-transcribed and detected during our qpcr experiments despite the fact that it corresponds to a chloroplast gene  <cit> .
however, preliminary analyses of a whole genome sequence of c. canephora revealed the presence of a cp47/like nuclear gene  <cit> .
interestingly, photosystem ii cp <dig> chlorophyll apoproteins encoding ests have also been reported to be expressed in c. arabica beans  <cit> , leaves infected by hemileia vastatrix  <cit>  and also in the cdna libraries  of drought-stressed coffee plants  <cit> , demonstrating increased expression of this gene under biotic and abiotic stress.
as cp <dig> and elip proteins are essential for the activity and protection of the photosynthetic apparatus  <cit> , the expression profiles reported here probably reflect a better photosynthetic and physiological status of iapar <dig> compared to rubi.
differential expression was also observed for the chitinase-encoding gene cachi <dig>  with higher expression in iapar <dig> than in rubi.
an opposite situation was observed with respect to the chitinase-encoding genes cachi <dig> and cachi <dig>  whose expression was reduced under drought conditions.
it is worth noting that the expression of these genes under drought conditions was always higher in iapar <dig> than in rubi.
these results also show that coffee chitinase-encoding genes responded in different ways to drought.
a large number of chitinase-encoding ests were identified in the bcgp project  <cit> , mainly in the sh <dig> cdna library of drought-stressed plants of c. arabica var.
catuai  <cit> , but also in the leaves of c. arabica infected by leaf rust  <cit> .
even though chitinases are defence-related enzymes induced by abiotic stress, some evidence also indicates their participation in tolerance to abiotic stress  <cit> .
even though the roles of pathogenesis-related proteins in abiotic stress are still not fully understood, dt transgenic plants over-expressing chitinase genes have been obtained  <cit> .
in that sense, the high level of expression for cachi <dig> in plagiotropic buds of iapar <dig> under both control and drought conditions could have an important function in drought tolerance.
arbutin is a phenolic glucoside  abundant in the leaves of many freezing- or desiccation-tolerant plants  <cit>  and also present in coffee fruits  <cit> .
in a previous study, down-regulation of the ccgas1gene encoding arbutin synthase was reported in leaves of c. canephora under drought conditions  <cit> .
the results presented here clearly demonstrated differential expression profiles for cagas <dig> between the two cultivars of c. arabica.
gene expression increased under drought conditions in iapar <dig> while the opposite was observed in rubi.
even though the presence of arbutin in coffee leaves has never been demonstrated, further analyses of this metabolite should be performed to investigate the role of this glucoside  in preventing cell damage in coffee subject to abiotic stresses.
the capp <dig> gene  also showed differential expression profiles, with higher expression in iapar <dig> than in rubi.
in higher plants, pp2s are sieve elements  very abundant in the phloem sap.
these proteins are believed to play an important role in the establishment of phloem-based defence mechanisms induced by insect attacks and feeding stress  <cit> , but also by wounding and oxidative conditions  <cit> .
the functions of pp <dig> proteins are still not clear but they could act by forming high molecular weight polymers to close  the sieve pores caused by external injuries mainly due to biotic stress  <cit> .
when arabidopsis was treated with hrpnea , the suppression of phloem-feeding activities by aphids was attributed to over-expression of the pp2-encoding gene atpp2-a <dig>  <cit> .
other studies showed that hrpn activated aba signalling, thereby inducing drought tolerance in arabidopsis thaliana  <cit> .
based on these results, the involvement of pp <dig> proteins in plant response mechanisms to abiotic stress can be hypothesized, for example by maintaining  the integrity of vessels under drought conditions by forming sieve plate filaments upon oxidation  <cit> .
in that case, higher synthesis of capp <dig> which would be expected to occur in iapar <dig> plagiotropic buds under drought conditions could play a role in drought-tolerance by reducing sap-flow in young leaves and consequently increasing the water use efficiency of this cultivar  <cit> .
other interesting results concerned the gene expression stability of the caaep <dig>  and caips <dig>  genes observed in iapar <dig> under control and drought conditions, whereas expression of both genes decreased under drought conditions in rubi.
plant cells use myo-inositol to synthesize a variety of low molecular weight compounds and sugar alcohols such as the galactinol, a key element in the formation of raffinose family oligosaccharides.
nishizawa et al.
<cit>  found that plants with high galactinol and raffinose contents were less susceptible to oxidative stress.
in c. arabica, up-regulation of cagols genes involved in galactinol biosynthesis was reported in leaves of plants subjected to severe drought  <cit> .
in addition, drought up-regulated the expression of mannose 6-phosphate reductase  in leaves of c. canephora  <cit>  and c. arabica  <cit> .
even though little is known about the biochemical mechanisms of drought tolerance in coffee, the accumulation of carbohydrates expected in leaves of drought-stressed plants as a consequence of the up-regulated expression of these genes, could play an important role in the genetic determinism of this phenotype in coffee  <cit> .
in addition to the previously described genes, our results also identified several orphan genes that presented differential expression profiles between the cultivars and treatments, such as caunk <dig>  caunk <dig> and caunk <dig> whose expression was highly induced under drought conditions in iapar <dig> and to a lesser extent in rubi.
orphan genes are also expected to interact specifically with the environment as a consequence of lineage-specific adaptations to that environment  <cit> .
interestingly, the expression profiles of the caunk <dig> and caunk <dig> orphan genes were very similar to those of type ii nsltp-encoding genes, with high expression mainly detected under drought conditions in plagiotropic buds of iapar <dig> but not in those of rubi.
up-regulation of ltp genes under drought conditions is well documented in higher plants .
lipid transfer proteins  are thought to be involved in the transfer of lipids through the extracellular matrix for the formation of cuticular wax  <cit> .
in fact, together with the lipophilic cutin polymer matrix, waxes enter in the composition of cuticle, which forms the first barrier between plants and environmental stresses by limiting non-stomatal water loss and gas exchanges, hence mitigating the effects of drought by controlling water loss associated with epidermal conductance  <cit> .
in nicotiana glauca, ltp genes are predominantly expressed in the guard and epidermal cells and are induced under drought conditions  <cit> , providing evidence that ltp play an important role in the development of drought tolerance.
even though the up-regulation of caltp genes observed under drought in plagiotropic buds of iapar <dig> cannot explain directly the greater thickness of leaf cuticle observed in this cultivar than in rubi, these results strongly suggested that lipid metabolism plays a major role in coffee drought tolerance.
as reported in other higher plants, our study also highlighted the differential expression of many genes encoding proteins known to be over-expressed under biotic stress , by drought.
the fact that our experiment was conducted with drought-stressed plants grown under uncontrolled  conditions, could explain such a situation.
however, it is also probable that these results reflect a biological reality since it is well known that crosstalk exists in higher plants between signalling pathways for biotic and abiotic stress responses  <cit> .
CONCLUSIONS
during this work, we produced some new transcriptomic information for c. arabica with a total of  <dig>  mbp of sequences assembled into  <dig>  clusters  from genes expressed in plagiotropic shoot apices enriched in meristems and primordium leaves in dt  and ds  cultivars grown under control and drought conditions.
major differences between these plants concerned their phenotypic behaviour  and transcriptome expression profiles.
differences between these plants affected genes of specific pathways such as those involved in abscisic acid biosynthesis, perception and transduction of drought stress, plant development and lipid metabolism.
in that sense, the present study increased the number of cgs potentially involved in the genetic determinism of drought tolerance firstly identified in c. canephora.
because c. arabica is an amphidiploid species , its transcriptome is a mixture of homologous genes expressed from these two sub-genomes in which c. eugenioides is assumed to express genes mainly for proteins involved in basal biological processes , while the c. canephora sub-genome is assumed to regulate arabica gene expression by expressing genes for regulatory proteins and adaptation processes  <cit> .
in this genetic context, it is possible that the characteristics of iapar <dig> that enable it to better withstand drought stress than rubi, really originated from the specific expression of c. canephora genes recently introgressed  in this cultivar of c. arabica  <cit> .
even though this study provides further indications about the way in which different coffee cultivars activate their transcriptomes, additional work is still required to understand how epigenetics and epistasis regulate gene expression in the different coffee sub-genomes  in c. arabica under drought conditions.
source of the plant materials and permissions
this work was carried out as part of the scientific cooperation project entitled “study of genetic determinism of drought tolerance in coffee”  approved between embrapa and cirad.
it complied with all institutional, national, or international guidelines.
in the frame of this project, field experiments were conducted at the cerrado agricultural research center  with all permissions of partners and in accordance with local legislation.
ethics approval and consent to participate
not applicable.
consent to publish
not applicable.
availability of supporting data
the reads were submitted to genbank and to the bioproject/ncbi database under the accession number prjna <dig>
additional files
additional file 1: table s <dig>  summary of blast2go automatic annotation of the transcriptome clusters.
additional file 2: figure s <dig>   complete bioinformatics pipeline of the transcriptome assembly and automatic annotation methods used in this work.
additional file 3: table s <dig>  summary of the deseq/ edger fold changes and p-values between the cultivars  and between control  and drought  conditions.
these tables also contain blast2go automatic annotation of the transcriptome clusters.
table lanes coloured in grey are related to clusters aligned to the new candidate genes tested by rt-qpcr .
abbreviations
estexpressed sequence tag
qpcrquantitative polymerase chain reaction
competing interests
the authors declare that they have no competing interests.
authors’ contributions
gcr and pm measured predawn leaf water potentials and harvested the samples.
fdb and tl performed meristem dissections, rna extractions and cdna synthesis.
lsm, mfc, gagp and rv were responsible for the bioinformatic processing of the data.
jlv, flm and ml performed the histology and microscopy analyses.
pm, aca and dp selected the candidate genes qpcr-analysed by fac, ngv, ked, jca and mgc.
gcr, aca and pm designed the study, drew up the experimental design and implemented it.
rv, mfc, gagp, aca and pm drafted the manuscript.
all the authors read and approved the final version of the manuscript.
the authors would like to thank dr aymbiré francisco almeida da fonseca from the incaper institute for kindly providing seeds of the rubi and iapar <dig> commercial cultivars of c. arabica.
the authors are also very grateful to drs antonio fernando guerra and omar cruz rocha  for their assistance during the field trial experiments.
the authors wish also to thank daphne goodfellow, as well as peter biggins and cécile fovet-rabot  for english revision of the manuscript.
funding
pm acknowledges financial support from cirad  and the cirad atp project “analysis of phenotypic plasticity in response to water constraints in perennial plants growing under different field conditions”.
aca acknowledges financial support from the brazilian coffee r&d consortium, finep and inct-café .
mfc and gagp acknowledge financial support from the center for computational engineering and sciences - fapesp/cepid .
the authors acknowledge the scholarships from the brazilian agencies capes , brazilian consortium of coffee research  and capes-cofecub project sv738- <dig> .

 coffee is the single most important tropical commodity traded worldwide and is a source of income for many developing countries in tropics. in the coffee genus, coffea arabica accounts for approximately 70 % of total production worldwide, estimated at million tons in. coffee production is subject to regular fluctuations mainly due to the natural biennial cycle but also caused by adverse climatic effects. among them, drought is a widespread limiting factor and affects flowering and bean development, hence coffee yield. marked variations in rainfall also increase bean defects and modify the biochemical composition of beans, hence the final quality of the beverage. periods of drought may become more pronounced as a consequence of global climate change and geographical coffee growing regions may shift considerably, leading to environmental, economic and social problems. in such a context, the creation of drought-tolerant coffee varieties has now become a priority for coffee research. genetic variability for drought tolerance exits in the coffee genus, particularly in coffea canephora but also in c. arabica. although molecular mechanisms of drought tolerance have been widely studied in model plants, they are less well understood in coffee sp. in a previous study analysing the effects of drought on gene expression, we recently identified a set of genes differentially expressed in the leaves of drought-tolerant and drought-susceptible clones of c. canephora grown in the greenhouse under control and drought conditions. in that case, the expression of genes encoding glycine-rich proteins, heat shock proteins, dehydrins, ascorbate peroxidase, as well as trans-acting factors, for example, increased under drought conditions. in coffea sp., est resources have been developed for various species and tissues including roots, leaves, and fruits. however, no genomic resources are available for shoot apices, which are considered as key organs for plant development by integrating several signals, such as environmental stimuli as well as hormones and transcription. on the other hand, next-generation sequencing provides new opportunities to study transcriptomic responses and to combine high-throughput sequencing with the functional annotation capacity of generated ests. in order to identify candidate genes involved in drought tolerance in coffee plants, we collected the shoot apices from drought-tolerant iapar and drought-susceptible rubi cultivars of c. arabica under control and drought conditions to generate libraries that were sequenced using the gs-flx titanium strategy. a reference full transcriptome was annotated and compared to pre-identify genes differentially expressed between cultivars and drought conditions. the transcription profiles of these genes were further analysed by qpcr in the plagiotropic buds of these plants. methods plant material we compared two cultivars of coffea arabica, the drought-susceptible rubi mg and the drought-tolerant iapar. rubi did not undergo recent introgression with c. canephora genomic dna, while iapar is the result of a cross between the timor hybrid ht832/ and the villa sarchi cultivar. field experiment seeds of these two commercial cultivars came from fruits harvested in may in the coffee experimental fields of the institute for research and rural assistance and germinated in greenhouse of this institute. five-month-old plantlets of the rubi and iapar were then planted in a field experiment at the cerrado agricultural research center under full-sunlight conditions in two blocks of plants for each cultivar. under the conditions of the cerrado climate, the rainfall pattern is divided into a dry season followed by a wet season that concentrates more than 80 % of annual precipitations. for each cultivar, one control block was irrigated while the drought block was not irrigated during the dry seasons. for the control condition, irrigation was supplied by sprinklers set up in the field in such a way that irrigation was uniform. soil water content was monitored using pr profile probes, and irrigation was applied regularly so as to maintain a moisture content above  cm h2o.cm- sampling for both cultivars and experiments, leaf predawn water potentials were measured once a week during the dry season of and only once in using a scholander-type pressure chamber in fully expanded leaves from the third pair from the apex of plagiotropic branches located in the upper third of the plant canopy. for sequencing, between and shoot apices were collected from three different plants at the end of the dry season from rubi and iapar under the control and drought conditions, and further dissected to isolate the shoot apex. for microscopic analyses, leaves identical to those used for Ψpd measurements were also collected from the same plants. at the end of the dry season, Ψpd were measured once for rubi and iapar plants under control and drought treatments, and shoot apices were collected for gene expression analyses.fig. tissue dissection of plagiotropic buds. a the plagiotropic buds were collected from plants during the dry season and used to extract rna for qpcr expression analysis. b meristem and leaf primordium dissected from plagiotropic buds harvested during the dry season and used to extract rna for pyrosequencing. the dotted circles show the position of meristem and leaf primordium. the same scale is used for both documents rna isolation, dna synthesis and 454-sequencing the plagiotropic buds were incubated for 5 min in the washing buffer and further incubated twice for 30 min under a vacuum in the fixation buffer then cooled to 4 °c. samples were stored in 75 % rnase-free ethanol. for the control and drought conditions, shoot apices of three different plants were separated from plagiotropic buds under a binocular microscope by dissection and then ground to powder in liquid nitrogen using a pestle and mortar. total rna was extracted using the nucleospin rna plant kit, including a dnase-i treatment. the quality and quantity of rna were checked with a bioanalyzer. the 1st strand cdna synthesis was performed using 1 μg total rna and the smarter™ pcr cdna synthesis kit. double-stranded dna was then produced for each library. for each sample, dna was nebulized to a mean fragment size of 650 bp, ligated to an adapter using standard procedures and then sequenced by performing two runs using gs-flx titanium which generated one million reads corresponding to more than 255 mb. transcriptome assembly and automatic annotation all 454-sequencing reads were inspected for low quality reads and adapters that were identified by ssaha software. a reference full transcriptome was then built using c. arabica reads originating from the present project and from the brazilian coffee genome project available in the genbank public database. the sanger and reads were submitted for a trimming pipeline using bdtrimmer software that was used to exclude ribosomal, vector, low quality and short sequences. all sequences were assembled using mira software. the contigs formed by only sanger reads were discarded from the full transcriptome assembly. the reference full transcriptome was annotated by blast2go software version using non-redundant protein, interpro and gene ontology databases. the same program was also used to group datasets in go according to the biological process. further details on the automatic annotation of all contigs are provided in additional file 1: table s the complete bioinformatic pipeline used for this work is described in additional file 2: figure s digital gene expression analysis the reference full transcriptome was also used to count all reads/libraries individually by parsing the ace file generated by mira software. the number of sequences anchored in each contig was subjected to differential expression analysis between the libraries using deseq and edger software in the r/bioconductor package. a unigene was considered as differentially expressed when it was identified in at least one software considering fold-change ≥  and p-value ≤ . the libraries were compared based on differentially expressed genes in iapar between c and d conditions, differentially expressed genes in rubi between c and d conditions, differentially expressed genes in the control library between rubi and iapar and differentially expressed genes in the drought library between rubi and iapar. further information about differentially expressed genes in all the libraries is given in additional file 3: table s functional annotation of differentially expressed genes the lists of differentially expressed genes in each analysis were separated into up and down regulated and subjected to go enrichment analysis to identify significantly enriched go slim terms using blast2go software and a p-value ≤ . selection of candidate genes cagas2 a cagrp2 a cah2a a cahsp3 a caips1 a cajamt1 a camas1 a capp2 a capsbb a casamt1 a casdc1 a caslp1 a castk1 a caunk1 c caunk2 b caunk3 b caunk4 b caunk5 b caunk6 b caunk7 b caunk8 b caunk9 b caunk10 c caunk11 b caunk12 b caunk13 b caunk14 b caunk15 b caunk16 b caunk17 b caltp1 d caltp2 d caltp3 d ltp d gene names were assigned based on the best blast hit obtained by comparing the coffee ests with public databases. c. canephora means coffee sequences that aligned with the candidate genes using blastx searches against nr/ncbi and filtration. genbank, atp and sgn accession numbers of coffee ests are also given, as well as the length of base pairs of amplicons. nf: no-hits found. the size of amplicons is based on the unigene.: candidate genes identified during this study.: orphan genes previously described and analysed in this study.: orphan genes with expression already been studied in leaves of dt and ds clones of c. canephora conilon.: ltp-encoding genes were previously described real-time quantitative pcr assays for qpcr experiments, plagiotropic buds containing shoot apices and small leaves were immediately frozen in liquid nitrogen after collection, and stored at -80 °c before being extracted and converted into single-strand cdna as previously described. real-time qpcr assays were carried out using the protocol recommended for the use of fast real-time pcr systems. dna preparations were diluted and tested by qpcr using cg primer pairs. rt-qpcr was performed with 1 μl of diluted ss-dna and  μm of each primer in a final volume of 10 μl with sybr green fluorochrome. the reaction mixture was incubated for 2 min at 50 °c, then for 5 min at 95 °c, followed by amplification cycles of 3 sec at 95 °c and finally for 30 sec at 60 °c. data were analysed using sds software to determine cycle threshold values. the specificity of the pcr products generated for each set of primers was verified by analysing the tm of amplified products. pcr efficiency was estimated using absolute fluorescence data captured during the exponential phase of amplification of each reaction with the equation e  =  -1) x. efficiency values were taken into account in all subsequent calculations. gene expression levels were normalized to expression levels of caubq as a constitutive reference. relative expression was quantified by applying the formula −ΔΔct where Δct target = ct target gene – ct reference gene and ΔΔct = Δct target – Δct internal calibrator, with the internal reference always being the rubi-control sample with relative expression equal to leaf histological analysis of cuticle mature leaves of the iapar and rubi genotypes were fixed for 48 h in 100 mm phosphate buffer at ph , supplemented with 1 % glutaraldehyde, 2 % paraformaldehyde, and 1 % caffeine, at room temperature. the samples were dehydrated and embedded in technovit resin according to the manufacturer’s recommendations. three-micrometer semi-thin sections were cut with glass knives on a leica rm microtome. the resulting sections were double stained according to buffard-morel et al.. briefly, polysaccharides were stained dark pink with periodic acid schiff and soluble proteins were stained blue with naphthol blue-black. sections were then mounted in mowiol. the slides were observed with a leica dm microscope under bright field or epifluorescent light. pictures were taken with a retiga 2000r camera and the images were processed with volocity.. cuticle thickness was measured with the freeware image j software. experiments were conducted on the “plate-forme d’histocytologie et imagerie cellulaire végétale ” using microscopes belonging to the montpellier rio imaging platform. the results are expressed as means of measured values. the data were statistically processed using an analysis of variance computer program, and the student-newman-keuls mean comparison test when the effect of the factor tested was found to be statistically significant. a probability level of p ≤  was considered significant for all the statistical analyses.  monitoring drought under field conditions in leaf predawn water potential values were similar in the leaves of irrigated rubi and iapar plants, ranging from - to -  mpa. this confirmed the unstressed status of these plants which were considered as the control in our experiment. at the same time, the Ψpd values decreased gradually during the dry season in the leaves of rubi and iapar under drought conditions reaching the lowest values at the end of the dry season. at that time, the less negative Ψpd values in iapar indicated that it had better access to soil water. the first rains then occurred and the Ψpd values of drought-stressed plants increased almost to those measured in irrigated plants, illustrating the complete recovery of stressed plants. in Ψpd was measured at the peak of the drought. under drought conditions, both rubi and iapar had similar Ψpd values that were more negative than those measured in indicating more severe drought stress in.fig. predawn leaf water potentials measured in plants of c. arabica. rubi and iapar cultivars were grown under control and drought conditions. Ψ pd values were measured once a week during the dry season. the time scale is in days and months. vertical bars are standard deviations and the dashed vertical line represents the harvest point of plagiotropic buds for rna extraction for sequencing and leaves for microscopic analyses. b Ψ pd of rubi and iapar plants measured at the end of the 2011dry season. in this case, Ψ pd values ranged from - to -  mpa for the control conditions, but were below the range of use of a scholander-type pressure chamber for drought conditions sequencing, assembly and annotation of the coffee shoot apex transcriptome the final reference assembly generated a total of,872 bp with coverage of x and clusters, corresponding to contigs and singletons. these data are composed of: clusters from sequences, exclusively; and hybrid clusters that contain reads, and at least one contig from sanger sequencing. the contigs formed by only sanger reads were discarded from the full transcriptome assembly. on average,  % and  % of the total raw data were discarded from sanger and respectively, due to low quality. after removing the adapters, these reads had a size of  bp. the statistical data for the sanger and reads are listed in table  table characteristics of reads used in this work statistics of all reads used in this work: public sanger reads and sequenced reads from two cultivars under two conditions. cultivars of c. arabica and treatments are indicated. the number of total reads, trimmed reads and average read length are indicated transcriptome annotation by blast2go using non-redundant protein and interpro databases resulted in transcriptome clusters with a known protein function, conserved proteins of unknown function, proteins identified by interpro only and unidentified proteins. the results of the digital gene expression analysis showed more differentially expressed genes in the cultivars rubi and iapar cultivars under drought conditions, totalling clusters, with clusters classified as up-regulated. under the control conditions, a few deg were found, corresponding to clusters. the comparison between control and drought conditions showed a prevalence of up-regulated genes and a total of deg in iapar with  % of total clusters, and clusters in rubi with  % of total clusters.table reads showing differential expression between cultivars and/or treatments differentially expressed genes were obtained with the r/bioconductor packages deseq and edger. total deg values mean the union of deseq and edger results. the calculation of percentage was based on total of clusters. cultivars of c. arabica and treatments are indicated the results of the gene ontology enrichment analysis are shown in fig.  and all go enrichment data are listed in additional file 1: tables s and additional file 3: table s for iapar the comparison of drought and control conditions identified over-represented go terms characterized by up-regulated genes involved in expression and translation processes and in the generation of precursor metabolites and energy. for rubi, a comparison of the rub-d/rub-c libraries revealed an over-representation of the following go terms which were up-regulated: protein metabolic process, response to stress and response to abiotic stimulus. when comparing both cultivars under drought conditions, go terms were identified related to increased enrichment of tropism for up-regulated genes and photosynthesis for down-regulated genes. under the control conditions, proteins of translational machinery were identified for up-regulated genes and photosynthesis for down-regulated genes.fig. gene ontology enrichment analysis on a list of differentially expressed genes up- and down-regulated under four conditions. the calculation of fold change was based on the ratio of: i59-d/i59-c; rub-d/rub-c; rub-c/i59-c; and rub-d/i59-d. the y axis indicates the number of genes normalized by the total number of genes used in each comparison from each library. cultivars of c. arabica and treatments are indicated expression profiles of candidate genes among the candidate genes identified in silico as presenting up- and down-regulation, expression profiles from of them were analysed by qpcr together with the expression of orphan genes and ltp genes. for all these genes, expression profiles were analysed in plagiotropic buds of rubi and iapar under control and drought conditions. these results are presented in separate sections below, according to the observed expression patterns. genes with induced expression under drought conditions twenty-five genes showing up-regulated expression profiles under drought conditions, mainly in iapar and to a lesser extent in rubi, were identified. this was observed for castk which encodes a putative oxidative stress response serine/threonine protein kinase with 87 % identity with a predicted protein of populus trichocarpa. in that case, expression of this gene was highly induced by drought in the dt cultivar iapar similar profiles were also observed for the casamt gene encoding a putative s-adenosyl-l-methionine-dependent methyltransferase and the orphan genes caunk and caunk the latter gene had no open reading frame but presented high identity with the sgn-u contig and also with various coffee ests mainly found in c. canephora cherries at early developmental stages.fig. expression profiles of genes up-regulated under drought conditions. gene expression was analysed in plagiotropic buds of rubi and iapar cultivars of c. arabica grown under control and drought conditions. the gene names are indicated in the histograms. transcript abundances were normalized using the expression of the caubq gene as the endogenous control. results are expressed using rub-c as the reference sample. values of three technical replications are presented as mean ± sd expression of the caslp gene encoding a putative protein homologous to a protein of nicotiana benthamiana containing a peptidase s8/subtilisin-related domain, was also higher in iapar than in rubi under drought conditions. a similar situation was observed for the camas1gene encoding a protein of amino acid residues sharing similarities with momilactone a synthase-like protein from vitis vinifera that contains a secoisolariciresinol dehydrogenase conserved domain. similar expression profiles, characterized by high up-regulation under drought conditions particularly in iapar were observed for the orphan genes caunk caunk caunk caunk and for capsbb and casdc encoding a putative protein related to the adenosylmethionine decarboxylase proenzyme of catharanthus roseus). expression of the caunk gene was also induced under drought conditions but without significant difference in expression between the two cultivars. interestingly, the expression profiles of orphan genes caunk caunk caunk caunk caunk and caunk were similar to that of hsp-encoding gene cahsp in the sense that gene expression was highly up-regulated under drought conditions in both cultivars. in the case of caunk it is worth noting that expression increased 145- and 88-fold under drought conditions in rubi and iapar respectively. under drought conditions, expression of the cagas gene encoding a putative protein homologous to the arbutin synthase from rauvolfia serpentina, was slightly increased in iapar but reduced in rubi. the cacab cachi and caelip genes encoding a photosystem ii light harvesting chlorophyll a/b binding protein of gardenia jasminoides, a class iii chitinase of c. arabica and an early light-induced protein of glycine max, respectively, showed similar profiles but with lower expression in rubi than in iapar under control and drought conditions. lastly, expression of the capp gene encoding a putative phloem protein of vitis vinifera increased under drought conditions in rubi but was quite stable in iapar under both conditions. expression of type ii nsltp genes the expression of type ii nsltp-encoding genes was also monitored using the primer pairs ltp-ft/ltp-r, ltp-ft/ltp-r and ltp-f100/ltp-r recognizing all homologous genes. no expression of nsltp genes was detected under the control conditions in both cultivars. however, expression of nsltp genes was highly up-regulated in iapar but not in rubi under drought conditions. it is worth noting that the caltp1-caltp and caltp genes were co-expressed in iapar and that the expression of cacc genes was slightly higher than that of cace genes.fig. expression of nsltp genes. expression of caltp1-caltp, caltp and all genes was analysed by qpcr in plagiotropic buds of rubi and iapar cultivars of c. arabica grown under control and drought conditions, using the ltp-ft/ltp-r ltp-ft/ltp-r and ltp-f100/ltp-r primer pairs, respectively. expression levels are expressed in arbitrary units of nsltp genes using the expression of the caubq gene as the endogenous control and rub-c as the reference sample. values of three technical replications are presented as mean ± sd drought influences leaf cuticle thickness leaf anatomical analyses were also performed, revealing that the abaxial epidermis of iapar had a thicker cuticle than rubi under drought conditions. there was also a strong interaction between genotype and drought conditions. for example, in the dt cultivar iapar the abaxial epidermis cuticle thickness greatly increased under drought conditions compared with the control treatment. however, no significant variation in abaxial epidermis cuticle thickness could be observed between the control and drought treatments for rubi leaves.fig. comparative analysis of leaf histological cross sections of iapar and rubi cultivars of c. arabica under control and drought conditions. samples were double stained with schiff and nbb and observed under wide field and fluorescent microscopy. le = lower epidermis. the white arrows indicate the fluorescent cuticle. values of leaf cuticle thickness are given in table  bars = 20 μm leaves of iapar and rubi cultivars of c. arabica grown under control and drought conditions were analysed to measure the cuticle thickness of the abaxial faces. values correspond to the average calculated from independent measurements. those marked with different letters are significantly different genes with reduced expression under drought conditions the qpcr experiments led to the identification of several genes whose expression was reduced under drought conditions. in both cultivars, expression of the orphan genes caunk and caunk and of the cadlp gene encoding a putative protein containing a dirigent-like protein domain homologous to the hypothetical protein of vitis vinifera, was greatly reduced under drought conditions. expression of the cachi gene encoding a protein homologous to the putative chitinase of catharanthus roseus, was 5-fold higher in iapar than in rubi under the control conditions but decreased under drought conditions. however, the expression level of the cachi gene was similar in iapar and rubi under drought conditions. for the genes cachi, caunk and cajamt, similar expression profiles were found. in these cases, drought reduced gene expression in both cultivars but expression levels were always higher in iapar than in rubi, particularly for cajamt fig. expression profiles of genes down-regulated under drought conditions. gene expression was analysed in plagiotropic buds of rubi and iapar cultivars of c. arabica grown under control and drought conditions. the gene names are indicated in the histograms. transcript abundances were normalized using the expression of the caubq gene as the endogenous control. results are expressed using rub-c as the reference sample. values of three technical replications are presented as mean ± sd gene expression levels of the cah2a, cagrp and caunk genes, were similar in rubi and iapar for the caaep and caips genes, gene expression remained high in iapar under both control and drought conditions, but decreased drastically in rubi under drought conditions. discussion in this study, we obtained mbp of sequences with longer reads from plagiotropic shoot apices enriched in meristems and primordium leaves of two cultivars of c. arabica under control and drought conditions. these sequences were assembled giving clusters with a mean size ≥ 300 bp each. these rnaseq data, which complement those already available in public databases for coffee ests, can be considered as innovative and relevant in the sense that they were produced from c. arabica tissues that have never previously been studied. the transcriptome annotation by blast2go provided information based on the nomenclature and organism of origin of genes in the ncbi/nr database, the enzyme family, a functional analysis of proteins from the interpro database, and metabolic functions, biological processes and cellular location from gene ontology. our results showed that a large percentage of transcriptome alignment had hits with known function, genes with unknown function both in the ncbi/nr database, and only hits in the interpro database, thereby enabling the identification of most genes. with this analysis, we identified genes related to coffea sp.. we also found genes from solanum sp., genes from populus trichocarpa, genes from vitis vinifera and genes from arabidopsis sp. thus, the transcriptome was aligned with several genes from different plant species and these genes may be conserved among these species, including coffea sp. on the other hand, our results also included “no-hit” genes, perhaps indicating the presence of unannotated or new genes. the comparisons of dna libraries undertaken during this work led to the identification of genes with differential expression profiles in silico between the drought-susceptible and drought-tolerant cultivars of c. arabica with drought conditions. the expression profiles of these genes, as well as those of other previously identified genes, were analysed by qpcr in plagiotropic buds taken from control and drought-stressed plants of rubi and iapar for most of the cgs identified during this work, in vivo gene expression profiles confirmed those deduced from in silico comparisons of dna libraries. for example, this was the case for the cahsp gene whose up-regulated expression under drought conditions can be considered as a “molecular control” of stress applied to the plants during this study and confirmed by leaf water potential measurements. many ests encoding putative hsps were also found in leaf cdna libraries of c. arabica and c. canephora plants grown under drought conditions, heat stress, leaf infection by hemileia vastatrix and also during bean development. our results also identified several genes differentially expressed in plagiotropic buds of iapar and rubi, as for the castk gene encoding a putative serine/threonine protein kinase containing a conserved domain of mitogen-activated protein kinases. these kinases are known to have a central role in the transduction of extra- and intracellular signals in plants, including cell division and differentiation, as well as in responses to various types of stress. in pisum sativum, there is evidence that the mapk cascade is involved in aba-regulated stomatal activity as well as aba-induced gene expression in the epidermal peels. in a recent study, shen et al. showed that the phosphorylation of oswrky protein by mapks is a key step in conferring drought tolerance in transgenic rice. according to our results, higher castk expression under drought conditions in iapar than in rubi could enhance the mapk cascade and therefore be involved in the drought tolerance of iapar in this cultivar, the over-expression of casamt under drought conditions is also particularly interesting because this sequence encodes a putative s-adenosyl-l-methionine-dependent methyltransferase related to the tumorous shoot development gene. in arabidopsis thaliana, tsd is a pleiotropic mutation that affects leaf, root and shoot meristem development. expression of a tsd2:: gus reporter gene has mainly been detected in meristems where this gene is essential for cell adhesion and coordinated plant development. the weaker expression of casamt in rubi than in iapar under drought conditions, points to the existence of major developmental differences between these two cultivars. the differential expression in rubi and iapar of the caslp gene encoding a putative subtilisin-like protein is also worth noting. in arabidopsis, the subtilisin-like serine-protease sdd gene was shown to be strongly expressed in stomatal precursor cells. in addition, sdd mutation increased leaf stomatal density while sdd over-expression led to the opposite phenotype with decreased sd. in c. arabica, maximum and minimum average stomatal densities were observed in full sunlight and shaded conditions respectively, providing evidence for the existence of plasticity for this characteristic in this coffee species. even though no sd were observed between rubi and iapar under moderate drought conditions, the caslp expression profiles presented here do not preclude the involvement of this gene in the genetic determinism of drought tolerance in coffee. another interesting response concerned the differential expression of the camas gene encoding a putative protein containing the conserved domain. this domain is also found in secoisolariciresinol dehydrogenase-like proteins catalyzing the nad-dependent conversion of -secoisolariciresinol to -matairesinol, like the arabidopsis aba protein considered to be one of the key regulators of aba biosynthesis. based on the camas expression profiles presented here, it is possible that aba synthesis was enhanced by drought in plagiotropic buds of iapar but not in those of rubi. this hypothesis is also reinforced by the fact that higher cajamt expression was observed in iapar than in rubi buds. indeed, in addition to well-known functions of jasmonates in plant defence mechanisms in response to biotic stress, recent studies also demonstrated that methyl jasmonate stimulates aba biosynthesis under drought conditions in panicles of oryza sativa. higher expression of casdc under drought conditions in iapar than in rubi is also worth noting because this enzyme catalyzes the synthesis of polyamines involved in stress tolerance in higher plants. in theobroma cacao, aba and drought induced the expression of tcsamdc increasing spermine and spermidine leaf contents correlated with changes in stomatal conductance. more recently, samdc over-expression in transgenic rice was also shown to facilitate drought tolerance. investigation of polyamine levels in plagiotropic buds and leaves of iapar and rubi would be of particular interest to see if these compounds are involved in drought tolerance in coffee. in mature plants, nuclear-encoded early-light inducible proteins accumulate in response to various stress conditions including aba or desiccation. these proteins are presumed to protect the chloroplast apparatus from photo-oxidation occurring after stomatal limitation of photosynthesis. in a recent study, transgenic plants of medicago truncatula over-expressing the dsp gene from craterostigma plantagineum were shown to be able to recover from water deprivation better than wild type plants, thereby reinforcing the idea of using elip-encoding genes to improve abiotic stress resistance in crops. our results clearly highlight the increased expression of the caelip, capsbb and cacab genes, respectively, under drought conditions. interestingly, the expression levels of all these genes were always higher in iapar than in rubi. these results are also in accordance with electronic northern experiments which showed high accumulation of elip and cab-encoding ests in cdna libraries of c. arabica and c. canephora subjected to drought. another surprising result concerned the capsbb gene that was reverse-transcribed and detected during our qpcr experiments despite the fact that it corresponds to a chloroplast gene. however, preliminary analyses of a whole genome sequence of c. canephora revealed the presence of a cp47/like nuclear gene. interestingly, photosystem ii cp chlorophyll apoproteins encoding ests have also been reported to be expressed in c. arabica beans, leaves infected by hemileia vastatrix and also in the cdna libraries of drought-stressed coffee plants, demonstrating increased expression of this gene under biotic and abiotic stress. as cp and elip proteins are essential for the activity and protection of the photosynthetic apparatus, the expression profiles reported here probably reflect a better photosynthetic and physiological status of iapar compared to rubi. differential expression was also observed for the chitinase-encoding gene cachi with higher expression in iapar than in rubi. an opposite situation was observed with respect to the chitinase-encoding genes cachi and cachi whose expression was reduced under drought conditions. it is worth noting that the expression of these genes under drought conditions was always higher in iapar than in rubi. these results also show that coffee chitinase-encoding genes responded in different ways to drought. a large number of chitinase-encoding ests were identified in the bcgp project, mainly in the sh cdna library of drought-stressed plants of c. arabica var. catuai, but also in the leaves of c. arabica infected by leaf rust. even though chitinases are defence-related enzymes induced by abiotic stress, some evidence also indicates their participation in tolerance to abiotic stress. even though the roles of pathogenesis-related proteins in abiotic stress are still not fully understood, dt transgenic plants over-expressing chitinase genes have been obtained. in that sense, the high level of expression for cachi in plagiotropic buds of iapar under both control and drought conditions could have an important function in drought tolerance. arbutin is a phenolic glucoside abundant in the leaves of many freezing- or desiccation-tolerant plants and also present in coffee fruits. in a previous study, down-regulation of the ccgas1gene encoding arbutin synthase was reported in leaves of c. canephora under drought conditions. the results presented here clearly demonstrated differential expression profiles for cagas between the two cultivars of c. arabica. gene expression increased under drought conditions in iapar while the opposite was observed in rubi. even though the presence of arbutin in coffee leaves has never been demonstrated, further analyses of this metabolite should be performed to investigate the role of this glucoside in preventing cell damage in coffee subject to abiotic stresses. the capp gene also showed differential expression profiles, with higher expression in iapar than in rubi. in higher plants, pp2s are sieve elements very abundant in the phloem sap. these proteins are believed to play an important role in the establishment of phloem-based defence mechanisms induced by insect attacks and feeding stress, but also by wounding and oxidative conditions. the functions of pp proteins are still not clear but they could act by forming high molecular weight polymers to close the sieve pores caused by external injuries mainly due to biotic stress. when arabidopsis was treated with hrpnea, the suppression of phloem-feeding activities by aphids was attributed to over-expression of the pp2-encoding gene atpp2-a. other studies showed that hrpn activated aba signalling, thereby inducing drought tolerance in arabidopsis thaliana. based on these results, the involvement of pp proteins in plant response mechanisms to abiotic stress can be hypothesized, for example by maintaining the integrity of vessels under drought conditions by forming sieve plate filaments upon oxidation. in that case, higher synthesis of capp which would be expected to occur in iapar plagiotropic buds under drought conditions could play a role in drought-tolerance by reducing sap-flow in young leaves and consequently increasing the water use efficiency of this cultivar. other interesting results concerned the gene expression stability of the caaep and caips genes observed in iapar under control and drought conditions, whereas expression of both genes decreased under drought conditions in rubi. plant cells use myo-inositol to synthesize a variety of low molecular weight compounds and sugar alcohols such as the galactinol, a key element in the formation of raffinose family oligosaccharides. nishizawa et al. found that plants with high galactinol and raffinose contents were less susceptible to oxidative stress. in c. arabica, up-regulation of cagols genes involved in galactinol biosynthesis was reported in leaves of plants subjected to severe drought. in addition, drought up-regulated the expression of mannose 6-phosphate reductase in leaves of c. canephora and c. arabica. even though little is known about the biochemical mechanisms of drought tolerance in coffee, the accumulation of carbohydrates expected in leaves of drought-stressed plants as a consequence of the up-regulated expression of these genes, could play an important role in the genetic determinism of this phenotype in coffee. in addition to the previously described genes, our results also identified several orphan genes that presented differential expression profiles between the cultivars and treatments, such as caunk caunk and caunk whose expression was highly induced under drought conditions in iapar and to a lesser extent in rubi. orphan genes are also expected to interact specifically with the environment as a consequence of lineage-specific adaptations to that environment. interestingly, the expression profiles of the caunk and caunk orphan genes were very similar to those of type ii nsltp-encoding genes, with high expression mainly detected under drought conditions in plagiotropic buds of iapar but not in those of rubi. up-regulation of ltp genes under drought conditions is well documented in higher plants. lipid transfer proteins are thought to be involved in the transfer of lipids through the extracellular matrix for the formation of cuticular wax. in fact, together with the lipophilic cutin polymer matrix, waxes enter in the composition of cuticle, which forms the first barrier between plants and environmental stresses by limiting non-stomatal water loss and gas exchanges, hence mitigating the effects of drought by controlling water loss associated with epidermal conductance. in nicotiana glauca, ltp genes are predominantly expressed in the guard and epidermal cells and are induced under drought conditions, providing evidence that ltp play an important role in the development of drought tolerance. even though the up-regulation of caltp genes observed under drought in plagiotropic buds of iapar cannot explain directly the greater thickness of leaf cuticle observed in this cultivar than in rubi, these results strongly suggested that lipid metabolism plays a major role in coffee drought tolerance. as reported in other higher plants, our study also highlighted the differential expression of many genes encoding proteins known to be over-expressed under biotic stress, by drought. the fact that our experiment was conducted with drought-stressed plants grown under uncontrolled conditions, could explain such a situation. however, it is also probable that these results reflect a biological reality since it is well known that crosstalk exists in higher plants between signalling pathways for biotic and abiotic stress responses.  during this work, we produced some new transcriptomic information for c. arabica with a total of mbp of sequences assembled into clusters from genes expressed in plagiotropic shoot apices enriched in meristems and primordium leaves in dt and ds cultivars grown under control and drought conditions. major differences between these plants concerned their phenotypic behaviour and transcriptome expression profiles. differences between these plants affected genes of specific pathways such as those involved in abscisic acid biosynthesis, perception and transduction of drought stress, plant development and lipid metabolism. in that sense, the present study increased the number of cgs potentially involved in the genetic determinism of drought tolerance firstly identified in c. canephora. because c. arabica is an amphidiploid species, its transcriptome is a mixture of homologous genes expressed from these two sub-genomes in which c. eugenioides is assumed to express genes mainly for proteins involved in basal biological processes, while the c. canephora sub-genome is assumed to regulate arabica gene expression by expressing genes for regulatory proteins and adaptation processes. in this genetic context, it is possible that the characteristics of iapar that enable it to better withstand drought stress than rubi, really originated from the specific expression of c. canephora genes recently introgressed in this cultivar of c. arabica. even though this study provides further indications about the way in which different coffee cultivars activate their transcriptomes, additional work is still required to understand how epigenetics and epistasis regulate gene expression in the different coffee sub-genomes in c. arabica under drought conditions. source of the plant materials and permissions this work was carried out as part of the scientific cooperation project entitled “study of genetic determinism of drought tolerance in coffee” approved between embrapa and cirad. it complied with all institutional, national, or international guidelines. in the frame of this project, field experiments were conducted at the cerrado agricultural research center with all permissions of partners and in accordance with local legislation. ethics approval and consent to participate not applicable. consent to publish not applicable. availability of supporting data the reads were submitted to genbank and to the bioproject/ncbi database under the accession number prjna additional files additional file 1: table s summary of blast2go automatic annotation of the transcriptome clusters. additional file 2: figure s complete bioinformatics pipeline of the transcriptome assembly and automatic annotation methods used in this work. additional file 3: table s summary of the deseq/ edger fold changes and p-values between the cultivars and between control and drought conditions. these tables also contain blast2go automatic annotation of the transcriptome clusters. table lanes coloured in grey are related to clusters aligned to the new candidate genes tested by rt-qpcr. abbreviations estexpressed sequence tag qpcrquantitative polymerase chain reaction competing interests the authors declare that they have no competing interests. authors’ contributions gcr and pm measured predawn leaf water potentials and harvested the samples. fdb and tl performed meristem dissections, rna extractions and cdna synthesis. lsm, mfc, gagp and rv were responsible for the bioinformatic processing of the data. jlv, flm and ml performed the histology and microscopy analyses. pm, aca and dp selected the candidate genes qpcr-analysed by fac, ngv, ked, jca and mgc. gcr, aca and pm designed the study, drew up the experimental design and implemented it. rv, mfc, gagp, aca and pm drafted the manuscript. all the authors read and approved the final version of the manuscript. the authors would like to thank dr aymbiré francisco almeida da fonseca from the incaper institute for kindly providing seeds of the rubi and iapar commercial cultivars of c. arabica. the authors are also very grateful to drs antonio fernando guerra and omar cruz rocha for their assistance during the field trial experiments. the authors wish also to thank daphne goodfellow, as well as peter biggins and cécile fovet-rabot for english revision of the manuscript. funding pm acknowledges financial support from cirad and the cirad atp project “analysis of phenotypic plasticity in response to water constraints in perennial plants growing under different field conditions”. aca acknowledges financial support from the brazilian coffee r&d consortium, finep and inct-café. mfc and gagp acknowledge financial support from the center for computational engineering and sciences - fapesp/cepid. the authors acknowledge the scholarships from the brazilian agencies capes, brazilian consortium of coffee research and capes-cofecub project sv738-.
their expression was monitored in plagiotropic buds, together with those of other candidates genes. using these data, candidate genes were selected and their differential expression profiles were confirmed by qpcr experiments in plagiotropic buds of iapar and rubi under drought conditions. candidate genecoffeedroughtdifferential gene expressionrna-seqreal-time pcr cirad atp project analysis of phenotypic plasticity in response to water constraints in perennial plants growing under different field conditionsbrazilian coffee r&d consortiumfinepinct-café center for computational engineering and sciences - fapesp/cepid 2013/08293-7pereira gonçalo amarante guimarães http://dx.doi.org/http://dx.doi.org/ /501100002322coordenação de aperfeiçoamento de pessoal de nível superiorcapes-cofecubsv738-12cotta michelle guitton issue-copyright-statement© the author 2016 as regards the genes up-regulated under drought conditions, specifically in the drought-tolerant iapar several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as aba and lipid metabolism, for example. in this study, we compared the molecular responses to drought in two commercial cultivars of coffea arabica grown in the field under control and drought conditions using the pyrosequencing of rna extracted from shoot apices and analysing the expression of candidate genes. it affects plant development, fruit production, bean development and consequently beverage quality. however, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. the comparison of dna libraries identified new candidate genes presenting differential expression between iapar and rubi and/or drought conditions. interestingly, the expression of lipid-transfer protein genes was also highly up-regulated under drought conditions in iapar this may have been related to the thicker cuticle observed on the abaxial leaf surface in iapar compared to rubi. drought is a widespread limiting factor in coffee plants. under drought conditions, up-regulated expression was observed in iapar but not in rubi for castk, casamt, caslp and camas. the full transcriptome assembly of c. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. genetic diversity for drought tolerance exists within the coffee genus. the identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee. pyrosequencing from shoot apices generated a total of mbp and reads enabling the identification of clusters.
 drought is a widespread limiting factor in coffee plants. it affects plant development, fruit production, bean development and consequently beverage quality. genetic diversity for drought tolerance exists within the coffee genus. however, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. in this study, we compared the molecular responses to drought in two commercial cultivars of coffea arabica grown in the field under control and drought conditions using the pyrosequencing of rna extracted from shoot apices and analysing the expression of candidate genes.  pyrosequencing from shoot apices generated a total of mbp and reads enabling the identification of clusters. these data included clusters exclusively from sequencing reads, along with hybrid clusters assembled with sequences. the comparison of dna libraries identified new candidate genes presenting differential expression between iapar and rubi and/or drought conditions. their expression was monitored in plagiotropic buds, together with those of other candidates genes. under drought conditions, up-regulated expression was observed in iapar but not in rubi for castk, casamt, caslp and camas. interestingly, the expression of lipid-transfer protein genes was also highly up-regulated under drought conditions in iapar this may have been related to the thicker cuticle observed on the abaxial leaf surface in iapar compared to rubi.  the full transcriptome assembly of c. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. using these data, candidate genes were selected and their differential expression profiles were confirmed by qpcr experiments in plagiotropic buds of iapar and rubi under drought conditions. as regards the genes up-regulated under drought conditions, specifically in the drought-tolerant iapar several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as aba and lipid metabolism, for example. the identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords candidate genecoffeedroughtdifferential gene expressionrna-seqreal-time pcr cirad atp project analysis of phenotypic plasticity in response to water constraints in perennial plants growing under different field conditionsbrazilian coffee r&d consortiumfinepinct-café center for computational engineering and sciences - fapesp/cepid 2013/08293-7pereira gonçalo amarante guimarães http://dx.doi.org/http://dx.doi.org/ /501100002322coordenação de aperfeiçoamento de pessoal de nível superiorcapes-cofecubsv738-12cotta michelle guitton issue-copyright-statement© the author 2016 