# Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit

Gabriel Sergio Costa Alves, Luana Ferreira Torres, Eveline Déchamp, Jean-Christophe Breitler, Thierry Joët, Frédéric Gatineau, Alan Carvalho Andrade, Benoît Bertrand, Pierre Marraccini, Hervé Etienne

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Gabriel Sergio Costa Alves, Luana Ferreira Torres, Eveline Déchamp, Jean-Christophe Breitler, Thierry Joët, et al. Differential fine-tuning of gene expression regulation in coffee leaves by Cc-DREB1D promoter haplotypes under water deficit. Journal of Experimental Botany, 2017, 68 (11), pp.3017-3031. 10.1093/jxb/erx166. hal-03377702

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# Differential fine-tuning of gene expression regulation in coffee leaves by CcDREB1D promoter haplotypes under water deficit

Article in Journal of Experimental Botany · July 2017

DOI: 10.1093/jxb/erx166

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RESEARCH PAPER

Differential fine-tuning of gene expression regulation in
coffee leaves by *CcDREB1D* promoter haplotypes under
water deficit

Gabriel Sergio Costa Alves<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,*, Luana Ferreira Torres<fnref n="2" />,<fnref n="3" />, Eveline Déchamp<fnref n="2" />, Jean-Christophe Breitler<fnref n="2" />, Thierry Joët<fnref n="4" />, Frédéric Gatineau<fnref n="5" />, Alan Carvalho Andrade<fnref n="1" />,<fnref n="6" />, Benoît Bertrand<fnref n="2" />, Pierre Marraccini<fnref n="1" />,<fnref n="5" />,† and Hervé Etienne<fnref n="2" />,‡

Received 8 December 2016; Editorial decision 25 April 2017; Accepted 25 April 2017

Editor: Roland Pieruschka, Forschungszentrum Jülich

**Abstract**

Despite the importance of the *DREB1D* gene (also known as *CBF4*) in plant responses to water deficit and cold stress, studies analysing its regulation by transgenic approaches are lacking. In the current work, a functional study of three *CcDREB1D* promoter haplotypes (named HP15, HP16 and HP17) isolated from drought-tolerant and drought-sensitive clones of *Coffea canephora* was carried out in plants of *C. arabica* stably transformed by *Agrobacterium tumefaciens* by analysing their ability to regulate the expression of the *uidA* reporter gene in response to water deficit mimicked by polyethylene glycol (–2.0 MPa) and low relative humidity treatments. A deletion analysis of their corresponding 5’-upstream regions revealed increased specificity of β-glucuronidase activity in the polyethylene glycol and low relative humidity treatments, with high expression in leaf mesophyll and guard cells in full-length constructs. RT-qPCR assays also revealed that the HP16 haplotype (specific to clone tolerant to water deficit) had stronger and earlier activity compared with the HP15 and HP17 haplotypes. As most of the *cis*-regulatory elements involved in ABA-dependent and -independent networks, tissue specificity and light regulation are common to these haplotypes, we propose that their organization, as well as the nucleic acid polymorphisms present outside these boxes, may play a role in modulating activities of *DREB1D* promoters in guard cells.

**Key words:** Coffee, *DREB1D* gene, genetic transformation, promoter haplotypes, *uidA* reporter gene, water deficit.

Abbreviations: ABA, abscisic acid; ABRE, ABA-responsive element; CBF, cold-binding factor; CRE, cis-regulatory element; DOF, DNA-binding one zinc finger; DRE, dehydration-responsive element; DREB, dehydration responsive element binding transcription factor; GUS, β-glucuronidase; ICE, inducer of CBF expression; PEG, polyethylene glycol; RH, relative humidity; SNP, single-nucleotide polymorphism; VPD, vapor pressure deficit.

<footnote n="1">¹ EMBRAPA Recursos Genéticos e Biotecnologia (LGM), Parque EB, 70770-917 Brasília, DF, Brazil</footnote>

<footnote n="2">² CIRAD, UMR IPME, F-34394 Montpellier, France</footnote>

<footnote n="3">³ Universidade Federal de Lavras, Departamento de Química, Laboratório Central de Biologia Molecular (LCBM), 37200-000 Lavras, MG, Brazil</footnote>

<footnote n="4">⁴ IRD, UMR DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier, France</footnote>

<footnote n="5">⁵ CIRAD, UMR AGAP, F-34398 Montpellier, France</footnote>

<footnote n="6">⁶ Embrapa Café, INOVACAFÉ, Campus UFLA, 37200-000 Lavras, MG, Brazil</footnote>

<footnote>* Present address: Laboratório de Microbiologia Interação Planta-Praga, Instituto de Ciências Biológicas UnB, Bloco I-1-35/8, Departamento de Biologia Celular, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, 70910-900, Brasília, DF, Brazil.</footnote>

<footnote>† Present address: CIRAD, UMR IPME, F-34394 Montpellier, France.</footnote>

<footnote>‡ Correspondence: herve.etienne@cirad.fr</footnote>
Introduction

Climate change is leading to increasingly extreme tempera-
tures and drought periods, which are major abiotic factors
affecting coffee production (DaMatta and Ramalho, 2006).
Recent modelling studies have delivered warnings on the
threat of severe droughts and high temperatures to coffee
production by increasing attacks by pests and pathogens
(Avelino et al., 2004; Jaramillo et al., 2011; Magrach and
Ghazoul, 2015). These climatic events, which already affect
coffee production, are expected to change the areas suitable
for coffee cultivation (Davis et al., 2012; Bunn et al., 2015;
Craparo et al., 2015). Drought is a limiting factor that affects
flowering and yield of coffee (DaMatta and Ramalho, 2006),
as well as bean development and biochemical composition
and hence the final quality of the beverage (Silva et al., 2005;
Vinecky et al., 2016). Increased [CO₂] in air is also a key factor
for coffee plant acclimation to high temperature, strengthen-
ing the photosynthetic pathway, metabolism and antioxidant
protection, and modifying gene transcription and mineral
balance (Ramalho et al., 2013; Martins et al., 2014, 2016;
Ghini et al., 2015; Rodrigues et al., 2016). In this context,
understanding the genetic determinism of coffee's adapta-
tion to climate change has become essential for creating new
varieties (Cheserek and Gichimu, 2012; van der Vossen et al.,
2015).

Within the *Coffea* genus, substantial genetic variability
exists in relation to drought tolerance, particularly in the
cultivated species *C. canephora* (Montagnon, 2000). This
species, also commercially known as ‘Robusta’, is diploid
(2n=2x=22), autogamous and phylogenetically separated into
distinct groups according to geographical origin in the inter-
tropical region of Africa (Cubry *et al.*, 2013). For instance,
the Congolese group contains subgroup 1 (SG1), originat-
ing from the coastal lowlands and relatively tolerant to water
deficit, while plants belonging to subgroup 2 (SG2) are more
sensitive to water deficit. In Brazil, breeding programs imple-
mented in the last decade on *C. canephora* Conilon (related
to SG1, Montagnon *et al.*, 2012) identified several drought-
tolerant (D<sup>T</sup>) and drought-sensitive (D<sup>S</sup>) clones, which were
intensively characterized (Lima *et al.*, 2002; Pinheiro *et al.*,
2004; Praxedes *et al.* 2006). Among their anatomical and
physiological traits, better root development and greater
water use efficiency were highlighted as important factors of
tolerance to water deficit in D<sup>T</sup> clones (Pinheiro *et al.*, 2005).

In order to investigate the molecular determinism of toler-
ance to water deficit in coffee, several of these *C. canephora*
clones (such as D<sup>T</sup> clone 14 and D<sup>S</sup> clone 22) were used to
identify candidate genes whose expression was induced by
drought stress (Marraccini *et al.*, 2011, 2012; Vieira *et al.*,
2013). Of those genes, *CcDREB1D* (encoding dehydration
responsive element binding transcription factor 1D) was of
outstanding interest since its expression was up-regulated
under conditions of water deficit in leaves of D<sup>T</sup> clone 14 but
not in those of D<sup>S</sup> clone 22 (Marraccini *et al.*, 2012; Vieira
*et al.*, 2013).

The *DREB* gene family includes key transcription factors
involved in responses to various abiotic stresses, such as water

deficit, cold and salt stress, regulating the expression of essen-
tial responsive genes (Lata and Prasad, 2011; Sakuma et al.,
2002). DREB factors act downstream of ABA-dependent
and -independent signal transduction pathways in abiotic
stress responses. Indeed, the overexpression of *DREB* genes
in several genetically engineered plants leads to up-regulation
of cold-regulated genes and osmotic stress-responsive genes,
resulting in increased abiotic stress tolerance (Agarwal et al.,
2006b). Several DREB transcription factors have also been
functionally characterized in model plants. In Arabidopsis
and rice, for instance, overexpression of *DREB1* and *DREB2*
have shown promising results in inducing tolerance to cold,
salt and water deficit (Novillo et al., 2004; Ito et al., 2006; Lim
et al., 2007; Kim et al., 2011). However, *DREB* genes have
been less extensively characterized in non-model (e.g. peren-
nial) plants, particularly due to laborious genetic transforma-
tion protocols often limiting plant regeneration.

Even though *DREB1/CBF* (cold-binding factor) gene
expression is mainly induced by cold, it was also reported
to be involved in the control of plant development and
response to other stress stimuli (Lata and Prasad, 2011). The
expression of *AtDREB1D* (also known as *AtCBF4*) is rap-
idly induced by water deficit but not by cold in Arabidopsis
(Haake et al., 2002). Expression of the *CBF4* gene was also
reported to be up-regulated in response to low temperatures,
water deficit, and salinity in different *Vitis* species (Xiao
et al., 2008; Zandkarimi et al., 2015) and in *Medicago trun-
catula* (Li et al., 2011). The fact that overexpression of the
*DREB1D/CBF4* gene increased tolerance to water deficit in
transgenic plants of Arabidopsis (Haake et al., 2002) and
*Glycine max* (Guttikonda et al., 2014) suggests that it plays
an important role in plant responses to abiotic stress.

Despite the importance of *DREB* genes in plant response
pathways to abiotic stress, a limited number of studies have
analysed the regulation of their corresponding promot-
ers. Promoter regions of *AtDREB1C* (Zarka et al., 2003),
*OsDREB1B* (Gutha and Reddy, 2008), *GmDREB3* (Chen et
al., 2009), and *AtDREB2C* (Chen et al., 2012) were shown
to be sufficient for regulating the transcription of the *uidA*
reporter gene by abiotic stresses in transgenic Arabidopsis.
More recently, Fang et al. (2015) also demonstrated that 1278
bp of the *FeDREB1* promoter from the common buckwheat
enhanced β-glucuronidase (GUS) activities in drought in
leaves of transgenic tobacco. However, functional characteri-
zation of the *DREB1D* promoter has not been reported. In
an attempt to better understand the tissular and environmen-
tal regulation of *CcDREB1D* promoter activity, as well as the
regulation of allele-specific expression depending on the hap-
lotype sequence, our study set out to (i) isolate and compare
the promoter haplotypes of *CcDREB1D* genes from D<sup>T</sup> clone
14 and D<sup>S</sup> clone 22 of *C. canephora*, (ii) evaluate their abil-
ity to regulate expression of the *uidA* reporter gene by moni-
toring GUS histochemical activity in transgenic plants of *C.
arabica* subjected to different abiotic stresses, and (iii) analyse
the expression of *uidA* and endogenous *CaDREB1D* genes in
leaves of these coffee plants.
# Materials and methods

## Plant material and growing conditions

D<sup>T</sup> clone 14 and D<sup>S</sup> clone 22 of *C. canephora* Conilon have been described previously (Marraccini *et al.*, 2011, 2012). For *Agrobacterium tumefaciens*-mediated transformation experiments, *C. arabica* var. Caturra plants were grown in *in vitro* conditions with a 12 h photoperiod (70 µmol m<sup>-2</sup> s<sup>-1</sup> light intensity) at 26 °C and 80% relative humidity (RH).

## DNA extraction and isolation of CcDREB1D promoter haplotypes

Leaves of D<sup>T</sup> clone 14 and D<sup>S</sup> clone 22 of *C. canephora* Conilon were used to extract genomic DNA and isolate *CcDREB1D* promoters. Genomic DNA was extracted from 20 mg of ground leaves as previously described by Cotta *et al.* (2014). For each coffee clone, *CcDREB1D* promoter haplotypes were amplified independently by PCR with high fidelity Taq Platinum® DNA polymerase according to the supplier's instructions (Invitrogen) (initial denaturation: 94 °C–2 min followed by 30 cycles of 94 °C–30 s, 68 °C–30 s, 72 °C–3 min and a final extension step of 72 °C–10 min) using 100 ng of genomic DNA and 0.2 µM of the DREB-F1/DREB-R1 primers (Table 1). Amplified products were cloned into the pCR2.1-TOPO vector (Invitrogen) and propagated in the *E. coli* DH5α strain. As *C. canephora* is a diploid species, recombinant plasmids were extracted from 12 independent *E. coli* colonies in order to identify

<table><caption>**Table 1.** List of primers used in this study</caption><thead><tr><td>Primer</td><td>Sequence</td></tr></thead><tbody><tr><td>DREB-F1<sup>a</sup></td><td>5' ACTCCTAGTAAGCGGCACGTTGTT 3'</td></tr><tr><td>DREB-R1<sup>a</sup></td><td>5' TGGCTTTGCAGGCATTGACTACG 3'</td></tr><tr><td>DREB-F2<sup>b</sup></td><td>5' TCGTCGATTCACAAGCACCCTCA 3'</td></tr><tr><td>DREB-R2<sup>b</sup></td><td>5' CCTTTTCGTGGTTGTCTCTTGACCT 3'</td></tr><tr><td>S-DREB<sup>c</sup></td><td>5' TAATCCCAAGCTTTGTCTGAAGT 3'</td></tr><tr><td>M-DREB<sup>d</sup></td><td>5' AAGAGAACAACAAGCTTCTTGT 3'</td></tr><tr><td>L-DREB<sup>e</sup></td><td>5' TCCTAGTAAGCTTCACGTTGT 3'</td></tr><tr><td>R-DREB<sup>f</sup></td><td>5' TGTTGAGAAATGGTTAGATCTTGAA 3'</td></tr><tr><td>GUS-F</td><td>5' GCACTAGCGGGACTTTGCAA 3'</td></tr><tr><td>GUS-R</td><td>5' CGCGAAGCGGGTAGATATCA 3'</td></tr><tr><td>DREBA09-F</td><td>5' CAATGCCTGCAAAGCCAATTA 3'</td></tr><tr><td>DREBA09-R</td><td>5' TTTTCCTGCCTGCACGTTTC 3'</td></tr><tr><td>GAPDH-F<sup>g</sup></td><td>5' TTGAAGGGCGGTGCAA 3'</td></tr><tr><td>GAPDH-R<sup>g</sup></td><td>5' AACATGGGTGCATCCTTGCT 3'</td></tr></tbody></table>

<sup>a</sup>Primers used to amplify HP15, HP16 and HP17 haplotype promoters from the *CcDREB1D* (*Cc02_g03430*) gene of *C. canephora* (Denoeud *et al.*, 2014).

<sup>b</sup>Internal primers used for sequencing to verify the sequence of *CcDREB1D* promoters.

<sup>c</sup>Forward primer used to amplify the short (S: +182/-608) fragment of the *CcDREB1D* promoter haplotype. The *Hind*III restriction site is italicized.

<sup>d</sup>Forward primer used to amplify the medium (M: +182/-966) fragment of the *CcDREB1D* promoter haplotype. The *Hind*III restriction site is italicized.

<sup>e</sup>Forward primer used to amplify the long (L: +182/-1.308) fragment of the *CcDREB1D* promoter haplotype. The *Hind*III restriction site is italicized.

<sup>f</sup>Reverse primer used to introduce the *Bg*III restriction site. Other primer pairs were used in RT-qPCR experiments to determine gene expression levels of *uidA* (GUS-F/R) and *CcDREB1D* (DREBA09-F/R).

<sup>g</sup>The primer pair GAPDH-F/R was used to amplify the transcripts of the *CaGAPDH* (glyceraldehyde-3-phosphate dehydrogenase) gene used as reference to standardize the results of RT-qPCR experiments.

the two haplotypes of *CcDREB1D* promoter regions for each clone that were further double-strand sequenced using M13F and M13R universal primers and internal DREB-F2 and DREB-R2 primers (Table 1).

## Bioinformatic analysis

The sequence of the unique gene *Cc02_g03430* (*CcDREB1D*: GSCOCP00020227001) from *C. canephora* (Denoeud *et al.*, 2014), available at Coffee Genome Hub (http://coffee-genome.org/), was analysed using the PlantPAN (http://plantpan.mbc.nctu.edu.tw, Chang *et al.*, 2008) and the PlantProm DB (http://www.softberry.com, Shahmuradov *et al.*, 2005) web interfaces. The DREB-F1/DREB-R1 primers (Table 1) were designed to amplify the region (–1308/+153 in Fig. 1) of *CcDREB1D* haplotypes from D<sup>T</sup> clone 14 and D<sup>S</sup> clone 22 that contained most of the *cis*-regulatory elements (CREs) supposed to be essential for their regulation by abiotic stress. The same web interfaces were used to predict the presence of CREs in the HP15, HP16 and HP17 haplotypes of the *CcDREB1D* promoters (Table 2). The significance of the putative CREs was evaluated through a maximum threshold of core similarity (equal to 1.0) and matrix similarity of 0.75.

## Construction of recombinant vectors

Binary vectors were generated by PCR using high fidelity Taq Platinum DNA polymerase amplification and recombinant plasmids harboring the HP15, HP16, and HP17 haplotypes of *CcDREB1D* promoters, as templates. The PCR reactions used the forward primers S-DREB, M-DREB, and L-DREB carrying the *Hind*III restriction site to amplify the short (S: +182/-608), medium (M: +182/-966), and long (L: +182/-1.308) fragments of the *CcDREB1D* promoter haplotypes, and the reverse primer R-DREB carrying the *Bg*II restriction site (Table 1). The position of the R-DREB primer made it possible to include the entire 5'-UTR region of the *CcDREB1D* gene in the final constructs. PCR reactions were carried out using 10 ng of plasmids, Taq Platinum DNA polymerase, and primers as previously described, except that the first ten amplification cycles were carried out at an annealing temperature of 60 °C to ensure primer mismatch (calculated with the PrimerQuest<sup>SM®</sup> software, Integrated DNA Technologies). The resulting PCR products were subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), digested with *Hind*III and *Bg*II and cloned in the pBI121 vector (Clontech, Palo Alto, CA, USA) double-digested by the same enzymes. Following ligation and transformation in *E. coli*, the recombinant constructs were named according to their haplotype (HP) of the *CcDREB1D* promoter and length (S: short, M: medium, and L: long), namely as pHP16S and pHP17S (+182/-608, relative to ATG of the *uidA* reporter gene), pHP16M (+182/-966), and pHP15L, pHP16L and pHP17L (+182/-1308) (Fig. 2B). Each recombinant vector was then transferred independently by electroporation into competent cells of the disarmed *A. tumefaciens* strain LBA1119. After each cloning step, recombinant vectors were systematically extracted with the Wizard® Plus SV Minipreps DNA Purification System and *CcDREB1D* promoter fragments were verified by double-strand sequencing (Genome Express, France).

## Agrobacterium tumefaciens-mediated transformation and regeneration of *C. arabica*

*A. tumefaciens* strain LBA1119 harboring the recombinant vectors was grown at 28 °C for 24 h in yeast extract mannitol agar (YMB) selective medium with rifampicin (25 mg l<sup>-1</sup>) and kanamycin (50 mg l<sup>-1</sup>). Transgenic lines of *C. arabica* were generated by *A. tumefaciens*-mediated transformation using 12-month-old embryogenic calli competent for transformation as previously described (Etienne, 2005; Ribas *et al.*, 2011). At least eight independent putatively transformed cell lines were obtained for the six PHP constructs, as well as for coffee plants transformed by the pBI121 (*CaMV35S:uidA*) and
<figure><img src="image_9.png" /><figcaption>**Fig. 1.** Consensus sequence of *CcDREB1D* promoter haplotypes. The consensus sequence contains all single nucleotide polymorphisms (SNPs, in gray) and insertion/deletions (INDELs, lower case underlined with horizontal brackets) found in the HP16 and HP17 haplotypes of *CcDREB1D* promoters. The nucleotides are numbered (on the right) in each lane using the first nucleotide (+1) of the *CcDREB1D* mRNA sequence (based on RNAseq data evidence available in the Coffee Genome Hub) as the start of numbering. CREs are identified by boxes with their corresponding name below except DOF/guard-cell (underlined), W-BOX (underlined and italics) and ABRE-LIKE (bold). Horizontal arrows indicate the primers (Table 1) used to amplify the full-length and truncated versions of coffee *CcDREB1D* promoter sequences (Fig. 2C). The TCA microsatellite GAAWTT unidentified motif and the putative TATA box are also indicated (bold and brackets). The 19 bp in italics upstream of the ATG (+174) of the β-glucuronidase corresponds to the multiple cloning site of the pBI121 vector.</figcaption></figure>

pBI101 (*uidA*-promoterless) vectors used as positive and negative controls, respectively. Each kanamycin-resistant callus regenerated cotyledonary embryos giving plantlets after 4–5 months that were cultivated on MS medium with active charcoal (1 g l⁻¹) under sterile conditions prior to testing for fast dehydration experiments. Once regenerated, DNA was extracted from leaf explants and successively tested by (i) PCR using the *nptII*-*kanamycin* and *uidA*-specific primers to confirm T-DNA integration in genomic DNA of *C. arabica*-transformed

plantlets, and (ii) RT-qPCR to select transgenic lines harboring a single insertion of T-DNA (data not shown) representing around 50% of T1 transgenic coffee lines (Ribas *et al.* 2011).

Abiotic assays used to study transformed plants of *C. arabica*

Regulation of the *CcDREB1D* promoter haplotypes was studied in primary transformants (T1) of *C. arabica* grown *in vitro* and
<caption>**Table 2.** *Cis-regulatory elements (CREs) present in the CcDREB1D promoter haplotypes*</caption>

The *cis*-regulatory elements present in the *CcDREB1D* promoter consensus sequence (Fig. 1) involved in osmotic- and cold-stress responsive gene expression were identified using PlantPAN (http://plantpan.mbc.nctu.edu.tw, Chang *et al.*, 2008) and the TSSP (http://www.softberry.com, Shahmuradov *et al.*, 2005) web interfaces. Experimental results supporting the involvement of CREs in the regulation of gene expression by abiotic stresses are also available in the literature (Ref.: 1, Sakuma *et al.*, 2002; 2, Yamaguchi-Shinozaki and Shinozaki, 2005; 3, Silva, 1994; 4, Plesch *et al.*, 2001; 5, Doherty *et al.*, 2009; 6, Higo *et al.*, 1999; 7, Chinnusamy *et al.*, 2007). Base abbreviations (IUPAC notation) are as follows: B=[not A], K=[G,T], M=[A,C], N=[any nucleotide], R=[A,G], V=[not T], W=[A,T], Y=[C,T]. TF: transcription factor. CREs were found either in sense (+) or antisense (−) DNA strands.

<table><thead><tr><th></th><th>CRE</th><th>Sequence</th><th>TF</th><th>Gene</th><th>Stress condition</th><th>Ref.</th><th>Sites</th><th>pHP15</th><th>pHP16</th><th>pHP17</th></tr></thead><tbody><tr><td rowspan="2">ABRE</td><td>ABRELATERD1</td><td>ACGTG</td><td>bZIP</td><td>erd1</td><td>Water deficit, ABA</td><td>6</td><td>-1272/-1268 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>ABRE-LIKE</td><td>BACGTGKM</td><td>bZIP</td><td>rd29</td><td>Water deficit, ABA</td><td>2</td><td>-557/-550 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">DRE</td><td>DRE</td><td>CCGNC</td><td>ERF/AP2</td><td>rd29A</td><td>Water deficit, cold</td><td>1</td><td>-347/-340 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>LTRECOREATCOR15</td><td>CCGAC</td><td>ERF/AP2</td><td>cor15A</td><td>Water deficit, cold</td><td>6</td><td>-267/-263 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">MYB</td><td>MYBCORE</td><td>CNGTTR</td><td>bHLH</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-1228/-1223 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>MYB1AT</td><td>WAACCA</td><td>bHLH (MYB15)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-1012/-1007 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">MYB2AT</td><td>MYB2AT</td><td>YAACTG</td><td>bHLH (MYB2)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-695/-690 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>MYB1AT</td><td>WAACCA</td><td>bHLH (MYB15)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>+9/+14 (-)</td><td>-</td><td>-</td><td>+</td></tr><tr><td rowspan="2">MYC</td><td>MYCCONSENSUSAT</td><td>CANNTG</td><td>bHLH (ICE1)</td><td>rd22</td><td>Water deficit</td><td>6</td><td>-1248/-1243 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>MYB2AT</td><td>YAACTG</td><td>bHLH (MYB2)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-1235/-1230 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">MYC</td><td>MYB2AT</td><td>YAACTG</td><td>bHLH (MYB2)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-575/-570 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>MYB1AT</td><td>WAACCA</td><td>bHLH (MYB15)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>+104/+109 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">MYC</td><td>MYB1AT</td><td>WAACCA</td><td>bHLH (MYB15)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-1273/-1268 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>MYB2AT</td><td>YAACTG</td><td>bHLH (MYB2)</td><td>rd22</td><td>Water deficit, ABA</td><td>6</td><td>-842/-837 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">ICE</td><td>ICE2</td><td>ACTCCG</td><td>bHLH (ICE)</td><td>CBF2/ DREB1C</td><td>Cold</td><td>7</td><td>-575/-570 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>ICE1</td><td>ACTCCG</td><td>bHLH (ICE)</td><td>CBF2/ DREB1C</td><td>Cold</td><td>7</td><td>-387/-382 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">CG-1</td><td>CG-1</td><td>VCGCGB</td><td>bHLH (CAMTA/SR1)</td><td>PAL1</td><td>CaM/Ca<sup>2+</sup> inducible</td><td>3</td><td>-347/-342 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>CG-1</td><td>VCGCGB</td><td>bHLH (CAMTA/SR1)</td><td>PAL1</td><td>CaM/Ca<sup>2+</sup> inducible</td><td>3</td><td>-1263/-1258 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">CM6</td><td>CM6</td><td>AAGATTGTCA</td><td>unknown</td><td>unknown</td><td>Water deficit, ABA</td><td>5</td><td>-1255/-1250 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>CM6</td><td>AAGATTGTCA</td><td>unknown</td><td>unknown</td><td>Water deficit, ABA</td><td>5</td><td>-148/-143 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">GT-1</td><td>GT-1</td><td>GRWAAW</td><td>bHLH</td><td>RBCS</td><td>Light-inducible</td><td>6</td><td>-1286/-1281 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>GT-1</td><td>GRWAAW</td><td>bHLH</td><td>RBCS</td><td>Light-inducible</td><td>6</td><td>-1179/-1174 (+)</td><td>-</td><td>-</td><td>+</td></tr><tr><td rowspan="2">DOF</td><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-1179/-1174 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-1017/-1013 (+)</td><td>+</td><td>+</td><td>-</td></tr><tr><td rowspan="2">DOF</td><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-1000/-996 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-968/-964 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">DOF</td><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-929/-925 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-925/-921 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">DOF</td><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-584/-580 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-529/-525 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td rowspan="2">DOF</td><td>DOF</td><td>WAAAG</td><td>ZF</td><td>KST1</td><td>Water deficit, ABA</td><td>4</td><td>-487/-483 (+)</td><td>+</td><td>+</td><td>+</td></tr></tbody></table>
<table><caption>**Table 2. Continued**</caption><thead><tr><th>CRE</th><th>Sequence</th><th>TF</th><th>Gene</th><th>Stress condition</th><th>Ref.</th><th>Sites</th><th>pHP15</th><th>pHP16</th><th>pHP17</th></tr></thead><tbody><tr><td rowspan="10">W-BOX</td><td rowspan="10">W-BOX</td><td rowspan="10">TTGAC</td><td rowspan="10">ZF</td><td rowspan="10">NPR1</td><td rowspan="10">Wounding</td><td rowspan="10">6</td><td>-236/-232 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>+49/+53 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>+94/+98 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>-1230/-1226 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>-552/-548 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>-506/-502 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>-482/-478 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>-403/-399 (-)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>-231/-227 (+)</td><td>+</td><td>+</td><td>+</td></tr><tr><td>+2/+6 (-)</td><td>+</td><td>+</td><td>+</td></tr></tbody></table>

subjected to the abiotic treatments described below. For each construct, 64 plantlets from eight independent transformation events were arranged in eight batches of eight plants. In each assay, each plant batch was subjected independently to water deficit for 0, 3, 6, 12, or 24 h, with 0 h (10.00 h, i.e. after 2 h of light exposure) corresponding to 'no stress conditions'.

### Fast dehydration

Genetically transformed *in vitro* plantlets with two to four pairs of leaves were placed in a laminar flow cabinet under a 0.49 m s⁻¹ air flow for 1 h until reaching a visible dehydrated state. Each pHP construct was composed of five plantlets corresponding to independent transformation events.

### Low RH assay (vapor pressure deficit)

A vapor pressure deficit (D) was induced by 9% RH at 27 °C under controlled conditions. A theoretical D was calculated applying the formula $D=[1-(RH/100)]×P_{WS}$ (kPa), where $P_{WS}$ (saturated vapor pressure)=3779 Pa at 28 °C. To create a 9% RH, 400 ml of KOH supersaturate solution was poured into the lower compartment of a 5 l bioreactor (Matis®, CID Plastiques, France) which remained sealed throughout the experiment. Plantlets were placed in the upper compartment of the bioreactor in 55 mm Petri dishes having their upper part exposed to the outside environment and radicles immersed in MS medium (Murashige and Skoog, 1962) through a small hole in the Petri dish cover. Seven plants were placed inside the bioreactor in the resulting 9% RH.

### Polyethylene glycol assay

Osmotic potential was fixed at -2.0 MPa by adding polyethylene glycol (PEG) (relative molecular mass 6000; Sigma-Aldrich) to the MS medium, at 25 °C, using the empirical equations defined by Michel and Kaufmann (1973). Batches of seven plantlets were placed over 60 mg of vermiculite fully imbibed with 40 ml of PEG diluted in MS solution in sterile plastic Magenta® boxes.

### GUS staining

Histochemical GUS assays were performed with different tissues (root, stem, apical meristem, and leaf) derived from the same plant as those immersed in GUS staining solution (100 mM sodium phosphate buffer, pH 7.2, 10 mM sodium EDTA, 0.1% Triton X-100, 1 mg ml⁻¹ 5-bromo-4-chloro-3-indolyl-D-glucuronic acid (Sigma-Aldrich), and 2.5 mM potassium ferrocyanidine). After infiltrating (vacuum for 2 × 10 min) the staining solution in plant tissues, the samples were incubated at 37 °C for 24 h, and then rinsed with water before image acquisition with a Retiga 2000R camera (G-Imaging Co., Wetzlar, Germany).

### Histology to reveal GUS in organs of transgenic coffee plants

Prior to observation, the GUS-stained samples were fixed (in 50% methanol and 10% acetic acid fixative) at 4 °C for 24 h, rinsed

with water and then dehydrated (10 min in 50% ethanol, 10 min in 70% ethanol and 10 min in 90% ethanol). After observation with a Nikon binocular SMZ 1500 loupe, samples were embedded in 6% agarose for subsequent sections in a Microm HM650V vibrating blade microtome. For bright field microscopy observation, 50 µm-thick leaf sections were examined using a DM600 Leica microscope (Leica Microsystems GmbH, Wetzlar, Germany) and photographed. GUS expression patterns appeared highly conserved for the eight lines transformed with the same promoter construct.

### Proportion of GUS-stained guard cells in PEG and low RH assays

For bright field microscopy, GUS-stained leaves were fixed (50% methanol and 10% acetic acid) at 4 °C for 24 h, rinsed with water and incubated for at least 3 days in clearing solution (chloral hydrate:glycerol:water (4:1:2, v/v/v) to remove all leaf pigments. Prior to observation, tissues were rinsed with 70% ethanol and assembled on microscope slides for photography as described before. The proportion of GUS-stained guard cells on the abaxial epidermis of coffee leaves was calculated to estimate *CcDREB1D* promoter activity. For each pHP construct, the same three independent transgenic lines were studied along the entire kinetics. The proportion of GUS-stained guard cells (*p*) was obtained by $p=x/n$, where *x* is the number of stained guard cells and *n* the total number of guard cells (=150) per leaf zone. These values were assessed in 24 × 36 mm areas distributed in six pre-delimited leaf zones. For each pHP construct, three leaves from plants of three independent transformation events were sampled for each time (0, 3, 6, 12, and 24 h) in the PEG and low RH assays, and the respective proportion means (from 450 guard cells) and standard deviations for each time point were calculated.

### RNA isolation

Total RNA was extracted from leaves frozen in liquid nitrogen that were further ground and treated as described previously (Breitler et al., 2016). RNA quantification was performed using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

### Real-time RT-PCR assays

PCR experiments were performed as previously described by Marraccini et al. (2012). Primers (Table 1) were designed using Primer Express software (Applied Biosystems) to determine gene expression levels of *uidA* (GUS-F/R) and *CcDREB1D* (DREBA09-F/R). Data were analysed using SDS 2.1 software (Applied Biosystems) to determine cycle threshold (Ct) values. The specificity of the PCR products generated for each set of primers was verified by analysing the Tm (dissociation) of amplified products. PCR efficiency (E) was estimated using absolute fluorescence data captured
<figure><img src="image_10.png" /><figcaption>**Fig. 2.** Haplotypes of *CcDREB1D* promoters of *C. canephora*. (A) *CcDREB1D* haplotypes found in drought-tolerant (D^T^) clone 14 (HP15/HP16) and drought-sensitive (D^S^) clone 22 (HP15/HP17) of *C. canephora*. Color code for haplotypes: HP15 (white), HP16 (black) and HP17 (gray). (B) Graphic representation of nucleotide variability detected in *CcDREB1D* promoter haplotypes. The x-axis corresponds to the bases of the HP15 sequence (Fig. 1). The y-axis corresponds to the frequency of polymorphic sites (S) observed in the HP16 (continuous line) and HP17 (dotted line) haplotypes compared with the HP15 sequence used as a reference. (C) Schematic representation of the *CcDREB1D* haplotypes analysed in transgenic plants of *C. arabica*. The schematic map of *CcDREB1D* (*Cc02_g03430*) is given in the upper part together with the DREB primers (F1, R1 in brackets, see Table 1) used to amplify the *CcDREB1D* promoter haplotypes. Plasmid names used for stable transformation of *C. arabica* are given for each construct, indicating the haplotype studied with its corresponding length (L, long; M, medium; S, short). The fragments were amplified using the forward primers (including the HindIII [AAGCTT] restriction site) corresponding to L-DREB (L, white square), M-DREB (M, white star) and S-DREB (S, white triangle) and the reverse primer R-DREB (including the BglII [AGATCT] restriction site) indicated by a black point and further cloned in front of the *uidA* reporter gene. pBI121 (CaMV35S:*uidA* gene construct) and pBI101 (*uidA* promoterless gene) were used as positive and negative controls of GUS enzymatic activities, respectively.</figcaption></figure>

during the exponential phase of amplification of each reaction with the equation $(1+E)^{10^{(-1/\text{slope})}}$ (Ramakers et al., 2003). Expression levels were calculated by applying the formula $(1+E)^{-\Delta\Delta C_t}$ where $\Delta C_{t,\text{target}} = C_{t,\text{targetgene}} - C_{t,\text{CaGAPDH}}$ and $\Delta\Delta C_t = \Delta C_{t,\text{target}} - \Delta C_{t,\text{references ample}}$, with the $T_0$ samples being used as references for each construct. Expression levels were normalized with the expression of the *CaGAPDH* gene (GB accession number GW445811 using primer pair GAPDH-F/R) as the endogenous control (Cruz et al., 2009).

## Results

### Comparison of DNA motifs and CREs between the different *CcDREB1D* promoter haplotypes isolated from drought-tolerant and drought-sensitive clones

The comparison of *CcDREB1D* promoter regions of *C. canephora* drought-tolerant (D^T^) clone 14 and drought-sensitive (D^S^) clone 22 revealed the existence of three haplotypes named HP15, HP16 and HP17, diverging from each other by several SNPs and INDELs (Fig. 1, and Supplementary Table S1 and Supplementary Fig. S1 at JXB online). The HP15 haplotype was common (without any SNPs) to both clones, while HP16 was specific to D^T^ clone 14 and HP17 specific to D^S^ clone 22 (Fig. 2A). Upstream of the first nucleotide (+1) of the *CcDREB1D* mRNA, a sliding window comparison of the haplotype sequences revealed two highly polymorphic domains (between base pairs -627/-806 and -932/-1121) concentrating most of the nucleotide variability (Fig. 2B), while the -115/-188 region was highly conserved between the three haplotypes, and the -188/-520 region was conserved between HP15 and HP16. Several CREs were also observed (Table 2) such as ABA-responsive (ABRE), dehydration-responsive (DRE), inducer of CBF expression region 2 (ICEr2), DNA-binding one zinc finger (DOF) binding sites (required for gene-specific expression in guard-cells), GT-1 binding sites (essential for light-inducible expression), the CG-1 element (also known as CAMTA: calmodulin binding transcription activator), and the conserved motif 6 (CM6), considered as a repressor of DREB1/CBF activation.

The comparison of *CcDREB1D* haplotypes revealed that the ICEr2 (+115/+120) and DOF (-1017/-1013) DNA boxes present in HP15 and HP16 were missing in HP17 (Table 2). However, HP17 displayed some elements, such as one MYBCORE (+9/+14) and one MYC (-340/-335) element, as well as two GT-1 binding sites (-489/-484 and -1179/-1174) not present in the HP15 and HP16 haplotypes (Table 2). Except for these differences, the HP15, HP16, and HP17 haplotypes had all CREs in common.

### Tissular activities of dehydration-induced *CcDREB1D* promoters in *C. arabica* transgenic plants

The activities of *CcDREB1D* promoter haplotypes were observed by comparing the GUS staining detected in *C. arabica* plants transformed by the short (pHP16S and pHP17S, Fig. 3A, B), the medium (pHP16M, Fig. 3C) and the full-length (pHP15L, pHP16L, and pHP17L, Fig. 3D–F) constructs and subjected to fast dehydration.

No staining was observed in dehydrated wild type plants and pBI101-transformed plants, whereas strong staining was seen in dehydrated plants transformed by pBI121 (Supplementary Fig. S2). In dehydrated pHP16S-transformed coffee plants, intense GUS staining was detected in root vascular tissues, while weak staining was observed in stem vascular tissues (Fig. 3B1, 2). In meristems, longitudinal-sections revealed weak GUS activity in leaf primordia and auxiliary
<figure><img src="image_11.png" /><figcaption>**Fig. 3.** Histochemical localization of GUS activity in different tissues of transgenic *C. arabica* subjected to dehydration. Columns 1: root; 2: stem; 3: meristem; 4: leaf. The tissues belong to plants regenerated from constructs pHP17S (A), pHP16S (B), pHP16M (C), pHP16L (D), pHP15L (E), and pHP17L (F). The histochemical localization of GUS activity in control plants (WT, pBI101, and pBI121) is given in Supplementary Fig. S2. The scale bars given for each image correspond to 30 µm, excepted in (E4) and (D4) (80 µm), and (E3) (300 µm). Arrows indicate GUS staining restricted to specific cells and tissues: leaf epidermis (le), guard cell (gc), parenchyma (p), vascular tissue (vt), and apical meristem (am).</figcaption></figure>

buds, while intense staining was observed in apical meristems (Fig. 3B3). Strong GUS activities were also detected in leaf epidermis, as well as in spongy and palisade parenchyma (Fig. 3B4). GUS activities in pHP17S-transformed plants were similar (at spatial, tissue, and cell-specific levels) to those observed in pHP16S-transformed plants (Fig. 3A2, 4), except in roots where no staining was seen (Fig. 3A1).

For the pHP16L, pHP15L and pHP17L constructs, no GUS staining was observed in roots, stems, and meristems (Fig. 3D1–3, E1–3 and F1–3, respectively). Microscopic analyses of leaf transverse sections revealed GUS staining in palisade and spongy parenchyma of pHP15L-transformed plants (Fig. 3E4), and in several guard cells and stomata of pHP16L-transformed plants (Fig. 3D4).

The effects of promoter length were observed by comparing GUS activities in plants transformed by the pHP16S, pHP16M, and pHP16L constructs (Fig. 3B, C, D, respectively). For example, GUS activities were detected in leaves in

pHP16M plants, particularly in the epidermis, in spongy and palisade parenchyma, and in the guard cells (Fig. 3C4) whereas no tissue specificity of staining was observed with pHP16S-transformed plants (Fig. 3B4). The greatest tissue specificity of GUS staining was shown for the plants transformed with the pHP16L construct, with histochemical staining observed in guard cells only (Fig. 3D4). The comparison of pHP17S- and pHP17L-transformed plants showed drastic reduction of GUS staining with the longer promoter (Fig. 3A and F, respectively).

*CcDREB1D promoters target uidA gene expression in guard cells of C. arabica under water deficit*

In order to fine-tune the GUS staining results previously observed during the fast-drying assay, transformed coffee plants were subjected to controlled water deficit mimicked by low RH (9%) and further analysed to investigate GUS coloration in abaxial leaf surfaces. As controls, no staining
was detected in plants transformed with the pBI101 vector (Fig. 4A) whereas strong staining was detected in guard and epidermis cells of leaves from pBI121-transformed plants (Fig. 4B). Under unstressed conditions, both epidermal cells and stomata were completely unstained in plants transformed by pHP17S, pHP17L, pHP16S, and pHP16L (Fig. 4C, E, G, I). After 24 h of reduced RH, GUS activities were clearly detected in guard cells of the same transgenic plants (Fig. 4D, F, H, J). GUS staining also appeared to be more intense in the colored stomata of pHP16L-transformed plants (Fig. 4J) than in those of pHP16S-transformed plants (Fig. 4H). For the three constructs, our results also clearly showed that in the same abaxial leaf area, stomata with stained guard cells coexisted with completely unstained stomata under a low RH, as observed for the HP16 haplotype (Fig. 4G, I). Longitudinal and cross sections of the pHP16L-transformed plants were also made to immunolocalize GUS proteins at the cellular level by using anti-GUS polyclonal antibody. This clearly detected GUS protein in guard cells with fluorescence signals that were highly concentrated in their thickened inner wall (Supplementary Fig. S3A). Fluorescence signals were not detected in all guard cells, but were observed in subsidiary cells, in spongy and palisade parenchyma cells, and in epidermis cells (Supplementary Fig. S3B).

*The activity of the HP16L CcDREB1D promoter was up-regulated by the low RH and PEG assays in leaves of transgenic C. arabica*

In order to see whether the nucleic acid polymorphisms existing in the three longest haplotypes (HP15L, HP16L, and

HP17L) of the *CcDREB1D* promoter influenced the regulation of these sequences, the activity of these promoters was evaluated by calculating the proportion of GUS-stained guard cells on leaf abaxial regions in a time course response to water deficit assays simulated by PEG (equivalent to -2.0 MPa) and to low RH assays (Fig. 5). For the pHP16L- and pHP17L-transformed plants, the proportion of GUS-stained guard cells increased in the first hours of PEG treatment, reaching 20% and 10% maximum on average after 12 and 6 h, respectively (Fig. 5). However, the proportion of GUS-stained guard cells remained relatively low and stable during the PEG assay for pHP15L-transformed plants. For the low RH assay, around 20% of guard cells were GUS stained after 24 h of treatment in pHP16L-transformed plants. This percentage was reached after 6 h in pHP15L-transformed plants and gradually decreased over the time course to reach 5% after 24 h of stress. In pHP17L-transformed plants, the proportion of GUS-stained guard cells increased slowly to reach a mean of 10% after 12 h of low RH or after 5 h of PEG treatment and decreased thereafter.

*uidA and CcDREB1D gene expression*

The monitoring of *uidA* gene expression in leaves of coffee plants transformed by pHP15L, pHP16L, and pHP17L and subjected to PEG treatment showed low variability for each construct at each time of the water deficit bioassay. Similar expression profiles were obtained for all the constructs, showing a gradual increase in *uidA* gene expression with PEG treatment, which reached maximum expression after 6 h (pHP16L and pHP17L) or after 12 h (pHP15L) and declined

<figure><img src="image_12.png" /><figcaption>**Fig. 4.** Histochemical localization of GUS activity in guard cells of *C. arabica* subjected to low air relative humidity (RH 9%). Guard cells visualized by bright field microscopy (x20 magnification) on the abaxial detached epidermis of coffee leaves. The explants were from coffee plants transformed by pBI101 (A, negative control), pBI121 (B, positive control), pHP17S (C, D), pHP17L (E, F), pHP16S (G, H), and pHP16L (I, J). Unstressed conditions for (C, E, G, I). Stressed conditions (9% RH) for (A, B, D, F, H, J). The scale bars in each image correspond to 30 µm.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Proportions of GUS-stained guard cells: water deficit-induced regulation of GUS activity in mature leaf stomata driven by *CcDREB1D* promoter haplotypes. Change of GUS-stained guard cells' proportions following 0, 3, 6, 12 and 24 h of exposure to PEG (equivalent to -2.0 MPa) and low air relative humidity (RH 9%) treatments in pHP15L-, pHP16L-, and pHP17L-transformed coffee plants. Box-and-whisker plots display variation in proportions of GUS-stained guard cells for each time assessed. The lower and upper hinges represent the first and third quartile respectively, the bold line represents the median, and the whiskers represent the smallest and greatest values.</figcaption></figure>

thereafter (Fig. 6A). The main differences observed between the constructs concerned relative expression values in the first hours of stress (3 and 6 h), with *uidA* gene expression being higher in pHP16L-transformed plants than in pHP15L- and pHP17L-transformed plants. As controls, expression of the *CcDREB1D* endogenous gene increased with PEG treatment, reaching a peak at 6 h and then decreased gradually afterwards (Fig. 6B).

Discussion

In our study, we investigated the responses of different haplo-
types of the *CcDREB1D* (*CBF4*) promoters of *C. canephora*
to PEG and low RH treatments by analysing their ability to
regulate the expression of the *uidA* reporter gene in stably
transformed *C. arabica* plants.

*Multiple CREs are involved in the regulation of*
*CcDREB1D promoter haplotypes*

The results presented here indicate that the HP15, HP16 and
HP17 haplotypes of *CcDREB1D* promoters harbored sev-
eral abiotic stress-responsive CREs in common, including
those involved in tissue-specific and light regulation. They
also contained ABRE and DRE elements, thereby suggest-
ing possible regulation of these promoters by both ABA-
dependent and -independent networks (Haake *et al.*, 2002;
Yamaguchi-Shinozaki and Shinozaki, 2005). Interestingly,
the co-occurrence in parallel strands and opposite direc-
tion of ABRE and DRE elements within 100 bp, identified
herein for all haplotypes, is the most active structural organi-
zation observed for these CREs in drought-responsive genes
(Mishra *et al.*, 2014). If we consider that DRE elements
share a common DNA motif with low-temperature respon-
sive element (LTRE), there could be a functional implica-
tion for coffee plant responses to cold stress, as observed
for homologous genes in several plants (Zhang *et al.*, 2004;
Ito *et al.*, 2006; Mao and Chen, 2012). This is reinforced by
the fact that the three *CcDREB1D* haplotypes reported here

<figure><img src="image_14.png" /><figcaption>**Fig. 6.** Expression profiles of the *uidA* and *CaDREB1D* genes in leaves of transgenic *C. arabica* during water deficit. Expression of the *uidA* (A) and *CaDREB1D* (B) genes was tested in leaves of coffee plants transformed by the pHP15L (white isobars), pHP16L (light gray isobars), and pHP17L (dark gray isobars) constructs and subjected to 0, 3, 6, 12, and 24 h osmotic stress (PEG treatment) by RT-qPCR experiments using the GUS-F/R and DREBA09-F/R primer pairs, respectively. Expression levels are indicated in relative quantification using the expression of the *CaGAPDH* gene as a reference. The results are expressed using T₀ samples as internal calibrators for each construct. The relative quantification values correspond to the mean of at least three biological repetitions analysed by three technical replicates±SD. The significance of expression level differences was evaluated using the pairwise Wilcoxon rank test (non-parametric test). Treatments sharing the same letter are not significantly different.</figcaption></figure>

also contain other CREs such as one ICEr2 box (except in
the HP17 haplotype) and eight MYC-related binding sites
(CANNTG), which are both motifs known to play a key
role in the regulation of DREB1/CBF genes by cold-stress.
Regarding MYC-related DNA motifs, two of them contained
the preferable target sequence CAAATG (-842/-837 for all
HPs and -340/-335 only for HP17) of the positive regulator inducer of CBF expression 1 (ICE1).

The three *CcDREB1D* haplotypes also contained several MYB-binding sites that can act as either an enhancer or repressor of promoter activity. One of them (MYB1AT: AAACCA) is a putative DNA binding site of the MYB15 *trans*-acting factor known to regulate negatively the expression of DREB1D/CBF genes (Agarwal *et al.*, 2006a). The remaining MYB boxes are potential positive regulators of DREB1/CBF expression, especially MYB2AT (YAACTG) binding sites required for activation of salt- and dehydration-responsive genes (Urao *et al.*, 1993; Abe *et al.*, 2003). *In silico* analyses of the *CcDREB1D* haplotypes also revealed the occurrence of several DOF-binding sites, already reported as key DNA motifs mediating guard cell-specific gene expression, and of several GT-1 binding sites essential for light-inducible expression, mainly located in the last 500 bp region (-921/-1183) of the *CcDREB1D* promoters (Plesch *et al.*, 2001; Galbiati *et al.*, 2008; Yang *et al.*, 2008; Gardner *et al.*, 2009; Cominelli *et al.*, 2011).

Modular organization of DOF and light-induced DNA elements may account for preferential expression of CcDREB1D promoter haplotypes in guard cells

Previous studies showed that *OsDREB1B*, *AtDREB1C*, *AtDREB2C*, and *FeDREB1* promoters contained all CREs within the proximal regions (up to 1 kb) of these promoters necessary for enhancing gene expression by several abiotic stresses (Zarka *et al.*, 2003; Gutha and Reddy, 2008; Chen *et al.*, 2009; Chen *et al.*, 2012; Fang *et al.*, 2015). The results of GUS activities presented here in transgenic coffee plants transformed by the short (S: +182/-608), medium (M: +182/-966), or full-length (L: +182/-1308) sequences of *CcDREB1D* haplotypes clearly revealed the importance of the CREs located in the -608/-1308 region in regulating the expression of the *uidA* reporter gene in a tissue-specific manner. Indeed, while ubiquitous and strong GUS activities were observed in different tissues of coffee plants transformed by the shortest constructs (pHP16S and pHP17S), fine-tuned regulation of the *uidA* reporter gene was observed in coffee plants transformed by the full-length constructs (pHP15L, pHP16L, and pHP17L), with GUS activities restricted to leaf mesophyll and guard cells under stressed conditions. In that case, the degree of *CcDREB1D* haplotype activity in guard cells also appeared to increase with the number of DOF-binding sites, from the short (six sites) to the full-length (11/12 sites) promoter regions. One possible explanation could be an increase in the numbers of regulatory sites recognized by *trans*-acting factors known in the longest constructs of *CcDREB1D* haplotypes. For example, of the five MYB DNA boxes found within the -608/-1308 region, three MYBCORE and one MYB2AT are positive regulators and one (MYB1AT) is a putative binding site of MYB15 *trans*-acting factor known to repress *DREB1/CBF* genes (Agarwal *et al.*, 2006a) but also to regulate guard cell stomatal closure under drought conditions (Ding *et al.*, 2009). The presence of such DNA boxes could explain the low GUS activities

observed in coffee plants transformed with the longest constructs (pHP15L, pHP16L, and pHP17L) compared with those observed in plants transformed with the shortest constructs (pHP16S and pHP17S). The increase in leaf-specific GUS activities observed from the short to the medium- and full-length constructs also tallied with the fact that cluster arrangements of DOF-binding sites are essential in determining guard cell-specific gene expression (Galbiati *et al.*, 2008; Yang *et al.*, 2008). However, this does not preclude the participation of other CREs functioning conjointly with DOF-binding sites to fine-tune the transcriptional regulation of gene expression in guard cells, as previously proposed in grape *VvMYB60*, potato *KST1* and cotton *GbSLSP* promoters, for example (Plesch *et al.*, 2001; Gardner *et al.*, 2009; Cominelli *et al.*, 2011; Galbiati *et al.*, 2011; Han *et al.*, 2013). Of the CREs that could play such roles, it is worth noting the presence of several GT-1 DNA binding sites in the *CcDREB1D* promoter haplotypes, with three of them (one in the medium (-608/-966) and two in the distal (-966/-1308) regions of the *CcDREB1D* promoters) closely located to DOF-binding elements. Therefore, GT-1 DNA binding sites could participate together with DOF elements in forming such clusters necessary for targeting guard cell-specific gene expression.

The *CcDREB1D* promoter haplotypes respond differentially to RH and PEG abiotic stress

Few studies have reported the existence of functional variation between haplotypes (or alleles) of plant promoters in regulating gene expression differentially (de Meaux *et al.*, 2005; Takeshima *et al.*, 2016). The results presented here regarding the proportion of GUS-stained guard cells measured in transgenic coffee lines subjected to low water potential (PEG treatment) clearly showed greater activity for the HP16 and HP17 full-length haplotypes compared with very low activity for the HP15L haplotype. However, the activity of all the full-length *CcDREB1D* haplotypes was up-regulated under low air relative humidity (RH 9%). In that case, a slight increase in GUS-stained guard cells was observed in pHP15L- and pHP17L-transformed plants, while this proportion increased slightly but regularly in pHP16L-transformed plants in the RH treatment. The different time courses of GUS-stained guard cells observed between the HP15, HP16, and HP17 haplotypes during the PEG and RH treatments clearly revealed differences in the fine-tuning of the regulation of these *CcDREB1D* promoters, which were undoubtedly related to the nucleic acid polymorphism identified between these sequences directly affecting CREs and/or altering their cluster organization (see discussion below). Our results also indicated that the PEG and RH treatments affected the time course of activity of the HP16 and HP17 haplotypes of *CcDREB1D* promoters differently. For example, GUS-stained guard cells were detected earlier in pHP16L- and pHP17L-transformed plants in response to the PEG treatment compared with the RH treatment. PEG treatment is known to mediate low $Ψ_w$ by reducing the osmotic potential of the nutrient solution, thereby mimicking low soil water potential that leads to a water deficit in the whole plant (Blum, 2014). Following the
perception of water deficit by roots (major ABA producing
organs), ABA would rapidly amplify the drought signal and
activate the *CcDREB1D* promoter in guard cells, probably
through the involvement of ABRE regulatory elements. The
fact that exogenous ABA up-regulated the expression of the
*uidA* and *CaDREB1D* gene, mainly in stomata guard cells of
pHP16L-transformed coffee plants, supports the idea that
this phytohormone plays a key role in coffee responses to
water deficit (Torres *et al.*, 2016). While ABA induces early
responses of *CcDREB1D* promoters by root-sensing water
deficit, an inverse drought stimulus (where water stress is
'sensed' by aerial parts rather than roots) is expected to delay
the response of the *CcDREB1D* promoter to water deficit. To
simulate such conditions, our transgenic coffee plants were
subjected to a water deficit of -2.0 MPa mimicked by the
low RH treatment, which is known to increase the leaf-to-air
water vapor pressure deficit (VPD), to generate evaporative
demand and, consequently, to elevate the transpiration rate
(Farooq *et al.*, 2009). The increase over time in GUS-stained
guard cells in pHP16L- and pHP17L-transformed plants
with the low RH (high VPD) treatment tends to support the
existence of such a mechanism. However, while the frequency
of GUS activity in guard cells increased gradually with a low
RH in pHP16L-transformed plants, it increased in the first
12 h only and then decreased slowly in pHP17L-transformed
plants, thereby demonstrating that these haplotypes differed
in activity and/or regulation.

*The nucleic acid polymorphisms existing in
CcDREB1D haplotypes are probably involved in
modulating promoter activities*

The comparison of HP15, HP16, and HP17 haplotype
sequences revealed that (i) they had most of the essential
CREs in common, and (ii) HP15 and HP16 were more closely
related than HP17 regarding INDEL-type polymorphisms.
By comparing CREs between HP16 and HP15, very few dif-
ferences were observed regarding the boxes known to recog-
nize key transcription factors involved in abiotic stress. The
fact that these haplotypes of *CcDREB1D* promoters are reg-
ulated in a different manner in controlling the expression of
the *uidA* reporter gene suggests that the nucleotide polymor-
phisms observed outside of the CREs might be responsible
for these differences, as could be the case for the 2 bp insertion
(AA: -1033/-1032) in HP16, with this INDEL being local-
ized close to GT1 (-1037/-1027) and DOF (-1017/-1013)
DNA binding sites. Several other polymorphisms, localized
close to essential CREs, were also identified mainly in the
polymorphic domains -627/-806 and -932/-1121. Within
the -627/-806 region, the presence of (i) MYB- and MYC-
like regulatory elements, respectively known as activator and
repressors of transcription by drought, and (ii) a tandem (×3)
repeat of the GAAWTT unidentified motif (-722/-739) only
present in the HP16 haplotype is worth noting. Compared
with the HP16 sequence, a G to A transition (in position
-1013) in the HP17 haplotype, leading to the loss of one DOF
domain, is also worth noting. Since DOF transcription fac-
tors are involved in the regulation of various plant processes

(Le Hir and Bellini, 2013), such as stomatal functioning, for
example (Gardner et al., 2009; Negi et al., 2013), this might
explain the differences in activity observed between these two
haplotypes under PEG and low RH assays.

Drought stress up-regulates the expression of
CcDREB1D haplotypes in transgenic plants of
C. arabica

The increase in GUS activities observed in the water deficit mimicking treatments in leaves of transgenic coffee plants transformed by the pHP15L, pHP16L, and pHP17L constructs was confirmed by monitoring expression of the *uidA* reporter gene. For all the haplotypes, *uidA*-specific transcripts were accumulated after 3–12 h of PEG treatment (maximum after 6–12h) and declined thereafter. Although similar in terms of profiles, differences in promoter strength were found, with the HP16L promoter being stronger and occurring sooner (significant expression after 3 h) than the other two haplotypes. It is worth noting that the accumulation of *uidA* transcripts followed quite well the expression profile of the endogenous *CaDREB1D* gene, which was also up-regulated in the PEG treatment. In that case, the quantity of *CaDREB1D* transcripts detected after 24 h of treatment was very weak and did not differ from that before water deficit treatment. *CaDREB1D* transcripts were almost barely detectable after 6 h. Our results strongly suggest that the transient expression of the *CaDREB1D* gene under water deficit directly reflected the transitory activity of *DREB1D* promoters.

Even though the expression of *DREB1/CBF* genes has been
commonly reported to be mainly induced by cold, our results
clearly indicated that the expression of the *DREB1D* genes
of *C. arabica* was rapidly induced by water deficit mimick-
ing treatments and that the up-regulated expression of these
genes was controlled at transcriptional level. These results
also showed that the different promoter haplotypes of the
*CcDREB1D* gene of the diploid *C. canephora* were correctly
recognized by the transcriptional machinery of the allotetra-
ploid species *C. arabica*.

Summarizing, the variations in intensities and staining
patterns between the three haplotypes tested during this
study suggested that HP16 (isolated from D<sup>T</sup> clone 14 of
*C. canephora*) had greater strength, as well as longer activ-
ity, under low RH conditions compared with HP15 (found in
both D<sup>T</sup> clone 14 and D<sup>S</sup> clone 22) and HP17 (isolated from
D<sup>S</sup> clone 22) haplotypes. These characteristics might explain
the higher stomatal conductance (g<sub>s</sub>), and consequently
the more efficient mechanisms in controlling the transpira-
tion rate observed under water deficit in D<sup>T</sup> clone 14 com-
pared with D<sup>S</sup> clone 22 (Marraccini *et al.*, 2012). Based on
the results of this study, work is now on-going to analyse the
genetic diversity of *DREB1D* promoters in other D<sup>T</sup> and D<sup>S</sup>
plants of *Coffea* species with the objective to identify molecu-
lar markers for breeding drought-resistant cultivars, and also
to study the effects of other types of abiotic stress (e.g. high
temperature and light intensities, cold stress, etc.) on the regu-
lation and activity of the *CcDREB1D* haplotypes.
## Supplementary data

Supplementary data are available at *JXB* online.

Fig. S1. Nucleic acid alignments of *CcDREB1D* haplotypes (HP15, HP16 and HP17) with the *CcDREB1D* consensus sequence (see Fig. 1).

Fig. S2. Histochemical localization (root, stem, meristem, leaf) of GUS activity in dehydrated control plants of *C. arabica*.

Fig. S3. Confocal laser scanning microscopy (CLSM) micrographs from leaf cross-sections with immunolocalization of GUS protein.

Table S1. List of nucleic acid polymorphisms found in the HP15, HP16, and HP17 haplotypes of *CcDREB1D* coffee promoters.

## Accession numbers

Sequence data for the HP15, HP16, and HP17 haplotypes of *CcDREB1D* promoters can be found in the GenBank database under accession numbers KM281308, KM281309, and KM281311, respectively.

## Acknowledgements

This work was carried out under the Embrapa-CIRAD scientific cooperation project entitled 'Genetic determinism of drought tolerance in coffee'. The authors acknowledge financial support from the Brazilian Consortium on Coffee R&D, Instituto Nacional de Ciência e Tecnologia—Café (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Fundação de Amparo à Pesquisa do Estado de Minas Gerais) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Comité Français d'Évaluation de la Coopération Universitaire et Scientifique avec le Brésil (Project 407–2012/University of Lavras and Montpellier SupAgro). The authors would like to thank Pr. Murilo Sversut Dias (University of Brasília-Department of Ecology) for his kind contribution of statistical and imaging analysis. We also thank Peter Biggins (CIRAD-DGDRS) for English revision of the manuscript.

## Author contributions

GSCA cloned and sequenced the *CcDREB1D* promoter haplotypes, made the constructs and analysed these sequences. GSCA, LFT, HE, and ED carried out the genetic transformation of *C. arabica*, applied abiotic stress, and carried out GUS staining and microscopy analyses with the help of FG. LFT extracted RNA samples used by J-CB to perform qPCR experiments and analysed the results together with TJ. HE, BB, ACA and PM designed the study, drew up the experimental design and implemented it. GSCA, ACA, TJ, PM, and HE wrote the article, which was approved by all the authors.

## References

**Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K.** 2003. *Arabidopsis* AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. The Plant Cell **15**, 63–78.

**Agarwal M, Hao Y, Kapoor A, Dong CH, Fujii H, Zheng X, Zhu JK.** 2006a. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. The Journal of Biological Chemistry **281**, 37636–37645.

**Agarwal PK, Agarwal P, Reddy MK, Sopory SK.** 2006b. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Reports **25**, 1263–1274.

**Avelino J, Willocquet L, Savary S.** 2004. Effects of crop management patterns on coffee rust epidemics. Plant Pathology **53**, 541–547.

**Blum A.** 2014. Genomics for drought resistance—getting down to earth. Functional Plant Biology **41**, 1191–1198.

**Breitler JC, Campa C, Georget F, Bertrand B, Etienne H.** 2016. A single-step method for RNA isolation from tropical crops in the field. Scientific Reports **6**, 38368.

**Bunn C, Läderach P, Pérez Jimenez JG, Montagnon C, Schilling T.** 2015. Multiclass classification of agro-ecological zones for Arabica coffee: an improved understanding of the impacts of climate change. PLoS ONE **10**, e0140490.

**Chang WC, Lee TY, Huang HD, Huang HY, Pan RL.** 2008. PlantPAN: Plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups. BMC Genomics **9**, 561.

**Chen H, Je J, Song C, Hwang JE, Lim CO.** 2012. A proximal promoter region of *Arabidopsis* *DREB2C* confers tissue-specific expression under heat stress. Journal of Integrative Plant Biology **54**, 640–651.

**Chen M, Xu Z, Xia L, Li L, Cheng X, Dong J, Wang Q, Ma Y.** 2009. Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, *GmDREB3*, in soybean (*Glycine max* L.). Journal of Experimental Botany **60**, 121–135.

**Cheserek JJ, Gichimu BM.** 2012. Drought and heat tolerance in coffee: a review. International Research Journal of Agricultural Science and Soil Science **2**, 498–501.

**Chinnusamy V, Zhu J, Zhu JK.** 2007. Cold stress regulation of gene expression in plants. Trends in Plant Science **12**, 444–451.

**Cominelli E, Galbiati M, Albertini A, Fornara F, Conti L, Coupland G, Tonelli C.** 2011. DOF-binding sites additively contribute to guard cell-specificity of *AtMYB60* promoter. BMC Plant Biology **11**, 162.

**Cotta MG, Barros LM, de Almeida JD, de Lamotte F, Barbosa EA, Vieira NG, Alves GS, Vinecky F, Andrade AC, Marraccini P.** 2014. Lipid transfer proteins in coffee: isolation of *Coffea* orthologs, *Coffea arabica* homeologs, expression during coffee fruit development and promoter analysis in transgenic tobacco plants. Plant Molecular Biology **85**, 11–31.

**Craparo ACW, Van Asten PJA, Läderach P, Jassogne LTP, Grab SW.** 2015. *Coffea arabica* yields decline in Tanzania due to climate change: Global implications. Agricultural and Forest Meteorology **207**, 1–10.

**Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros LMG, Romano E, Grossi-de-Sá MF, Vaslin M, Alves-Ferreira M.** 2009. Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Molecular Breeding **23**, 607–616.

**Cubry P, De Bellis F, Pot D, Musoli P, Leroy T.** 2013. Global analysis of *Coffea canephora* Pierre ex Froehner (Rubiaceae) from the Guineo-Congolese region reveals impacts from climatic refuges and migration effects. Genetic Resources and Crop Evolution **60**, 483–501.

**DaMatta FM, Ramalho JDC.** 2006. Impacts of drought and temperature stress on coffee physiology and production: a review. Brazilian Journal of Plant Physiology **18**, 55–81.

**Davis AP, Gole TW, Baena S, Moat J.** 2012. The impact of climate change on indigenous Arabica coffee (*Coffea arabica*): predicting future trends and identifying priorities. PLoS ONE **7**, e47981.

**de Meaux J, Goebel U, Pop A, Mitchell-Olds T.** 2005. Allele-specific assay reveals functional variation in the *chalcone synthase* promoter of *Arabidopsis thaliana* that is compatible with neutral evolution. The Plant Cell **17**, 676–690.

**Denoeud F, Carretero-Paulet L, Dereeper A, et al.** 2014. The coffee genome provides insight into the convergent evolution of caffeine biosynthesis. Science **345**, 1181–1184.

**Ding Z, Li S, An X, Liu X, Qin H, Wang D.** 2009. Transgenic expression of *MYB15* confers enhanced sensitivity to abscisic acid and improved drought tolerance in *Arabidopsis thaliana*. Journal of Genetics and Genomics **36**, 17–29.

**Doherty CJ, Van Buskirk HA, Myers SJ, Thomashow MF.** 2009. Roles for *Arabidopsis* CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. The Plant Cell **21**, 972–984.

**Etienne H.** 2005. Somatic embryogenesis protocol: Coffee (*Coffea arabica* L. and *C. canephora* P.). In: Jain SM, Gupta PK, eds. Protocols of somatic embryogenesis in woody plants. Dordrecht: Springer, 167–179.
Fang ZW, Xu XY, Gao JF, Wang PK, Liu ZX, Feng BL. 2015. Characterization of *FeDREB1* promoter involved in cold- and drought-inducible expression from common buckwheat (*Fagopyrum esculentum*). *Genetics and Molecular Research* **14**, 7990–8000.

Farooq M, Wahid A, Kobayashi N, Fujita D, Basra SMA. 2009. Plant drought stress: effects, mechanisms and management. *Agronomy for Sustainable Development* **29**, 185–212.

Galbiati M, Matus JT, Francia P, Rusconi F, Cañón P, Medina C, Conti L, Cominelli E, Tonelli C, Arce-Johnson P. 2011. The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress. *BMC Plant Biology* **11**, 142.

Galbiati M, Simoni L, Pavesi G, Cominelli E, Francia P, Vavasseur A, Nelson T, Bevan M, Tonelli C. 2008. Gene trap lines identify *Arabidopsis* genes expressed in stomatal guard cells. *The Plant Journal* **53**, 750–762.

Gardner MJ, Baker AJ, Assie JM, Poethig RS, Haseloff JP, Webb AA. 2009. GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression. *Journal of Experimental Botany* **60**, 213–226.

Ghini R, Torre-Neto A, Dentzien AFM, et al. 2015. Coffee growth, pest and yield responses to free-air CO₂ enrichment. *Climatic Change* **132**, 307–320.

Gutha LR, Reddy AR. 2008. Rice *DREB1B* promoter shows distinct stress-specific responses, and the overexpression of cDNA in tobacco confers improved abiotic and biotic stress tolerance. *Plant Molecular Biology* **68**, 533–555.

Guttikonda SK, Valliyodan B, Neelakandan AK, et al. 2014. Overexpression of *AtDREB1D* transcription factor improves drought tolerance in soybean. *Molecular Biology Reports* **41**, 7995–8008.

Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ. 2002. Transcription factor CBF4 is a regulator of drought adaptation in *Arabidopsis*. *Plant Physiology* **130**, 639–648.

Han L, Han YN, Xiao XG. 2013. Truncated cotton subtilase promoter directs guard cell-specific expression of foreign genes in tobacco and *Arabidopsis*. PLoS ONE **8**, e59802.

Higo K, Ugawa Y, Iwamoto M, Korenaga T. 1999. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Research **27**, 297–300.

Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. 2006. Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. *Plant & Cell Physiology* **47**, 141–153.

Jaramillo J, Muchugu E, Vega FE, Davis A, Borgemeister C, Chabi-Olaye A. 2011. Some like it hot: the influence and implications of climate change on coffee berry borer (*Hypothemus hampei*) and coffee production in East Africa. PLoS ONE **6**, e24528.

Kim JS, Mizoi J, Yoshida T, et al. 2011. An ABRE promoter sequence is involved in osmotic stress-responsive expression of the *DREB2A* gene, which encodes a transcription factor regulating drought-inducible genes in *Arabidopsis*. *Plant & Cell Physiology* **52**, 2136–2146.

Lata C, Prasad M. 2011. Role of DREBs in regulation of abiotic stress responses in plants. *Journal of Experimental Botany* **62**, 4731–4748.

Le Hir R, Bellini C. 2013. The plant-specific Dof transcription factors family: new players involved in vascular system development and functioning in *Arabidopsis*. *Frontiers in Plant Science* **4**, 164.

Li D, Zhang Y, Hu X, Shen X, Ma L, Su Z, Wang T, Dong J. 2011. Transcriptional profiling of *Medicago truncatula* under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses. *BMC Plant Biology* **11**, 109.

Lim CJ, Hwang JE, Chen H, Hong JK, Yang KA, Choi MS, Lee KO, Chung WS, Lee SY, Lim CO. 2007. Over-expression of the *Arabidopsis* DRE/CRT-binding transcription factor DREB2C enhances thermotolerance. *Biochemical and Biophysical Research Communications* **362**, 431–436.

Lima ALS, DaMatta FM, Pinheiro HA, Totola MR, Loureiro ME. 2002. Photochemical responses and oxidative stress in two clones of *Coffea canephora* under water stress. *Environmental and Experimental Botany* **47**, 239–247.

Magrach A, Ghazoul J. 2015. Climate and pest-driven geographic shifts in global coffee production: implications for forest cover, biodiversity and carbon storage. PLoS ONE **10**, e0133071.

Mao D, Chen C. 2012. Colinearity and similar expression pattern of rice DREB1s reveal their functional conservation in the cold-responsive pathway. PLoS ONE **7**, e47275.

Marraccini P, Freire LP, Alves GS, et al. 2011. RBCS1 expression in coffee: *Coffea* orthologs, *Coffea arabica* homeologs, and expression variability between genotypes and under drought stress. *BMC Plant Biology* **11**, 85.

Marraccini P, Vinecky F, Alves GS, et al. 2012. Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of *Coffea canephora*. *Journal of Experimental Botany* **63**, 4191–4212.

Martins LD, Tomaz MA, Lidon FC, DaMatta FM, Ramalho JC. 2014. Combined effects of elevated [CO₂] and high temperature on leaf mineral balance in *Coffea* spp. plants. *Climatic Change* **126**, 365–379.

Martins LD, Silva MJ, Reboredo FH, et al. 2016. Protective response mechanisms to heat stress in interaction with high [CO₂] conditions in *Coffea* spp. *Frontiers in Plant Science* **7**, 947.

Michel BE, Kaufmann MR. 1973. The osmotic potential of polyethylene glycol 6000. *Plant Physiology* **51**, 914–916.

Mishra S, Shukla A, Upadhyay S, et al. 2014. Identification, occurrence, and validation of DRE and ABRE cis-regulatory motifs in the promoter regions of genes of *Arabidopsis thaliana*. *Journal of Integrative Plant Biology* **56**, 388–399.

Montagnon C. 2000. Optimisation des gains génétiques dans le schéma de sélection récurrente réciproque de *Coffea canephora* Pierre. PhD Thesis, Montpellier University.

Montagnon C, Cubry P, Leroy T. 2012. Coffee *Coffea canephora* Pierre genetic improvement: Acquired knowledge, strategies and perspectives. *Cahiers de l'Agriculture* **21**, 143–153.

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. *Physiologia Plantarum* **15**, 473–497.

Negi J, Moriwaki K, Konishi M, et al. 2013. A Dof transcription factor, SCAP1, is essential for the development of functional stomata in *Arabidopsis*. *Current Biology* **23**, 479–484.

Novillo F, Alonso JM, Ecker JR, Salinas J. 2004. CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in *Arabidopsis*. *Proceedings of the National Academy of Sciences, USA* **101**, 3985–3990.

Pinheiro HA, DaMatta FM, Chaves ARM, Fontes EPB, Loureiro ME. 2004. Drought tolerance in relation to protection against oxidative stress in clones of *Coffea canephora* subjected to long-term drought. *Plant Science* **167**, 1307–1314.

Pinheiro HA, DaMatta FM, Chaves AR, Loureiro ME, Ducatti C. 2005. Drought tolerance is associated with rooting depth and stomatal control of water use in clones of *Coffea canephora*. *Annals of Botany* **96**, 101–108.

Plesch G, Ehrhardt T, Mueller-Roeber B. 2001. Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression. *The Plant Journal* **28**, 455–464.

Praxedes SC, DaMatta FM, Loureiro ME, Ferrão MAG, Cordeiro AT. 2006. Effects of long-term soil drought on photosynthesis and carbohydrate metabolism in mature robusta coffee (*Coffea canephora* Pierre var. kouillou) leaves. *Environmental and Experimental Botany* **56**, 263–273.

Ramakers C, Ruijter JM, Deprez RH, Moorman AF. 2003. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. *Neuroscience Letters* **339**, 62–66.

Ramalho JC, Rodrigues AP, Semedo JN, et al. 2013. Sustained photosynthetic performance of *Coffea* spp. under long-term enhanced [CO₂]. PLoS ONE **8**, e82712.

Ribas AF, Dechamp E, Champion A, Bertrand B, Combes MC, Verdeil JL, Lapeyre F, Lashermes P, Etienne H. 2011. *Agrobacterium*-mediated genetic transformation of *Coffea arabica* (L.) is greatly enhanced by using established embryogenic callus cultures. *BMC Plant Biology* **11**, 92.

Rodrigues WP, Martins MQ, Fortunato AS, et al. 2016. Long-term elevated air [CO₂] strengthens photosynthetic functioning and mitigates the impact of supra-optimal temperatures in tropical *Coffea arabica* and *C. canephora* species. *Global Change Biology* **22**, 415–431.

Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K. 2002. DNA-binding specificity of the ERF/AP2 domain of *Arabidopsis* DREBs, transcription factors involved in dehydration- and
cold-inducible gene expression. Biochemical and Biophysical Research Communications **290**, 998–1009.

Shahmuradov IA, Solovyev VV, Gammerman AJ. 2005. Plant promoter prediction with confidence estimation. Nucleic Acids Research **33**, 1069–1076.

Silva EA, Mazzafera P, Brunini O, Sakai E, Arruda FB, Mattoso LHC, Carvalho CRL, Pires RCM. 2005. The influence of water management and environmental conditions on the chemical composition and beverage quality of coffee beans. Brazilian Journal of Plant Physiology **17**, 229–238.

Silva OC. 1994. CG-1, a parsley light-induced DNA-binding protein. Plant Molecular Biology **25**, 921–924.

Takeshima R, Hayashi T, Zhu J, et al. 2016. A soybean quantitative trait locus that promotes flowering under long days is identified as *FT5a*, a *FLOWERING LOCUS T* ortholog. Journal of Experimental Botany **67**, 5247–5258.

Torres LF, Déchamp E, Alves GSC, Diniz LEC, Paiva LV, Breitler J-C, Andrade AC, Marraccini P, Etienne H. 2016. Influence of abiotic stresses in phenotypic expression of transgenic plants of *Coffea arabica* under action *CcDREB1D* promoter. In: 26th International Conference on Coffee Science. Kunming, China: Association for Science and Information on Coffee (ASIC), B2.

Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. 1993. An *Arabidopsis myb* homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. The Plant Cell **5**, 1529–1539.

van der Vossen H. Bertrand B, Charrier A. 2015. Next generation variety development for sustainable production of arabica coffee (*Coffea arabica* L.): A review. Euphytica **204**, 243–256.

Vieira NG, Carneiro FA, Sujii PS, et al. 2013. Different molecular mechanisms account for drought tolerance in *Coffea canephora* var. Conilon. Tropical Plant Biology **6**, 181–190.

Vinecky F, Davrieux F, Mera AC, et al. 2016. Controlled irrigation and nitrogen, phosphorous and potassium fertilization affect the biochemical composition and quality of Arabica coffee beans. Journal of Agricultural Science, doi: 10.1017/S0021859616000988.

Xiao H, Tattersall EA, Siddiqua MK, Cramer GR, Nassuth A. 2008. CBF4 is a unique member of the CBF transcription factor family of *Vitis vinifera* and *Vitis riparia*. Plant, Cell & Environment **31**, 1–10.

Yamaguchi-Shinozaki K, Shinozaki K. 2005. Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends in Plant Science **10**, 88–94.

Yang Y, Costa A, Leonhardt N, Siegel RS, Schroeder JI. 2008. Isolation of a strong *Arabidopsis* guard cell promoter and its potential as a research tool. Plant Methods **4**, 6.

Zandkarimi H, Ebadi A, Salami SA, Alizade H, Baisakh N. 2015. Analyzing the expression profile of *AREB/ABF* and *DREB/CBF* genes under drought and salinity stresses in grape (*Vitis vinifera* L.). PLoS ONE **10**, e0134288.

Zarka DG, Vogel JT, Cook D, Thomashow MF. 2003. Cold induction of *Arabidopsis* CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature. Plant Physiology **133**, 910–918.

Zhang X, Fowler SG, Cheng H, Lou Y, Rhee SY, Stockinger EJ, Thomashow MF. 2004. Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant *Arabidopsis*. The Plant Journal **39**, 905–919.