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Mol Genet GenomicsDOI 10.1007/s00438-014-0864-y

OrIGInal PaPer

Homeologous genes involved in mannitol synthesis reveal unequal contributions in response to abiotic stress in Coffea arabica

Kenia de Carvalho · Carmen L. O. Petkowicz · Getulio T. Nagashima · João C. Bespalhok Filho · Luiz G. E. Vieira · Luiz F. P. Pereira · Douglas S. Domingues

received: 6 november 2013 / accepted: 6 May 2014 © Springer-Verlag Berlin Heidelberg 2014

aimed to investigate the transcriptional responses of genes involved in mannitol biosynthesis and catabolism in C. ara-bica leaves under water deficit, salt stress and high temper-ature. Mannitol concentration was significantly increased in leaves of plants under drought and salinity, but reduced by heat stress. Fructose content followed the level of mannitol only in heat-stressed plants, suggesting the partitioning of the former into other metabolites during drought and salt stress conditions. Transcripts of the key enzymes involved in mannitol biosynthesis, CaM6PR, CaPMI and CaMTD, were modulated in distinct ways depending on the abiotic stress. Our data suggest that changes in mannitol accumu-lation during drought and salt stress in leaves of C. arabica are due, at least in part, to the increased expression of the key genes involved in mannitol biosynthesis. In addition, the homeologs of the Coffea canephora subgenome did not present the same pattern of overall transcriptional response, indicating differential regulation of these genes by the same stimulus. In this way, this study adds new information on the differential expression of C. arabica homeologous genes under adverse environmental conditions showing that abiotic stresses can influence the homeologous gene regula-tion pattern, in this case, mainly on those involved in man-nitol pathway.

Keywords Mannitol · Phosphomannose-isomerase · Mannose-6-phosphate reductase · Mannitol dehydrogenase · Water deficit · Salt stress · High temperature · Homeologous genes

AbbreviationsPMI Phosphomannose-isomeraseM6Pr Mannose-6-phosphate reductaseMTD Mannitol dehydrogenaseGaPDH Glyceraldehyde-3-phosphate-dehydrogenase

Abstract Polyploid plants can exhibit transcriptional modulation in homeologous genes in response to abiotic stresses. Coffea arabica, an allotetraploid, accounts for 75 % of the world’s coffee production. extreme tempera-tures, salinity and drought limit crop productivity, which includes coffee plants. Mannitol is known to be involved in abiotic stress tolerance in higher plants. This study

Communicated by S. Hohmann.

Electronic supplementary material The online version of this article (doi:10.1007/s00438-014-0864-y) contains supplementary material, which is available to authorized users.

K. de Carvalho · l. G. e. Vieira · l. F. P. Pereira · D. S. Domingues (*) laboratório de Biotecnologia Vegetal, Instituto agronômico do Paraná, londrina, Paraná CeP 86047-902, Brazile-mail: doug@iapar.br

K. de Carvalho · J. C. Bespalhok Filho Programa de Pós-Graduação em agronomia (Produção Vegetal), Universidade Federal do Paraná, Curitiba, Paraná CeP 80035-050, Brazil

C. l. O. Petkowicz Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Setor de Ciências Biológicas, Curitiba, Paraná CeP 81531-980, Brazil

G. T. nagashima Área de ecofisiologia, Instituto agronômico do Paraná, londrina, Paraná CeP 86047-902, Brazil

l. G. e. Vieira Universidade do Oeste Paulista, Presidente Prudente, São Paulo CeP 19067-175, Brazil

l. F. P. Pereira embrapa Café, Brasília, Distrito Federal CeP 70770-901, Brazil

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eF1 elongation factor 1eF1α elongation factor 1-alphaUBQ10 Polyubiquitin 10Ca Coffea arabicaCc Coffea canephora

Introduction

allopolyploidy is evolutionary and mechanistically com-pelling. The process involves the reconciliation of two or more sets of diverged genomes and regulatory interactions. recent findings show that duplicate gene pairs may dis-play homeolog expression bias, where the two homeologs are expressed unequally, often varying among tissues (Yoo et al. 2013). The contributions of homeologous transcripts to the pattern of total expression level have not been fully explored. Studies involving this mechanism have been reported in few species such as cotton (Dong and adams 2011), Spartina (Chelaifa et al. 2010), coffee plants (lash-ermes et al. 2014) and wheat (Chagué et al. 2010), in sev-eral tissues and experimental conditions (liu and adams 2007; Stamati et al. 2009; Dong and adams, 2011).

environmental factors influence nearly every aspect of a plant’s function throughout its life cycle. In fact, a Food and agricultural Organization (FaO) report stated that only 3.5 % of global land area is not affected by some envi-ronmental constraint (Cramer et al. 2011). In response to changing and often unfavorable conditions, a complex and overlapping network of molecular machinery must regu-late plant responses to these conditions. Stress perception in plants initiates signal transduction events that lead to expression of specific genes and generation of “protecting” metabolites (Barkla and Pantoja 2011).

Plants can overcome abiotic stresses through intracel-lular accumulation of organic compatible solutes that act as osmoprotectants, such as quaternary ammonium com-pounds, proline, oligosaccharides and polyols (rathina-sabapathi 2000). Polyols are the reduced forms of aldoses and ketoses that can be found in all living organisms (noiraud et al. 2001). Mannitol is the most common polyol in nature and has been observed in more than 100 vascular plant species of several families including the rubiaceae (rogers et al. 1999). In plants, mannitol is synthesized through fructose-6-phosphate isomerization by the action of the phosphomannose-isomerase enzyme (PMI), form-ing mannose-6-phosphate, which is converted to mannitol-1-phosphate by the activity of the key enzyme naDPH-dependent mannose-6-phosphate reductase (M6Pr). Then, the mannitol-1-phosphate is further converted to mannitol by the activity of a non-specific phosphatase (Song et al. 2010). This sugar alcohol can be translocated through the phloem to heterotrophic sink tissues, where it may be either

stored or oxidized to mannose by naD+-dependent manni-tol dehydrogenase (MTD) and used as a carbon and energy source (Conde et al. 2011). Plant cells can convert mannose to mannose-6-phosphate by a non-specific hexokinase, which in turn may cause inhibition of phosphoglucose isomerase, causing a block in glycolysis and depletion of inorganic phosphate that is required for aTP production (Goldsworthy and Street 1965; Herold and lewis 1977). However, in some higher plants, endogenous PMI and/or M6Pr can catalyze the conversion of mannose-6-phos-phate to fructose-6-phosphate and mannitol-1-phosphate, respectively (Privalle 2002; Gao and loescher 2003).

The role of mannitol as osmoprotectant, reactive oxygen species scavenger, protein and membrane structure stabilizer and photosynthetic apparatus protector under abiotic stress conditions is well documented in several species (Shen et al. 1997; Chan et al. 2011) including celery, where mannitol represents approximately 50 % of total translocated carbo-hydrates (everard et al. 1997; Song et al. 2010). The balance of these roles is due to a precise coordination of mannitol synthesis and catabolism (Stoop et al. 1996; Seckin et al. 2009; Cheng et al. 2009). Mannitol concentration increases when plants are exposed to low water potential and accu-mulation is regulated by inhibition of competing pathways and decreased mannitol consumption and catabolism (Stoop et al. 1996). The production of mannitol may also con-fer higher carbon utilization efficiency (Stoop et al. 1996), increased oxidative stress tolerance (Williamson et al. 1995; Keunen et al. 2013), and salt stress tolerance (Zhifang and loescher 2003; Sickler et al. 2007). also, since the osmopro-tectors synthesis usually occurs in mature leaves, an increase of sugar rate in sink tissues is certainly associated with an increase of these molecules transport. On the other hand, this sugar alcohol has also been reported to have effects under biotic stress conditions, facilitating infection and survival of pathogens (Delavault et al. 2002; Vélëz et al. 2008).

Coffee is one of the most valuable international agricul-tural commodities, and its cultivation, processing, transpor-tation and marketing provide employment for millions of people worldwide. Coffee production is subject to regular oscillations that can be explained by the natural biennial cycle, but also by the adverse effects of climatic conditions. among them, drought, salt stress and high temperatures are key factors affecting coffee plant development and produc-tion (DaMatta and ramalho 2006; Dos Santos et al. 2011). In addition, as a result of global climate change, periods of drought may become more pronounced, and the sustain-ability of total production, productivity and coffee quality may become more difficult to maintain (assad et al. 2004).

Coffea arabica is an allotetraploid (2n = 4x = 44) derived from a recent interspecific hybridization (approxi-mately one million years ago) between two diploid spe-cies: Coffea eugenioides and Coffea canephora (Vidal et al.

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2010; Yu et al. 2011). Homeolog expression can change in response to abiotic stresses and no single unifying factor has been reported, suggesting that each allopolyploid plant species has its own regulatory mechanism (Chaudhary et al. 2009; Zhu et al. 2011). In this way, the extent of homeolo-gous gene expression changes in response to abiotic stresses remains poorly studied. In C. arabica, previous studies con-firmed the existence of homeologous genes with differential expression (Vidal et al. 2010; Marraccini et al. 2011), but this characteristic is not a general feature of all C. arabica genes (Vidal et al. 2010; Combes et al. 2012).

Breeders usually attribute abiotic and biotic stress toler-ance to the “expression” of C. canephora traits contained in C. arabica. as previously observed for rBCS1 in C. arabica, the transcriptional contribution of each homeolog may be affected by abiotic stress (Marraccini et al. 2011). Therefore, it is important to add new information on the expression levels of homeologs from the two subgenomes [C. eugenioides (CaCe) and C. canephora (CaCc)] in dif-ferent abiotic stress conditions (Bardil et al. 2011). Con-sidering that mannitol is a primary photosynthetic product (Chan et al. 2011) and that drought, salinity and high tem-peratures damage the photosynthetic apparatus, the eluci-dation of the role of mannitol in protecting against abiotic stress might be important for yield enhancement of coffee plants under sub-optimal environmental conditions.

Here, we investigate the role of mannitol in the leaves of C. arabica plants submitted to drought, salinity and high temperature by measuring fructose and mannitol lev-els, analyzing the transcriptional pattern of the key genes involved in mannitol metabolism (CaM6PR, CaPMI and CaMTD), and assessing the contribution of C. canephora homeolog transcripts under each abiotic stress.

Materials and methods

Plant material and abiotic stress treatments

Coffea arabica cv. IaPar 59 plants with standardized growth were used for all experiments. The basic procedures for abiotic stress treatments followed Dos Santos et al. (2011), Carvalho et al. (2013a) and lima et al. (2013). Bio-logical replicates were represented by three pools of leaves from the same pair (second plagiotropic pair) and each pool consisted in two pairs of leaves from three different plants. The leaves were immediately immersed in liquid n2 and stored at −80 °C until the assays were performed.

For the drought experiment, leaves from 24-month-old plants with uniform growth were used in all analyses. Plants were grown in a greenhouse at 25 °C on a 12-/12-h day/night cycle. To monitor plant water status, leaf discs of approxi-mately 2 cm2 were collected and placed in thermocouple

psychrometer chambers (model C-30, Inc., Wescor) assem-bled with a datalogger (model Cr-7, Campbell Scientific, Inc.) for determination of leaf water potential (Ψw). leaves were sampled based on water potential. The treatments were established as: irrigated (Ir; Ψw = −1.34 MPa), moderate stress (MS; Ψw = −2.4), severe stress (Se; Ψw = −4.5 MPa) and recovery (reC, 72 h after re-irrigation). Moderate and severe stresses were achieved approximately 12 and 23 days after irrigation suppression, respectively.

The salt stress experiment was carried out using leaves of 18-month-old coffee plants grown in the same condi-tions as in the drought stress experiment. To avoid osmotic shock, plants were irrigated with 50 mM naCl in the first day of the experiment. From the third day up to the end of the experiment, the plants were daily irrigated with 150 mM naCl. leaves were harvested as following: day 0 (control without addition of naCl), day 4, 6 and 12 after adding 150 mM naCl.

In the heat-stress experiment, we used leaves of 18-month-old coffee plants that were maintained in a growth chamber for 7 days at 24 °C, controlled photoperiod (12:12 h) and under a photosynthetic photon flux density of approximately 400 mmol m−2 s−1 for acclimatization. In sequence, the chamber temperature was raised to 37 °C for 5 days. To avoid water deficit, plants were irrigated daily to pot-capacity during all the heat-stress period. Samples were collected at four evaluation stages: non-stressed control (plants maintained 7 days in growth chamber at 24 °C), day 1, 3 and 5 (at 37 °C).

Quantification of fructose, mannose and mannitol

leaves collected from coffee plants under stress treat-ments were lyophilized and submitted to an extraction process for obtaining low molecular weight oligosaccha-rides according to albini et al. (1994). Fructose, mannose and mannitol were determined by high performance liquid chromatography (HPlC) using a Shimadzu system (Japan) equipped with CBM-10a interface module, CTO-10a col-umn oven, lC-10aD pump and with a rID-10a refrac-tive index detector. a Supelcogel Ca column (Supelco_ USa), 30 cm × 7.8 mm and Supelcogel Ca pre-column, 5 cm × 4.6 mm were used. The HPlC-column was eluted with water at a flow rate of 0.5 ml min−1 at 80 °C. Differ-ences in carbohydrates concentration in each abiotic stress were analyzed by one-way analysis of variance (anOVa), taking P < 0.05 as significant according to Student t test.

Identification of candidate genes involved in mannitol biosynthesis

To identify the M6Pr-encoding gene, the annotated Bra-zilian Coffee Genome Project database (http://www.lge.ib

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i.unicamp.br/coffea/; Mondego et al. 2011) was searched using the keyword ‘naDPH-dependent mannose 6 phos-phate reductase’. all eSTs obtained for M6PR, designated here as CaM6PR, were assembled using ClC (Main Work-bench 4.1.1) software. each contig was compared with individual protein sequences deposited in the nCBI data-base (national Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and the sequences were indi-vidually analyzed to verify similarity and identity. analyses of a C. arabica BaC library (Cação et al. 2013) identified CaM6PR as a single-copy gene.

To obtain the PMI gene, initial search was carried out using the sequence from the Arabidopsis thaliana (at1g67070; Maruta et al. 2008) ortholog in The arabidop-sis Information resource (TaIr) databases (http://www.arabidopsis.org/). In the case of MTD (CaMTD), we used the sequence of the Apium graveolens ortholog (GenBank accession aF067082.1), previously mentioned by Mustorp et al. (2008). The protein sequences of PMI and MTD were used as queries in a TBlaSTn against Coffea spp. eSTs from dbeST (nCBI) database. These sequences were also used as queries in the Brazilian Coffee Genome Project database (Mondego et al. 2011). In all cases, genes were represented by only one assembled eST contig in the data-base, mostly expressed in leaves (Online resource 1).

Coffea canephora homeolog identification

To study the individual contribution of C. canephora home-ologs to the overall expression level of each gene in C. ara-bica, we used the same approach of Vidal et al. (2010) and Marraccini et al. (2011), which is based on the TaqMaMa assay (li et al. 2004). This method relies on differential amplification based on Single nucleotide Polymorphisms detected between C. arabica and C. canephora derived eSTs. To this purpose, the sequences for M6PR, PMI and MTD obtained from the Brazilian Coffee Genome Project database were used to obtain C. arabica and C. canephora contigs. These contigs were then submitted to Blast2 Sequences tool (nCBI), with default parameters, to detect SnPs.

Phylogenetic analysis

For phylogenetic analyses, sequences for M6PR, PMI and MTD were obtained from complete plant genomes in Phy-tozome database (http://www.phytozome.net/) and used as queries for a TBlaSTn search. For PMI and MTD phy-logenetic analysis, we also used sequences from Maruta et al. (2008), Barakat et al. (2009) and Conde et al. (2011), respectively.

all protein sequences selected were aligned using MUSCle, and a phylogenetic tree was constructed with

the aligned protein sequences by the maximum likeli-hood method. To assess the confidence limits in phyloge-netic analyses, bootstrapping was carried out (500 trials) to assess the support for individual nodes. all analyses were developed in MeGa version 5 (Tamura et al. 2011). In the case of MTD, subclasses were classified according to Barakat et al. (2009).

rna isolation, purification and cDna synthesis

Total leaf rna was isolated from leaves by a modified pro-cedure of Chang et al. (1993). rna samples were purified using the Pure link Micro to Midi Total rna Purification System (Invitrogen), their integrity examined by 1 % agarose gel electrophoresis and then treated with Dnase (rnase-free, Invitrogen). rna concentration and purity were determined using a nanoDrop® nD-100 spectrophotometer (Thermo Scientific) and the absence of genomic Dna contamination was confirmed by conventional PCr using GAPDH primers (data not shown). Complementary Dna (cDna) was syn-thesized by a SuperScript III reverse Transcriptase (Invitro-gen) following the manufacturer’s instructions using 5 µg of total rna in a final volume of 20 µl. The final cDna prod-ucts were diluted tenfold prior to use in qPCr.

Primer design and amplification efficiency

Primers were designed using Primer express v. 3.0 (applied Biosystems) to obtain amplicons of approxi-mately 100 base pairs with Tm of 60 ± 2 °C. amplicons were sequenced and analyzed using BlaST to verify spec-ificity. Primers for total gene expression were named here as CaM6PR, CaPMI, CaMTD and primers specific for C. canephora subgenome were named as CaM6PRCc, CaP-MICc and CaMTDCc (Online resource 2). The three allelic-specific primer pairs were designed to amplify sequences in the same gene region of the primers used to assess overall gene expression.

The efficiency of each primer pair was calculated using a standard curve generated from a serial dilution of cDna using E = [10(−1/slope) − 1]. The cDna used to this analysis was from leaves of non-treated plants. Four serial dilutions were set up to determine quantification cycle (Cq) and reac-tion efficiencies for all primers pairs. Standard curves were generated for each primer pair using the Cq value versus the logarithm of each cDna dilution factor. For qPCr data normalization, the reference genes were selected according to Carvalho et al. (2013a).

qPCr and data analysis

The transcript level for each gene was detected by qPCr (7500 Fast real-Time PCr System, applied Biosystems)

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using SYBr Green PCr Master Mix (applied Bio-systems). The reaction mixture contained 12.5 µl of 2 × SYBr Green master mix, 1 µl of each primer (10 µM), 1 µl of cDna 1:10 diluted and Milli-Q water to a total vol-ume of 25 µl. Thermal conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 60 s. Melting curves were analyzed to verify the presence of a single product including a negative control. all reactions were performed in triplicate for each of the three biological replicates in all treatments studied. Dissociation analyses and fragment sequencing confirmed their identity with the target genes.

relative expression levels of the genes were expressed by applying the formula (1 + E)−ΔΔCt, where ΔCt tar-

get = Ct target gene–Ct geometric average of reference genes and ΔΔCt = ΔCt target–ΔCt internal calibrator, similarly to Mar-raccini et al. (2012). The internal calibrator always was the onset of each stress treatment, with relative quantification equal to 1. normalization used the geometric average of two reference genes specific to each abiotic stress as rec-ommended by Carvalho et al. (2013a). CaGAPDH and CaUBQ10 were used to normalize relative expression in the drought stress experiment (Cruz et al. 2009), while CaEF1 and CaUBQ10 were used for salt stress and CaGAPDH and CaEF1α for heat-stress experiments (Carvalho et al. 2013a). Significance was determined using anOVa and Tukey’s post hoc test. an asterisk indicates a significant difference relative to day 0 in each stress (P < 0.05).

We also evaluated C. canephora homologs contribu-tions through the ratio of Ca/CaCc, where Ca represents total gene expression and CaCc represents the specific amplification of C. canephora homeologous genes within the C. arabica genome. The basic procedures for the calculation of Ca/CaCc ratio followed Marraccini et al. (2011). Ca/CaCc ratio corresponded to (1 + E)−ΔCt, where ΔCt = CtmeanCa−Ctmean CaCc, with e as the efficiency of gene amplification. The Ct data were normalized with the reference genes appropriated for each specific abiotic stress condition.

Results

Carbohydrate quantification

Mannitol and fructose levels in the coffee leaves varied depending on the nature and intensity of stress (Fig. 1). On the other hand, mannose was not detected in any of the cof-fee leaf samples investigated in this study (data not shown).

Mannitol and fructose concentrations in coffee leaves were altered during water deficit (Fig. 1a). The mannitol content increased to similar levels during moderate and severe stresses when compared to normal water supply

conditions. after re-irrigation, mannitol concentrations declined to significantly lower levels than those observed on day 0. The fructose content observed in the leaves of drought-stressed plants was lower as compared to manni-tol, showing a slight but significant increase during moder-ate stress and a later reduction at severe drought and recov-ery conditions (Fig. 1a).

Similarly, there was a considerable increase in manni-tol content in the leaves of coffee plants submitted to salt stress (Fig. 1b). On the other hand, fructose levels showed a significant decline until day 6, when there was a small increase at the end of the stress period (day 12). With the exception of day 0, the mannitol content in the leaf tissue was higher compared with fructose.

In contrast to drought and salinity conditions, mannitol and fructose concentrations were substantially reduced in both heat-stress sampling periods. no significant differ-ences were observed for the content of either sugar between day 3 and 5 (Fig. 1c).

Phylogenetic analysis

The deduced amino acid sequences of C. arabica M6PR, PMI and MTD were aligned with sequences from several plant genomes and maximum likelihood trees were drawn for each gene. In the M6PR and PMI trees, a separation between monocot and eudicot sequences was observed (Fig. 2a, b).

The classification obtained with the MTD tree showed that CaMTD was grouped into class II (Fig. 2c), a sub-class previously reported by Barakat et al. (2009) that represents genes that are related to mannitol degrada-tion. These authors identified a C. canephora sequence in class I (Fig. 2c), but genes grouped into class I are related to cinnamyl alcohol dehydrogenases involved in lignin biosynthesis.

Transcriptional analysis of CaM6PR, CaPMI and CaMTD

each primer pair, described in Online resource 2, had its specificity confirmed by the identification of a single frag-ment with the predicted molecular size on an agarose gel and by the observation of a single peak in the melting curve analyses of qPCr. Primers for amplification of the C. canephora subgenome were tested using C. canephora and C. eugenioides Dna and cDna to confirm the allele-specific amplification (Fig. 3 and Online resource 2). The amplification efficiencies for all primer pairs ranged from 89 to 108 % (Online resource 2).

The relative expression patterns of CaM6PR, CaPMI and CaMTD were assayed by qPCr, revealing differen-tial regulation upon drought stress (Fig. 4a). CaM6PR and CaPMI presented an equivalent transcriptional

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pattern, characterized by mrna up-regulation under moderate and severe stress. This modulation was more pronounced for CaM6PR, which was approxi-mately 20-fold higher than CaPMI during these periods (Fig. 4a). CaMTD was down-regulated in response to water deficit, with the leaves maintaining similar tran-script levels during moderate and severe stress and pre-senting a small increase at the end of the experiment. Interestingly, the mrna abundance of CaM6PR and

CaPMI was lower after re-irrigation than in the non-stressed condition (Fig. 4a).

Under salt stress, the fold increase in CaM6PR mrna level was much less than that observed for drought stress; CaM6PR became strongly up-regulated only after 12 days of salt stress imposition. CaPMI presented the highest expression level at day 6 with a great reduction at the end of the experiment. CaMTD showed an increase in mrna abundance on the first days of the stress, but its expression was markedly down-regulated by progressively severe stress established over 12 days of salinity treatment (Fig. 4b).

The expression profiles of CaM6PR, CaPMI and CaMTD were distinct during heat stress. CaM6PR was conspicuously down-regulated from day 1 of stress imposi-tion. On the other hand, CaPMI was first up-regulated, after which the expression returned to the non-stressed level. a steep increase in the abundance of CaMTD mrna was observed at the beginning of the heat-stress treatment, and similarly to the other genes, was down-regulated with more severe stress (Fig. 4c).

Coffea canephora-specific transcriptional pattern

qPCr analysis specific to C. canephora subgenome alleles was performed for the three abiotic stresses (Fig. 4d–f). The ratio between Ca/CaCc was also evaluated. low val-ues indicate high contribution of C. canephora homeolog (Table 1).

although different expression values were observed, CaM6PRCc presented the same pattern during drought stress when compared to the overall expression of CaM6PR, i.e., a noticeable increase in transcriptional lev-els during moderate and severe stress and a later decline after the plants were re-irrigated (Fig. 4d), while CaPMICc and CaMTDCc expression also increased upon water stress, albeit on a smaller scale (Fig. 4d). The Ca/CaCc expression of CaM6PR and CaMTD illustrates the high contribution of C. canephora homeologs in response to drought (Table 1).

allele-specific transcription of CaM6PRCc in plants sub-mitted to salinity showed a great increase in the last day of the experiment (day 12), while the highest expression of CaPMICc and CaMTDCc occurred at day 6 (Fig. 4e). These results are consistent with the Ca/CaCc ratio presented in Table 1, which shows a higher contribution from C. canephora homeologs at those stress levels.

When the coffee plants were submitted to high tempera-ture, as compared to drought and salt stress, homeolog tran-scriptional analysis displayed reduced CaM6PRCc mrna abundance (Fig. 4f). an important observation is that for the three abiotic stresses CaPMICc shows the lowest Ca/CaCc ratio, which shows that the C. canephora allele was mainly responsible for gene expression under this severe stress condition (Table 1).

Fig. 1 Mannitol and fructose concentrations in leaves of C. ara-bica cv. IaPar-59 submitted to a drought stress—non-stressed (day 0), moderate stress (Me), severe stress (Se) and recovery (reC); b salt stress s—non-stressed (day 0) and 4, 6 and 12 days after the beginning of salt stress; c heat stress—non-stressed (day 0), moder-ate stress (day 3) and severe stress (day 5). Values are mean ± SD (n = 3). Different letters represent significant differences between means at P < 0.05, determined by Student’s t test

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Fig. 2 Maximum likelihood trees based on protein sequences of M6PR (a), PMI (b) and MTD (c). Bootstrap values over 50 % are indicated for each branch divergence. GenBank accession numbers

are indicated, except for Arabidopsis thaliana, where genomic loci are listed. Sequences marked with (asterisk) were retrieved from phy-tozome

Fig. 3 amplification products from Coffea species using primers designed for total gene expression and for C. canephora subgenome expression using Dna and cDna of non-stressed plants. Primers were speci-fied in Online resource 2. M molecular weight standard, Ca C. arabica, Cc C. canephora, Ce C. eugenioides

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Discussion

There is great interest in dissecting the processes that are involved in the sugar sensing and response pathways that allow plants to acclimate to the constantly changing envi-ronment, but the dual function of sugars as nutrients and

signaling molecules significantly complicates the analyses of the mechanisms involved (rolland et al. 2001).

Mannitol, an osmoprotectant soluble sugar alco-hol, is synthesized during stressful conditions and is by far the most abundant polyol in plants (loesher et al. 1987). Previous studies have shown that an increased

Fig. 4 Total C. arabica (Ca) genome and C. canephora (Cc) sub-genome expression profiles for mannose-6-phosphate reductase (M6Pr), phosphomannose-isomerase (PMI) and mannitol dehydro-genase (MTD) during drought (a, d), salt (b, e) and heat (c, f) stress

imposition examined by qPCr. non-stressed (day 0), moderate stress (Me), severe stress (Se) and recovery (reC) (a). The value 1 was attributed to the lowest expression

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accumulation of mannitol following drought and salin-ity conditions improved plant tolerance to stresses (Williamson et al. 2002; abebe et al. 2003; Sickler et al. 2007), but there is no physiological evidence of the impact of this accumulation on tolerance to these stresses in coffee plants. Thus, mannitol accumula-tion during stress imposition would suggest a relation between this sugar and stress tolerance. Studies involv-ing the synthesis and accumulation of mannitol in coffee have only been reported during fruit development in C. canephora and C. arabica (rogers et al. 1999), which revealed maximum concentration peaks of 0.18 µg g−1 for C. canephora and 0.70 µg g−1 for C. arabica, both in the early stages of fruit development.

In this study, the high mannitol levels observed in C. arabica leaves are most likely due to the higher photosyn-thetic activity in this tissue as compared to immature green fruits, which may also explain the non-detection of man-nitol in ripe coffee fruits (rogers et al. 1999). Our data also showed that after plants were recovered from drought stress, mannitol content was drastically reduced in the cof-fee leaves (Fig. 1a). The fact that mannitol production is an alternative pathway for the carbohydrates produced by pho-tosynthesis may explain this decline, as the energy used in mannitol biosynthesis can be diverted to various other met-abolic processes involved in recovery from environmental stresses, such as synthesis of proteins like ribulose-l,5-bisphosphate carboxylase/oxygenase (rubisco), the rate-limiting enzyme in photosynthesis, which is inactivated by abiotic stress conditions (Sakamoto and Murata 2002).

The mannitol content in coffee leaves during stress conditions was at a higher level than fructose, most likely because fructose, in addition to being a precursor to man-nitol, is used heavily in other metabolic processes. The high mannitol concentrations observed in this study con-trast with the results observed by Karacas et al. (1997) using wild-type Nicotiana tabacum plants, where mannitol presented the lowest accumulation among other osmolytes after the application of naCl treatments. However, other studies have shown that mannitol provides protection by acting as compatible solute, by protecting the hydration shell around membranes and proteins and by scavenging reactive oxygen species even at very low levels of accumu-lation (Galinski 1993; Shen et al. 1997).

It is also worth mentioning that, as a general pattern, the mannitol content of the coffee leaves increased upon drought and salt stress, while fructose presented a clear decline after stress imposition. In contrast, there was a decrease in the concentration of both sugars in response to the heat-stress treatment. Several factors may have contrib-uted to the observed differences in carbohydrate content after the stresses. according to Goldschmidt and Huber (1992), when photosynthesis is reduced due to stressful conditions, the accumulation of carbohydrates is highly variable and can be regulated by the levels of photosyn-thetic metabolites, not necessarily by the action the final products themselves (Geiger 1987; Bagnall et al. 1988; Foyer 1988). In addition, the differences observed in man-nitol and fructose content in the coffee leaves at day 0 in the different experiments can be explained by the differ-ences in plant developmental stages. Plants subjected to the water deficit experiment presented higher sugar levels as compared to the younger plants used in the other two stress treatments. another important point is that coffee plants used for the heat-stress experiment were exposed to lower radiance when compared to the greenhouse condi-tions, where the drought and salinity experiments were car-ried out. Further studies on the patterns of hexose synthesis in plants of different developmental stages during abiotic stresses of different magnitudes would help to elucidate these variables.

altogether, our results show that mannitol accumulates highly in C. arabica leaves in response to water deficit and salinity conditions, but not to heat stress. The compara-tively lower accumulation of mannitol in the coffee leaves during heat stress, even for only a short period, could be expected because mannitol is a direct product of photosyn-thesis and rapid inhibition of photosynthesis occurs at high temperatures due to the reduced rates of ruBP regeneration caused by disruption of electron transport activity (Salvucci and Crafts-Brandner 2004).

Bolouri-Moghaddam et al. (2010) hypothesized that the synergistic interaction of sugars (or sugar-like compounds)

Table 1 relative contributions of C. canephora subgenome M6PR, PMI and MTD genes under various abiotic conditions measured by the ratio of Ca/CaCc

Treatments Ca/CaCc

M6PR PMI MTD

Drought stress

Day 0 37.08 1.42 1,771.69

Me 16.42 1.64 28.96

Se 14.65 1.72 42.09

reC 28.57 1.87 265.88

Salt stress

Day 0 120.10 2.46 838.59

Day 4 199.51 2.28 1,499.27

Day 6 53.17 2.13 38.85

Day 12 36.83 2.51 333.66

Heat stress

Day 0 130.99 1.79 22.32

Day 1 41.53 4.08 361.50

Day 3 45.60 0.84 33.77

Day 5 18.02 1.51 5.83

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and phenolic compounds forms part of an integrated redox system, quenching rOS and contributing to stress toler-ance, especially in tissues or organelles with high soluble sugar concentrations. It has also been proposed that the protective effects of soluble sugars against oxidative stress may be attributed to indirect signaling effects, triggering the production of specific rOS scavengers (Couée et al. 2006; ramel et al. 2009). In addition, lowering naDH and/or naDPH appears to have beneficial effects on mitochon-drial respiration, energy charge, and possibly on reducing rOS (Bartels 2001). Because mannitol synthesis involves an aldo–keto reductase and the use of a reductant, such a process may, at least in part, contribute to enhanced abiotic stress tolerance (Zhifang and loescher 2003).

The presence of mannose was not detected in any of the coffee leaf samples. Some studies showed that this sugar can be toxic to plant cells (Song et al. 2010), primarily due to the sequestration of inorganic phosphate (Herold and lewis 1977; Stein and Hansen 1999). Mannose is an unus-able carbon source for several plant species; this sugar is phosphorylated by a hexokinase to mannose-6-phosphate, which blocks glycolysis by the inhibition of phosphoglu-coisomerase, resulting in a severe inhibition of growth (Degenhardt et al. 2006). One way to avoid the accumula-tion of this sugar is through the isomerization of mannose-6-phosphate into fructose-6-phosphate by the action of phosphomannose-isomerase (PMI). Fructose-6-phosphate is then used in glycolysis (Maruta et al. 2008). Conse-quently, high levels of PMI and/or M6Pr are present in mannose tolerant species (Song et al. 2010). In addition, mannose can repress the transcription of genes required in photosynthesis and the glyoxylate cycle (Graham et al. 1997). Thus, plants can overcome this issue through the conversion of mannose into mannitol or fructose.

The phylogenetic analysis performed on the three genes revealed that only the CaM6PR and CaPMI sequences were grouped into two distinct clades, reflecting the divergence between monocots and dicots (Fig. 2a–c), which suggests that the three genes suffered distinct impacts of selection, with CaMTD being the least conserved. CaMTD was clas-sified, according to Barakat et al. (2009), in a group that was previously related to genes that may function under stress conditions.

Previous studies on the role of mannitol in abiotic stress tolerance were focused mainly on salt stress toler-ance (Zhifang and loescher 2003). Here, we showed that CaM6PR, CaPMI and CaMTD were regulated in distinct ways depending on the abiotic stress, specifically drought, salinity and high temperature. CaM6PR was drastically up-regulated by drought stress and high levels of mannitol were detected in leaf tissues. although these studies have provided evidence that mannitol acts primarily as a com-patible solute in plants (Chan et al. 2011), no increased

protection against water deficit was observed in arabidop-sis M6PR transformants (Sickler et al. 2007), indicating that the plant cells did not lower the osmotic potential suf-ficiently for protection against dehydration. However, this same work reported that the transgenic plants presented increased tolerance to salinity and suggested that mannitol synthesis may be limited by low PMI activity. In our case, CaM6PR transcript abundance and mannitol content during salt stress were lower than in leaves of plants under water deprivation, indicating that coffee plants have different responses to drought and salinity.

Interestingly, CaPMI followed the same modulation as CaM6PR, confirming the tight regulation of these two genes during drought stress. On the other hand, mannitol was present at lower levels as compared to fructose when coffee plants were submitted to high temperature, with the exception of day 1. also, at day 1 of the heat stress CaMTD presented high transcript levels, which is consistent with the reduction in mannitol content (Figs. 1c, 4c). However, it seems plausible that the mannitol biosynthetic pathway is subject to other regulatory controls besides gene expres-sion, such as changes in enzyme activity in the absence of transcriptional changes. Considering the complex regula-tion involved in gene expression, it is plausible to observe some differences between gene transcriptional pattern and metabolite accumulation, as it was previously observed in maize (Zhao et al. 2004), pondweed (Harada et al. 2005), coffee plants (dos Santos et al. 2011) and citrus plants (Campos et al. 2011; Carvalho et al. 2013b). although pre-vious studies have provided evidence that mannitol acts primarily as a compatible solute in plants, the production of this sugar alcohol can have far-ranging effects on other metabolic pathways involved in the response to abiotic stresses (Chan et al. 2011).

The up-regulation of M6PR by water deficit in C. canephora and C. arabica genotypes was recently reported (Marraccini et al. 2012; Freire et al. 2013), however, the relationship between gene expression and mannitol con-centration in coffee during abiotic stresses has not yet been investigated. In other plant species, the transcriptional pro-file of this gene was analyzed by northern blot analysis (Delavault et al. 2002; Zhifang and loescher 2003), but the effects of abiotic stresses or the specific subgenome con-tribution to gene expression were not considered in these studies. In addition to total gene expression of M6PR, PMI and MTD, the present study also evaluated the contribu-tion of the C. canephora homeologs based on the fact that, in an allotetraploid, two divergent genomes merged in a common nucleus can contribute either equally or dispro-portionately to the transcriptome (Chaudhary et al. 2009; Combes et al. 2012). In addition, the divergence between the subgenomes indicates that a mechanism may be pre-sent to prevent C. arabica genome homogenization by

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avoiding recombination between CaCc and CaCe subge-nomes (Vidal et al. 2010). The differential expression of homeologous genes has been reported and increasingly studied in allopolyploid species such as wheat (Mochida et al. 2003), cotton (Udall et al. 2006; Hovav et al. 2008; Zhu et al. 2011) and coffee (Vidal et al. 2010; Marraccini et al. 2011; Combes et al. 2012). The results of our study showed that the relative contribution of homeologs to the transcriptome varied according to the abiotic stress applied (Fig. 4d–f; Table 1). In salt and heat stress, homeologs of C. canephora expression levels were similar to those observed for total gene expression. This was also observed by Bardil et al. (2011) and Marraccini et al. (2011) in stud-ies with coffee plants submitted to high temperatures and water deficit, respectively.

Vidal et al. (2010) proposed that the C. canephora sub-genome within the C. arabica transcriptome is more asso-ciated with expressing genes coding for regulatory pro-teins, while C. eugenioides subgenome expression appears to be more closely linked with basal processes. a differ-ent result was reported by Petitot et al. (2008) for genes involved in coffee defense responses to biotic stress. These authors reported that CaWRKY1a and CaWRKY1b, genes that code for transcription factors, were concomitantly expressed, and both homeologous genes contributed to the overall transcriptional level. Therefore, our allele-specific transcriptional expression of CaM6PRCc, CaPMICc and CaMTDCc showed a distinct participation of subgenomes in transcriptional pattern of C. arabica (Table 1). Moreover, through the ratio results observed in Table 1, PMI presented the higher ratio values among the three genes and for all abiotic stresses, indicating a higher contribution of the C. canephora homeolog. These results are consistent with the fact that, in general, as reported in the studies mentioned above, the C. canephora subgenome is frequently reported to be more involved in the expression of genes related to abiotic stresses.

Unlike in non-mannitol producing species, mannitol seems to have important functions in the response of cof-fee plants to water deficit. even considering that it is still not clear whether stress-induced accumulation of man-nitol directly increases tolerance to adverse conditions, or just participates in broader and complex mechanisms by acting synergistically with other strategies employed for defense against environmental stressors, our study on man-nitol accumulation and transcriptional evaluation of genes involved in the metabolism of this polyol under three dif-ferent abiotic stresses provides new information about mannitol regulation.

In this way, homeologs of the C. arabica subgenomes do not contributed equally to mannitol metabolism dur-ing drought, salt and heat stresses and since many impor-tant crops are also polyploid (i.e., wheat, canola, potato,

sugarcane and cotton), this study may help to design bio-technological strategies for improving crops tolerance to various adverse growth conditions once the expression changes shown here are involved in stress responses.

Acknowledgments We thank Silvia Graciele Hülse de Souza for plant material, nathalia Volpi e Silva for initial annotation, Viviane Yumi Baba for help in statistical analyses and CaPeS for the schol-arship. This work was supported by grants from FIneP, Fundação araucária and Consórcio Pesquisa Café. luiz Gonzaga esteves Vieira and luiz Filipe Protasio Pereira are CnPq research fellows. Douglas Silva Domingues is Fundação araucária research fellow.

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