Identifying water stress-response mechanisms in citrus by in silico transcriptome analysis

STRESS RESPONSES

RESEARCH ARTICLE

Identifying water stress-response mechanisms in citrus by in silico transcriptome analysis

Gisele A.M. Torres; Marcos A. Gimenes; Vicente E. de Rosa Jr.; Vera Quecini

Centro de Pesquisa e Desenvolvimento de Recursos Genéticos Vegetais, Instituto Agronômico de Campinas, Campinas, SP, Brazil

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ABSTRACT

Water deficit is one of the most critical environmental stresses to which plants are submitted during their life cycle. The evolutionary and economic performance of the plant is affected directly by reducing its survival in the natural environment and its productivity in agriculture. Plants respond to water stress with biochemical and physiological modifications that may be involved in tolerance or adaptation mechanisms. A great number of genes have been identified as transcriptionally regulated for water deficit. EST sequencing projects provide a significant contribution to the discovery of expressed genes. The identification and determination of gene expression patterns is important not only to understand the molecular bases of plant responses but also to improve water stress tolerance. In our citrus transcriptome survey we have attempted to identify homologs to genes known to be induced and regulated under water stress conditions. We have identified 89 transcripts whose deduced amino acid sequences share similarities with proteins involved in uptake and transport of water and ion, 34 similar to components of the osmolyte metabolism, 67 involved in processes of membranes and proteins protection and 115 homologs of reactive oxygen species scavenger. Many drought-inducible genes identified are known to be regulated by development, salt, osmotic and low temperature. Their possible roles in specific or general mechanisms of water stress citrus responses are discussed.

Key words: environmental stress, CitEST, data mining, tolerance mechanisms, water deficit.

Introduction

Water stress is one of the most critical environmental stresses that plants are exposed to. It affects both the evolutionary and the economic performance of a plant by directly reducing its survival in the natural environment and its productivity in agriculture. The International Water Management Institute estimates that by the year 2025, one third of the world population will inhabit regions of severe water scarcity. Moreover, the use of fresh water for irrigation competes with its use for human consumption, and the increased yields obtained with the employment of irrigation water have been predicted to be unsustainable (IWMI, 2005). The disruption of the plant water status and its detrimental effects on the plant performance are common features of several abiotic stresses, thus, likely to be present in the vast majority of agricultural zones.

In perennial species, seasonal variation in environmental conditions may influence water relationships. In Citrus trees, the large canopy and low hydraulic conductivities of the stem and the root contribute to high water deficit (Moreshet et al., 1990). Moreover, at midday, a transient water deficit is a characteristic of citrus (Cohen et al., 1997) and correlated to reduced photosynthesis rates (Brakke and Allen, 1995). Although temporary water deficit periods occur in many of cultivated regions, irrigation is rarely employed for citrus species in Brazil (Ortolani et al., 1991).

The environmental amounts of water available to plants may decrease due to drought, altered ion content and water uptake caused by salinity or cellular dehydration induced by the formation of extracellular ice during freezing stress (Verslues et al., 2006). Therefore, dehydration, osmotic stress, salinity and, to some extent, cold stresses have been treated as a group of factors imposing alterations in the plant water status (Vinocur and Altman, 2005; Verslues et al., 2006). Water stress and dehydration are general terms to indicate a mild form of water deficit, a physiological condition where the water status of the plant body undergoes minor changes (Bray, 1997). The most severe form of water stress is desiccation, which consists in the loss of most protoplasmic "free" or "bulk" water, forcing the plant cells to survive exclusively with "bound water", that is, the water associated to the cell matrix (Ramanjulu and Bartels, 2002). As a consequence of water stress, citrus canopy growth decreases whereas the root system appears to sustain less damage (Lloyd and Howie, 1989). Water stress may reduce leaf expansion and even lead to premature abscission or senescence of adult leaves (Munns, 2002).

Throughout evolution the sessile nature of plants has led to the development of adaptive strategies to cope with environmental stresses. An essential feature of these adaptive strategies is that they must be elicited in response to an external stimulus, providing utilization of resources when needed and preventing energy waste in the absence of the stress. Plant cells have evolved mechanisms to perceive distinct environmental signals, to integrate them and to modulate the expression of the required genes to respond accordingly. Plants are able to cope with water deficit via two general mechanisms: i) stress avoidance - by producing seeds before the establishment of the drought conditions or by developing specific morphological adaptations, such as leaf surfaces less prone to transpiration losses, reduced leaf area, sunken stomata, increased root length and density (Ramanjulu and Bartels, 2002) and, ii) stress tolerance - which consists of coordinated physiological and biochemical alterations at cellular and molecular levels, such as the accumulation of late embryogenesis-abundant (LEA) proteins associated with activity of the antioxidant system of the cells.

The molecular bases of water stress tolerance remains unknown. Candidate genes induced by water-deficit stress in plants which are relatively sensitive to cellular dehydration have been identified and characterized, mainly in the model plant Arabidopsis thaliana (Vinocur and Altman, 2005; Verslues et al., 2006). The investigated plant systems have been shown to have common molecular and physiological components in a wide range of tolerance levels, indicating a major role for spatial and temporal gene expression regulation in water stress resistance (Ramanjulu and Bartels, 2002; Taji et al., 2004).

The primary site for the detection of water stress in plants remains unknown (Ramanjulu and Bartels, 2002). In yeast and bacterial cells, well-characterized osmosensors are responsible for detection of osmotic stress. Osmosensors are members of the broad class of two-component systems, consisting of a histidine kinase sensor and an intracellular response-regulator which is responsible for relaying the phosphorylation signal to the next component of the pathway, leading to transcriptional regulation of gene expression (Wurgler-Murphy and Saito, 1997). A homolog to the yeast osmosensor SLN1 has been recently characterized in Arabidopsis, AtHK1 (Urao et al., 1999). Microarray whole-genome expression profiles in A. thaliana have shown water stress-induced alteration in the transcription of several sensor- and response regulator-like genes as early as 15 min after the onset of water deprivation stress (Seki et al., 2002). The transcriptional regulation of two component system-like genes is similarly affected under osmotic and salt stress; however, the functional significance of these findings remains unknown (Seki et al., 2002).

In eukaryotic cells, biotic and abiotic stresses trigger the production of reactive oxygen species (ROS) which causes the oxidation of cellular components and ultimately, cell death. The activation of oxidative stress signaling is evolutionarily conserved from yeast to mammals and involves mitogen-activated protein kinase (MAPK) cascades. Similarly, in plants, the production of ROS is induced by environmental, mechanical and biological stress conditions (Inzé and van Montagu, 1995). The observation of changes in the protein phosphorylation status of plants submitted to water deficit conditions indicates the involvement of reversible phosphate relay in the regulation of drought stress signal transduction (Pastori and Foyer, 2002). Several MAPKs are components of dehydration- and abscisic acid-induced signal transduction in plants (Mikolajczyk et al., 2000). Thus, ROS-triggered signal transduction via a MAPK-based cascades induces the expression of detoxification and stress protection genes, such as heat shock proteins (HSP), glutathione-S-transferases (GST), peroxidases, superoxide-dismutases and pathogenesis-related (PR) proteins, protecting the plant from damage (Kovtun et al., 2000). Consistently, the constitutive expression of a heterologous MAPK kinase kinase (MAPKKK) from tobacco induced both drought (Shou et al., 2004a) and freezing (Shou et al., 2004b) tolerance in transgenic maize.

Recent microarray studies in Arabidopsis indicate that a set of genes is involved in promoting tolerance, whereas another set responds to water deficit stress (Seki et al., 2002). The genes responding to dehydration can be categorized into two distinct classes: i) early-response genes - in seconds or minutes, and ii) late-response genes - over hours, days or even weeks (Ramanjulu and Bartels, 2002). This temporal separation demonstrates distinct roles in the stress response; the early genes could provide initial protection and amplification of the signal transduction pathway while the late ones could be involved in adaptation to the stress condition. On the other hand, the manipulation of genes involved in protection and maintenance of cell components structure and cellular functions has been the major target of attempts to produce plants showing enhanced stress tolerance.

During late stages of embryogenesis in dico- and monocotyledonous plants, the cells undergo a severe dehydration process and simultaneously accumulate LEA proteins. Other stress situations, such as low temperatures, increased salinity and exogenous ABA treatment also induce LEA proteins which indicate their involvement in the general cellular protective system against water loss (Cuming, 1999). Molecularly, they are characterized by a biased amino acid composition: high hydrophilicity and high solubility in water. LEA proteins can be divided into five groups according to their predicted biochemical properties and motif similarity (Ingram and Bartels, 1996; Cuming, 1999). LEA-like proteins were the most abundant transcripts found in the dehydration transcriptome of the bryophyte Tortula ruralis, demonstrating the importance of this class of proteins in the adaptive acquirement of tolerance to dehydration, as well as in the cellular rehydration repair response (Oliver et al., 2004). Moreover, drought and salinity tolerance have been increased in rice (Xu et al., 1996) and wheat (Sivamani et al., 2000) with the introduction and expression of a heterologous LEA protein HVA1 from barley.

Aquaporins are members of a family of water channel proteins involved in the facilitation of its transport along transmembrane water potential gradients, thus regulating the hydraulic conductivity of membranes and water permeability (Maurel and Chrispeels, 2001). Several aquaporin-coding genes are upregulated by dehydration in Arabidopsis (Yamaguchi-Shinozaki et al., 1992), tomato (Fray et al., 1994) and C. plantagineum through the ABA-dependent and independent pathways (Mariaux et al., 1998). However, the transcriptional regulation of aquaporin genes is complex with several hierarchic levels of control, and is responsive to both water deficit and numerous environmental and physiological factors (Maurel and Chrispeels, 2001; Tournaire-Roux et al., 2003; Jang et al., 2004).

The integrity of photosynthetic structures, especially of membrane-associated proteins, after a period of water stress is a crucial mechanism in desiccation tolerant plants (Godde, 1999; Bartels and Salamini, 2001). In C. plantagineum, three genes that are highly induced upon the onset of water deficit encode chloroplast-localized stress proteins (DSP). Two of them, DSP22 and DSP34, are thylakoid-associated and one, DSP21, is localized in the stroma (Schneider et al., 1993; Alamillo and Bartels, 2001). Similarly stress-induced chloroplast protection proteins were found in potato (Pruvot et al., 1996; Rey et al., 1998) and in whole-genome expression profile in T. ruralis submitted to rehydration (Oliver et al., 2004).

A common strategy for protection against water deficit in many organisms is the accumulation of compatible solutes or osmolytes. Osmolytes are only synthesized in response to osmotic stress and are biochemically inert in the cell, exclusively helping to maintain the osmotic balance necessary for growth and cellular metabolism under dehydration (Bray et al., 2000). Besides their role in osmotic adjustment, osmolytes might also be involved in other protective mechanisms, such as ROS scavenging (Hong et al., 2000).

The increased synthesis of osmolytes induced under water-stress conditions is caused by modulation of the expression and activity of key regulatory enzymes in their biosynthetic pathways (Ramanjulu and Bartels, 2002). In plants, sugars, polyols, proline, quaternary ammonium compounds and tertiary sulfonium compounds are often found to function as osmolytes. In citrus, the osmotic adjustment under salt stress is mostly dependent upon accumulation of proline and inorganic ions (Arbona et al., 2005).

The accumulation of soluble sugars is a common feature of the desiccation process, in both desiccation-tolerant and desiccation-susceptible plants. Sugars have a role in osmotic adjustment, but also have indirect protective effects, such as protein stabilization (Carpenter et al., 1990). In the desiccation-tolerant plant C. plantagineum, dehydration induces the conversion of 2-octulose, an eight-carbon sugar, to sucrose (Bianchi et al., 1991). This conversion correlates to increases in the gene expression for sucrose synthase (SUS) and sucrose phosphate synthase (SPS) (Ingram et al., 1997; Kleines et al., 1999), which are considered key enzymes of sucrose synthesis/metabolism. Under conditions of dehydration/osmotic stress, the expression of genes coding for SUS isoforms is upregulated in several plants (Pelah et al., 1997; Déjardin et al., 1999). Similarly, antisense expression of the SPS coding sequence in potato plants completely suppressed the water stress-induced stimulation of sucrose synthesis (Geigenberger et al., 1999). Thus, SUS and SPS in plants are crucial steps in the acclimation process of dehydration. Highly soluble sugars, such as the polyfructose molecules fructans, are involved in plant and bacterial adaptation to osmotic stress. Transgenic tobacco and sugar beet plants, overexpressing the gene SacB that codes for a levan sucrase from Bacillus subtillis, accumulated higher levels of fructans and performed better than the untransformed controls under water deficit conditions (Pilon-Smits et al., 1995; Pilon-Smits et al., 1999). Similar results were obtained with transgenic tobacco plants overexpressing a gene encoding the trehalose synthase subunit (TPS1) of the yeast trehalose synthase enzyme (Holmstrom et al., 1996) and bacterial trehalose-6-phosphate synthase and trehalose-6-phosphate-phosphatase genes (Pilon-Smits et al., 1998). The function of trehalose, a non-reducing disaccharide of glucose, in desiccation is hypothesized to involve the stabilization of membrane proteins and lipids and its use as a reserve metabolite. The accumulation of the methylated sugar alcohol, D-ononitol, in transgenic tobacco plants overexpressing the IMT1 gene from Mesembryanthemum crystallinum, has led to increased salt and drought tolerance (Sheveleva et al., 1997).

The regulation of the levels of proline in plants under water stress conditions is simultaneously controlled by upregulation of the P-5-C synthase (P-5-CS) gene and downregulation of the proline dehydrogenase gene (ProDH) (Yoshiba et al., 1997). Furthermore, a proline and glycine betaine transporter (LeProT1) has been shown to be induced in tomato plants submitted to scarce water conditions (Schwacke et al., 1999). Despite the complexity of the control of proline levels, transgenic tobacco and rice plants with higher proline levels due to the overexpression of P-5-CS gene, had higher biomass production under water stress conditions (Kavi Kishor et al., 1995; Zhu et al., 1998).

Water shortage, like other biotic and abiotic stresses, causes the accumulation of enzymes responsible for the oxidative cellular defense system, such as superoxide dismutase, ascorbate, peroxidases, catalases, glutathione-S-transferases and glutathione peroxidases (Kovtun et al., 2000). Other proteins involved in the repair of damaged cellular components have also been shown to be induced under water deficit conditions (Seki et al., 2001; Oliver et al., 2004).

The effect of individual genes involved in water stress tolerance is minimal. Molecular switches and regulatory genes have been proposed to be a better means to increase plant tolerance to water restrictive conditions (Ramanjulu and Bartels, 2002). Thus, one of the most successful strategies for plant modification for enhanced drought tolerance is based on the manipulation of genes coding for transcription factors and/or signaling partners that directly protect plant cells against water deficit. Furthermore, the majority of transcripts identified in association to plant stress responses are regulated by this condition instead of being de novo synthesized. For citrus plants submitted to water deficit during cyclic periods, as for other perennial species, it is important to explore constitutive gene expression that could be induced under constraint conditions.

Thellungiella halophila, an extremophyle plant displaying tolerance to high salinity, low humidity and freezing, exhibits higher pre-stress concentrations of several compounds that have been shown to have protective functions in osmotic imbalance, a common component between water- and salt-stress (Hasegawa et al., 2000). The regulated expression of constitutive genes under stress conditions indicates further posttranscriptional regulation and may represent an early protection of the plant against water constraint. Drought is often interconnected to various environmental stresses that may induce similar cellular damage. As a consequence, similar cell signaling pathways are activated and oxidative stress is frequently induced causing protein denaturation (Smirnoff, 1998; Shinozaki and Yamaguchi-Shinozaki, 2000).

The plant organ submitted to stress conditions is another relevant aspect. Considered the hidden part of the plant, the root system is primordial to citrus physiology under water constraints, not only due to the provision of water and mineral nutrients but also as a storage organ. Roots accumulate carbohydrates in the winter and play a critical role in exporting them to developing fruitlets during early stages of fruit set. However, gene expression modulations and changes in physiological parameters, related to cell water status, are slighter in roots than in shoots (Schena et al., 1995; Seki et al., 2002; Torres et al., 2006).

Thus, in order to cope with water deficit, the plant induces modifications of its physiological state and metabolic pathways using two major categories of responses: i) uptake and transport of water and ions, and ii) protection of membranes and proteins. These genes are known to be constitutively expressed and could actively participate in improving plant tolerance subject to water stress. The aim of this study was to survey citrus EST databases to identify components presenting similarity to genes functionally related to the fore mentioned classes of water stress responses that could represent interesting candidates for transgenic analyses.

Material and Methods

Database searches and alignments

Homologs of functionally characterized genes involved in dehydration responses were identified in BLAST searches (Altschul et al., 1997) against EST contig sequences from the citrus index databases at CitEST. These consisted of approximately 176,200 ESTs obtained from the sequencing of 53 citrus libraries. Data validation was performed by tBLASTx and tBLASTn searches with BLOSUM80 scoring matrix of the retrieved citrus sequence against the databases at NCBI built inside the CitEST project. The resulting alignments were filtered by a threshold e-value of 1e^-15 for the hits and further analyzed. Validated sequences were translated and protein (deduced amino acid) alignments were performed using ClustalX (Thompson et al., 1997). When necessary, alignments were manually adjusted using Lasergene MegAlign (DNASTAR, Madison, WI, USA).

Motif analysis and in silico characterization

The identified citrus homologs were further investigated for the presence and sequence conservation of recognizable functional domains: described in several protein analysis and gene function databases (European Bioinformatics Institute - European Molecular Biology Laboratory - EMBL-EBI, Expert Protein Analysis System - ExPaSy of the Swiss Institute of Bioinformatics - SIB, and Protein Families - Pfam).

Phylogenetic analysis

The putative functionality of the citrus genes in comparison to their homologs from model systems was assessed by genetic distance and phylogenetic studies. Phylogenetic analyses were performed using distance and parsimony methods in the software PAUP* 4.0b10, using the software default parameters. Resampling bootstrap trees containing 1000 random samples were constructed using PSIGNFIT software and ClustalX (Thompson et al., 1997).

In silico gene expression analysis

Qualitative gene expression profiling was performed by in silico analyses of the citrus EST database through the generation of a relational matrix between the number of ESTs corresponding to a determined gene in a given library and normalizing the result to the number of reads of the library. Gene expression patterns of EST contigs and libraries were determined by hierarchical clustering, based on Spearman Rank correlation matrix, using Cluster and Tree View software packages (Eisen et al., 1998) and cluster results were shown as their average expression pattern. The expression profile matrix was ordered accordingly and displayed in grayscale.

Results and Discussion

We have performed extensive BLAST and key word searches of the citrus transcriptome to identify homologs of the genes involved in responsive mechanisms to water deficit in citrus. We have searched for transcripts whose deduced amino acid sequences share similarity to proteins involved in uptake and transport of water and ions, osmolyte metabolism, processes of membrane and protein protection and reactive oxygen species scavenging. In CitEST databases, 305 assembled sequences and EST singlets sharing significant sequence identity with functionally characterized proteins were identified and analyzed (Table 1).

Ion transporters

We have identified 63 sequences in the citrus transcriptome showing significant deduced amino acid homology to functionally characterized ion transport-associated proteins (Table S1): 10 EST contigs and eight singlets are similar to ATPases involved in ion transport and H⁺-exchange; whereas 24 EST contigs and 21 singlets show anion- and cation-binding and transporting functional motifs.

The families of Calcium (ACA), H⁺ (AHA) and H⁺/Na⁺ exchanger (NHX) ATPases are approximately equally represented in citrus analyzed databases, including in libraries derived from non-stressed and non-infected tissues. The family of vacuolar A type H⁺-ATPases (VHA-A) is characterized by lower expression levels in several model species. However, we were able to identify two ESTs from C. sinensis libraries that are highly similar to members of the family. Several ESTs containing motifs responsible for K⁺ and Ca⁺² transport were identified in citrus species. Interestingly, a gene encoding a putative Cu⁺²-transporter was found in the citrus genome database (Table S1). The elevated sequence similarity of the deduced amino acid sequence from the citrus EST, with previously characterized haloacid dehalogenase-like hydrolases, indicate a role in heavy metal detoxification (Himelblau and Amasino, 2000).

Ion transporters are associated with water loss-derived secondary stresses (Verslues et al., 2006) and several developmental processes including embryogenesis and fruit development involve cellular dehydration (Ramanjulu and Bartels, 2002). Interestingly, in citrus libraries derived from whole plants submitted to water stress, no significant increase in the frequency of ion transporter-like reads was observed. However, libraries obtained from the initial three stages of fruit development presented a high frequency of anion and cation transporter-related ESTs (Figure 1). Reads showing sequence similarity to monovalent cation transporters, such as K⁺-transporting proteins and H⁺-ATPases, are highly induced in the first and second stages of fruit development in C. sinensis and C. reticulata. ESTs related to divalent cation transporters were more frequent in libraries derived from later stages of fruit development in C. reticulata; whereas, in C. sinensis, they were more frequent in the first and third stage, and were virtually absent from second stage-derived libraries (Figure 1). These observations suggest that in citrus, developmentally induced dehydration is responsible for the most significant changes in ion transporter gene expression. This indicates that these proteins may have secondary roles in environmentally induced-water loss protection.

Water channels

Water channel proteins are responsible for the transmembrane flow of water across the lipid bilayer plasma membrane, so they are involved in several aspects of plant-water relations. The proteins have a conserved pore-forming structure throughout evolution, consisting of six membrane spanning helices and two loops containing asparagines-proline-alanine (NPA) motifs (Figure 2A) and are members of large membrane intrinsic protein (MIP) families in plants (Chaumont et al., 1998; Johanson et al., 2001). In citrus, we have identified 18 EST contigs and eight EST singlets sharing deduced amino acid sequence homology with functionally characterized MIP family members (Table S2). Amino acid sequence alignment shows significant sequence conservation at the six membrane spanning helices and at the two NPA motifs between the citrus and Arabidopsis proteins (Figure 2B).

From the total 26 MIP-like proteins, 15 were more related to plasma membrane-associated water channels (PIP), three to tonoplast channels (TIP), five to nodulin-like proteins (NIP) and, three to the newly described family of small basic intrinsic proteins (SIP). Phylogenetic analysis indicates that the citrus MIP family has perhaps undergone an intraspecific duplication process, due to the presence of divergent citrus-exclusive PIP branch (Figure 2C). In Arabidopsis, the majority of the PIPs and some TIPs are constitutively highly expressed, whereas NIPs are categorized as rare transcripts (Alexandersson et al., 2005). In citrus, a relatively high frequency of NIP and NIP-like transcripts were identified (Table S2), suggesting that in spite of the high level of sequence conservation between Arabidopsis and citrus MIP proteins, they may be submitted to distinct expression regulation. Dehydration transcriptionally and translationally down-regulates PIP genes in Arabidopsis (Alexandersson et al., 2005); however, we were unable to identify changes in the frequency of MIP-like reads in citrus libraries submitted to environmental and developmental processes that trigger water loss. Thus at this point, the role of water channel proteins in desiccation protection in citrus remains unclear.

Osmolytes

Analyses of EST databases from citrus species revealed the presence of several transcripts which showed sequence similarity to genes encoding enzymes involved in the production of the osmolytes and osmoprotectants most commonly found in model plant species in response to stress (Table S3). These observations suggest the presence of extensive conservation in osmolyte and osmoprotectant metabolism between citrus and model plants. However, we were unable to identify genes coding for components involved in the metabolism of rare plant osmolytes or compounds associated to fungi- and bacteria osmoregulators such as those associated to production of DMSP, choline-O-sulfate or D-ononitol (Table S3).

Drought-induced osmotic stress causes detrimental changes in cellular components, which can be prevented by a wide range of metabolites, including amino acids (e.g. proline), quaternary and other amines (e.g. glycine-betaine and polyamines) and a variety of sugars and sugar alcohols (e.g. mannitol and trehalose). These metabolic changes were not evident in citrus transcriptome analyses, since the frequency of reads showing sequence similarity to transcripts involved in osmoprotection responses remained unaffected in libraries derived from drought-stricken tissues (data not shown). Thus, extensive conservation in osmolyte metabolism is observed in citrus, although its role in water stress protection remains to be established.

Several components of the betaine biosynthetic pathway were identified in citrus transcriptome, including the enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) (Table S3). Citrus species CMO and BADH are highly similar to the enzymes from other organisms, including at catalytic sites (Table S3). This provides evidence for the production of glycine betaine rather than PRO- or Ala-betaines in citrus. Two singlet reads showed extensive similarity to CMO, whereas three BADH-like transcripts were found. The low frequency of betaine metabolism-related transcripts suggests that the pathway is expressed at low levels and/or that these genes are specifically upregulated by biotic and abiotic stresses, although such an induction was absent from drought-derived tissues. Alternatively, the low frequency of betaine biosynthesis transcripts could correspond to low levels of the compound in planta. Proline is biosynthetically derived from the amino acid L-glutamate and its direct precursor is the imino acid (S)-D1-pyrroline-5-carboxylate (P5C). Transcripts showing sequence homology to genes encoding enzymes responsible for proline biosynthesis and degradation were identified in citrus species transcriptome, including homologs to P5C synthase (P5CS), P5C reductase (P5CR) and pyrroline-5-carboxylate reductase (PROD) (Table S3). We have also identified transcripts showing similarity to the coding sequence of enzymes involved in sugar biosynthesis (Table S3). In citrus, the later category comprises trehalose synthesis enzymes (T6PP and T6PS) and mannose specific enzyme M6PR, along with sucrose metabolism components (enzymes SUSY and SPS) and one invertase (fructofuranosidase) that is likely to be involved in fructan metabolism (Table S3). Similarly to that observed for the betaine pathway, a role for transcripts showing sequence similarity to components of proline and osmolyte sugar metabolism in drought tolerance remains to be established.

Metabolic engineering of abiotic stress tolerance employs two general strategies; the first one aiming to increase the production of specific desired compounds or reduction in the levels of unwanted toxic compounds (Capell and Christou, 2004). However, modulation of a single enzymatic step is usually regulated by cellular systems that tend to restore homeostasis, thus limiting the potential of this approach (Vinocur and Altman, 2005). Alternatively, targeting multiple steps of the same pathway has been proposed as a means to control metabolic fluxes in a more predictable manner (Konstantinova et al., 2002). The lack of correlation between the frequency of transcripts showing sequence conservation to members of osmolyte and osmoprotectant metabolism and drought-induced responses in citrus suggests that these protective mechanisms remain unsaturated under water shortage. This indicates potential for metabolic engineering.

Heat-shock proteins

Following heat stress, the amount of cellular proteins is diminished. However, some proteins are accumulated under those conditions and are thus called 'heat shock proteins' (HSP). The HSPs are ubiquitous and present high sequence conservation. They are present in all cellular compartments and their classification follows their kDa molecular mass: HSP60, HSP70, HSP90, HSP100 and the small HSP, which range from 15 to 30 kDa (Vierling, 1991).

Distinct proteins belonging to HSP classes have been related to plant water stress responses. In this study, we have identified 47 sequences presenting similarity to heat shock proteins in the citrus transcriptome (Table S4): one contig reveals homology to a chaperonin from HSP60 class; 18 contigs and one singlet show high similarity to HSP70; six contigs and three singlets, to HSP90; five contigs and three singlets to HSP100; and finally, 10 contigs are similar to small HSP (sHSP). Most of the sequences are derived from HSP70 class (38%) followed by ESTs encoding proteins from HSP90 class (34%) (Figure 3).

HSP70 are essential in helping to prevent the aggregation and assisting in the folding of proteins under normal and stress conditions (Sung et al., 2001a). They also play a regulatory role in stress-associated gene expression (Lee and Schöffl, 1996). Plant HSP70 genes are encoded by a highly conserved multigene family and are localized in several cellular compartments (Sung et al., 2001b; Wang et al., 2004). HSP70s are known to be differentially regulated in response to developmental stages and to a wide range of stresses. Several studies suggest the association of HSP70 to other stress-related responses of plants. These proteins could coordinate to prevent cellular damage and to re-establish cellular homeostasis. In citrus species, they could represent interesting targets for biotechnological manipulations aimed at improving plant tolerance to water deficit.

HSP90 family members have been isolated from animals and plants. They encode structurally related proteins ranging from 80 to 90 kDa. HSP90 is one of the major species of molecular chaperones that requires ATP for its functions (Wang et al., 2004). Three putative ATP-binding motifs are highly conserved among members of hsp90 gene family. Hsp90 genes are developmentally regulated in plants (Koning et al., 1992; Marrs et al., 1993; Krishna and Gloor, 2001) and their expression is induced upon stress conditions in both prokaryotes and eukaryotes. HSP90 proteins are distinct from many other molecular chaperones due to their interaction with steroid hormone receptors and signaling kinases (Young et al., 2001). Thus, it is not surprising that they were found in high proportion in our transcriptome survey. Furthermore, Hsp90 acts as part of a multichaperone system together with Hsp70 and cooperates with several co-chaperones (Wang et al., 2004).

Comparing the data from control and water-stressed libraries of Citrus limonia roots, we have found alterations in the expression levels of five sequences related to HSP. A putative cytosolic HSP70 (AT3G12580) and a chaperonin containing TCP-1 domain belonging to HSP60 class present high levels of expression that diminish in response to water stress. The other three sequences (a HSC70-1 homolog; a sequence similar to an HSP81-2 and another encoding to a putative HSP100) were not detected in control plants and are induced in stressed roots following water deficit.

The heat shock cognate proteins (HSC70) are expressed in normal growth conditions and may or may not be induced in response to stress (DeRocher and Vierling, 1995; Miernyk, 1997; Sung et al., 2001a). In Arabidopsis thaliana seedlings and bean plants, the expression of homologs of hsc70 genes was induced after dehydration (Kiyosue et al., 1994; Torres et al., 2006). Interestingly, the transcript encoding an HSC70 was induced during the light period in untreated roots and in a manner probably independent of ABA biosynthesis (Torres et al., 2006). Thus, HSC70 homologs appear as promising candidates for functional analyses. The early expression changes verified in other plant species may be an indication of a mechanism capable of responding rapidly to water constraint.

We could also identify a contig encoding a chaperonin with very high similarity to a protein isolated from Bruguiera sexangula. It has a chaperone activity in vitro and is capable of improving salt stress tolerance when expressed in E. coli (Yamada et al., 2002). BsCCTa has three highly conserved domains: an equatorial domain that contains the ATP-binding site; an apical domain that binds to peptides; and an intermediate domain. By expression studies in E. coli, the authors verified an increased salt tolerance and identified a region of 218 amino acids as responsible and sufficient to improve stress tolerance. This region is equally conserved in the citrus contig (Table S4), which also presents a decrease in mRNA expression under water deficit conditions. A detailed protein expression study would provide further information about its role in water stress. At this point, assuming a high turnover rate, there are indications that these transcripts play an important role in cellular protection.

HSP81-2 is a member of HSP90 family that is expressed abundantly in root apical meristem of Arabidopsis and is induced by NaCl (Yabe et al., 1994). The hsp90 gene family was characterized in A. thaliana plants and calli in response to heat and heavy metals (Milioni and Hatzopoulos, 1997). Although a similar induction profile for all six genes in response to stress conditions was observed, the results were specifically dependent on the kinetic experiment analysis. Furthermore, a combination of heat and drought could induce a more rapid change in hsp90 genes expression. In Citrus, it would be interesting to analyze the profile of transcript levels of hsp90 mRNAs in response to the kinetics of water stress in different organs.

Genes coding for HSP100 have been isolated from various plant species and can be also termed 'Clp' proteins due to their sequence similarity to E. coli ClpA (Gottesman et al., 1990). They are found in all cellular compartments and are hypothesized to participate in proteolysis regulation, protein translocation and acquired thermotolerance. Thermotolerance in yeast was associated to higher stability of membrane proteins which may be the result of the protective role of HSPs (Swan, 1997). Under water stress conditions, a similar function could also be required. In Phaseolus lunatus leaves, a ClpB homolog has been identified as mediating the response of the chloroplast to heat stress. Surprisingly the citrus ESTs were present in libraries derived from roots. The discrimination between mitochondrial and plastidial transit peptides remains uncertain and some proteins have been demonstrated to be dually targeted (Peeters and Small, 2001; Zhang and Glaser, 2002). Based on mitochondrial proteome analyses, Heazlewood et al. (2004) demonstrated that only one half of the proteins were correctly predicted. Thus, these data require future investigations to clarify the specific role of ClpB in roots and the subcellular location of this protein.

Comparing data from healthy leaves of six citrus species (Figure 4), we have observed a great number of reads corresponding to HSP81-2, one of the sequences already described as differentially expressed in response to water deficit. In Citrus aurantium, we were unable to detect BiP homologs. This protein belongs to HSP70 family and is targeted to endoplasmic reticulum. Its overproduction in transgenic tobacco plants enhanced their tolerance to water deficit (Alvim et al., 2001). BiP plays a role in translocation/retranslocation, folding and assembly of ER proteins and its expression could be regulated by various environmental conditions (Noh et al., 2003). Exclusively in C. aurantifolia and C. aurantium, we have observed high levels of expression of small heat shock proteins (HSP18.2 and HSP17.9, respectively). The sHSP were directly implicated in improving plant tolerance to water stress (Sun et al., 2002). This kind of differential regulation in both species could be an interesting tool for future studies on the mechanisms of drought tolerance. Finally, in C. aurantifolia and Poncirus trifoliata we have found a remarkably distinct pattern of expression of mtHSP70. Bean mtHSP70 homologs were found in the outer mitochondrial membrane facing the cytosol and equally in the mitochondrial matrix, playing a role in protein translocation (Vidal et al., 1993). Mitochondria perform a variety of functions in eukaryotic cells, notably responding to cellular signals as oxidative stress. The precise and eventually specific roles of this protein in C. aurantifolia and Poncirus trifoliata remain to be elucidated.

LEA proteins

LEA proteins were initially described as being present during the late period of seed development that is accompanied by dehydration. They are characterized by low molecular weight ranging mainly from 10 to 30 kDa and above 30 kDa (Hong-Bo et al., 2005) and most of them constitute a more widespread group called "hydrophilins" (Garay-Arroyo et al., 2000). First studied in developing cotton seeds (Dure and Croudh, 1981), LEA proteins have been detected in several plant species (Close, 1996; Han and Kermode, 1996; Chen et al., 2003). The proteins are regulated not only during seed development but also upon most diverse environmental conditions. However, the knowledge of the biochemical functions of LEA proteins is still incomplete (Bartels and Salamini, 2001) but some of them certainly contribute to improving plant drought tolerance (Babu et al., 2004; Hara et al., 2004).

In citrus, we have identified nine EST contigs and nine EST singlets sharing deduced amino acid sequence homology to LEA protein coding genes (Table S5). From the total 18 LEA-like proteins, six were more related to COR19, one to CsDHN, two to LEA group 1, three to LEA group 4, four to PgEMB8, one to LEA group 5 and one to the recently identified PsLEAm.

The most frequent sequences correspond to homologs of LEA 14-A coding genes followed by LEAs from group 1. These sequences were mainly found in libraries obtained from fruits in different developmental stages in both C. sinensis and C. reticulata species (Figure 5). In C. sinensis, a defined pattern of changes in LEA14-A and COR19 family genes expression was not observed. In contrast, in C. reticulata, we have observed an increase in LEA-14A expression during the fruit maturation process. This fact indicates that these genes are down regulated throughout fruit maturation stages, which are known to be accompanied by dehydration.

Moreover, in citrus seed-derived libraries, we have observed generally high levels of expression of LEA-coding genes. These genes could exert specific roles related to seed development and the imposition of a desiccation step. A novel mitochondrial LEA protein has been identified in pea seeds (Grelet et al., 2005). The corresponding mRNAs are responsive to maturation of seeds and to water deficit. Conversely, they are down-regulated during seed germination. We identified transcripts sharing sequence similarity to this putative mitochondrial LEA exclusively in Poncirus trifoliata seeds. Therefore, it would be interesting to analyze its profile of expression under different conditions.

On the other hand, Cor19 genes and the CsDHN gene were absent from seed libraries. Cor19 and dehydrin genes in general may be involved in response to several environmental stresses and to development stages. The kinetics of the response and their regulation varies according to the species and to the stress applied. Recently, Hara et al. (2004) demonstrated that CuCOR19 acts as radical scavenger and may reduce the oxidative damage induced by water deficit in Citrus unshiu. Generally, as dehydrin genes are expressed during water stress responses in plants, it is speculated that they protect plants from damage caused by cell desiccation. Therefore their specific role in citrus drought tolerance remains to be clarified.

Soluble protein 1

Soluble protein 1 (SP1) has been described as a member of a novel class of plant stress-response proteins. Initially isolated from an expression library of water-stressed aspen plants, SP1 homologs are widespread over a range of different organisms (Wang et al., 2002). In citrus transcriptome survey, we have identified one EST contig and one singlet with high similarity to Populus tremula, Arabidopsis thaliana and Oryza sativa SP1-coding ESTs (Table S6). Citrus sequences have conserved Phe residues at four positions and the consensus motif "K-F-WG-D" located in the middle portion of the sequences. Aspen SP1 transcripts are expressed under non-stressed conditions and are also induced upon water and hypo-osmotic stress. These transcripts are also maintained at high levels during stress recovery (Wang et al., 2002). The identified sequences of SP1 homologs in citrus were present in different parts of the plant, such as leaves, bark and fruits. Interestingly, SP1-like mRNAs were present in Poncirus trifoliata plants infected with Citrus tristeza virus. Nevertheless, SP1 is stress-related and has high thermostability as small heat shock proteins (sHSP), it does not present amino acid sequence nor function similarity in stress protection (Dgany et al., 2004). Therefore, Wang et al. (2006) have described aspen SP1 as a remarkably resistant protein. It is boiling-stable and resistant to proteases, organic solvents and high levels of ionic detergent. However, at this point its function and involvement in repair of cellular damage remains to be elucidated.

Reactive oxygen species-scavenging enzymes

Aerobic organisms utilize oxygen as electron receptors during respiration. Under optimal conditions, there is the reduction of O₂ to H₂O, following the reception of four electrons. However, when O₂ receives one, two or three electrons, reactive oxygen species (ROS) are formed (Levine, 1999). ROS have been demonstrated to have signaling function in several environmental responses and developmental processes, including biotic and abiotic stress responses, allelopathic plant-plant interactions, cell division and elongation, and programmed cell death (Apel and Hirt, 2004; Foyer and Noctor, 2005). Moreover, normal cell metabolism constantly generates ROS; thus, their basal levels are tightly controlled. In Arabidopsis thaliana, the ROS gene network comprises at least 152 genes, such as the scavenging enzymes (superoxide dismutases - SODs, ascorbate peroxidases - APXs, catalases - CATs, glutathione peroxidases - GPX, and peroxiredoxins - PRPX) and enzymes involved in ascorbate-glutathione cycle (monodehydroascorbate reductase - MDAR, dehydroascorbate reductase - DHAR and glutathione reductase - GR) (Mittler et al., 2004).

Under water stress conditions, the plants may activate the antioxidant-defense system to control ROS overproduction (Bartels, 2001). Nonetheless, this activation is dependent on the plant species, the developmental stage, the time and intensity of stress conditions, as on the radicals formed and the cellular compartment localization (Levine, 1999; Bowler and Fluhr, 2000).

In citrus transcriptome analysis, we have identified 115 transcripts that share sequence conservation to Arabidopsis ROS metabolism (Table S7 to S12): 23 SOD-like (Table S7), 46 plant APX/PRPX (Table S8), six MDAR, 10 DHAR, four GR, five GST (Table S9), four AOX (Table S10), 15 CAT (Table S11) and two transcripts sharing sequence similarity to potato thioredoxin (Table S12).

In citrus transcriptome, copper/zinc SOD (CDS) transcripts were the most abundant subfamily (48%) of this class of ROS scavenging enzymes, whereas iron (FDS) and manganese (MDS) were less frequent (30% and 22%, respectively) (Table S7). The deduced amino acid sequence of citrus species SOD transcripts is highly similar to the Arabidopsis proteins, especially for FDS- and MDS-like sequences (Figure 6). Eight citrus transcripts showed extensive conservation of the deduced amino acid sequence to Arabidopsis APX proteins (Table S8). Interestingly, the sequence conservation between citrus and Arabidopsis AtGPX family is less significant, although a higher number of homologous transcripts was identified, representing 57% of all citrus peroxidase-like mRNAs (Table S8).

A high ratio of reduced peroxidized ascorbic acid and glutathione is believed to be essential for the proper scavenging of ROS in cells. It is maintained by glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR) using NADPH as reducing power (Mittler, 2002). APX catalyses the reduction of H₂O₂ with simultaneous oxidation of ascorbate generating monodehydroascorbate (MDHA) (Yoon et al., 2004) or dehydroascorbate (DHA). MDAR and DHAR have auxiliary functions in the maintenance of proper ascorbate concentration in cells by reducing the MDHA and DHA radical directly to ascorbate (Mittler et al., 2004). MDAR reduces the MDHA to ascorbate at the expense of a NAD(P)H and DHAR reduces DHA to ascorbate using glutathione (GSH) as a reducing agent, resulting in GSSG that is reduced to GSH by glutathione reductase (GR) using NADPH (Allen, 1995). Glutathione is a ubiquitous tripeptide that is synthesized via two ATP-dependent reactions. In plants, most of glutathione exists in the reduced form (GSH) and the content of the oxidized form (GSSG) normally does not exceed 10% of the total GSH content. Six transcripts showed extensive conservation of the deduced amino acid sequence to Pisum sativum MDAR, 10 to Arabidopsis DHAR, and four to Arabidopsis GR (Table S9).

Glutathione S-transferases (GST) are a family of multifunctional, dimeric enzymes that catalyze the nucleophilic attack of glutathione on lipophilic compounds with electrophilic centers. In plants, it has been reported mainly associated with herbicide detoxification, although they could respond to diverse environmental stresses, notably dehydration (Marrs, 1996). In citrus EST database, we were able to identify four EST contigs and one singlet coding for GST homologs (Table S9).

Alternative oxidase (AOX) mediates the conversion of O₂ to H₂O in a one-step reaction coupled with ATP production. This enzyme is thought to function as a homodimeric protein and is encoded by multigene families in several plant species (Whelan et al., 1995; Finnegan et al., 1997; Ito et al., 1997; Saisho et al., 1997). Expression of AOX genes is developmentally and environmentally regulated (Vanlerberghe and McIntosh, 1997). In our citrus EST search, we have found three EST contigs and one singlet presenting similarity to Arabidopsis AOX transcripts (Table S10). One contig and one singlet related to AOX1A and two contigs related to AOX2. The expression of AOX homologs was not affected in water stress-derived libraries. In tobacco- and Arabidopsis- transgenic plants, AOX exerts a role in diminishing the oxidative stress, evidenced by changes at transcriptional levels (Maxwell et al., 1999; Saisho et al., 2001). Thus, in order to fully characterize citrus AOX, more studies are necessary.

Catalase multigene family in A. thaliana consists of three genes (CAT1, CAT2 and CAT3) encoding individual subunits, which associate to form at least six isozymes that are readily resolved by non-denaturing gel electrophoresis (McClung, 1997). Catalase is a tetramer and catalase activity gels reveal that three isozymes are detectable throughout the Arabidopsis life cycle (Salomé and McClung, 2002). CAT2 and CAT3 are clock-regulated and CAT1 is not (Michael and McClung, 2002).

All three mRNAs are detectable in freshly imbibed seeds, although the pattern of mRNA relative abundance varies among the three genes during early germination (McClung, 1997). Arabidopsis transgenic plants with high levels of tolerance to chilling and oxidative stresses presented an induction of transcript levels and the activity of CAT1. CAT1 appears to be an important responsive gene to oxidative stress and to be induced in water deficit-tolerant plants (Hsieh et al., 2002a; Hsieh et al., 2002b). In our citrus transcriptome survey, we have identified one EST contig that is highly related to CAT1, 10 EST contigs and two singlets similar to CAT2 and finally, one EST contig homologous to CAT3 (Table S11). Although a great number of EST coding for catalase in citrus ESTs libraries is available, we were unable to observe a bias in these transcript levels in response to stresses or developmental conditions. As observed in Arabidopsis (Salomé and McClung, 2002), CAT2 transcripts were the most abundant in citrus libraries.

Thioredoxins are small proteins containing a Cys-Gly-Pro-Cys active site domain that is able to reduce disulfide bridges on target proteins (Eklund et al., 1991). They exert a general role in enzyme activity regulation via thiol redox control. CDSP32 is a recently identified new thioredoxin highly induced under drought and oxidative stress conditions (Rey et al., 1998; Broin et al., 2000). Lines lacking CDSP32 are more susceptible to photoxidative treatments (Broin et al., 2002). CDSP32 is a critical component in the defense system against lipid peroxidation in the photosynthetic apparatus (Broin and Rey, 2003). In the present citrus data mining effort, we have found two EST contigs coding for homologs of CDSP32 (Table S12). Reads similar to CDSP32 were absent from citrus water-stressed libraries. However, a slight down-regulation of these transcripts was observed in C. reticulata, C. sinensis and Poncirus trifoliata that are infected with CVC or CTV. Its role in a general plant defense mechanism prompts a detailed investigation.

Recently, ROS signals have been demonstrated to possess a certain degree of specificity and selectivity, which allows them to act efficiently in a variety of developmental processes and environmental responses (Gadjev et al., 2006). The chemical nature of ROS and/or their subcellular site of production could be critical for the specificity and selectivity of these signals. During abiotic stress conditions, ROS accumulation has been hypothesized to consist in toxic stress by-products and signal transduction molecules responsible for the activation of defense mechanisms (Mittler, 2002). Dehydration-induced transcription of ROS scavenging proteins has been demonstrated in the desiccation-tolerant plant Craterostigma plantagineum and in the model system A. thaliana (Mittler, 2002) and is thought to be mediated by abscisic acid signaling (Zhang et al., 2006). ABA and ROS treatment induce the expression of antioxidant genes and all activities of the antioxidant enzymes catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase via a mitogen-activated protein kinase (MAPK)-mediated pathway (Zhang et al., 2006).

Our survey of citrus transcriptome has identified cDNAs showing sequence similarity to ROS scavenging enzymes in the majority of the libraries available in the CitEST database, including those from distinct developmental stages, pathogen-attacked tissues and water stress. In C. sinensis and C. reticulata, peroxidase-like reads are identified by BLAST searches and are equally frequent in libraries obtained from fruit developmental stages, pathogen-attacked and water stressed tissue. Interestingly, in P. trifoliata, the frequency of peroxidase homologs appears to be biased; glutathione (ATGPX) and L-ascorbate (APX) homologs were identified in fruit development libraries, whereas peroxiredoxin (PRPX) and APX-like sequences were found in libraries obtained from pathogen-attacked tissues (Figure 7). Water stress appears to induce the transcription of homologs of all three peroxidase families in P. trifoliata, with a smaller prevalence of APX-like mRNAs (Figure 7). Thus, at this point a consistent association between a specific sub-set of oxidative stress metabolism enzymes and water stress-induced ROS scavenging in citrus remains to be established, although APX-like genes appear to be suitable candidates for responding to environmentally caused water loss.

Concluding Remarks

Water stress is arguably the most serious constraint to agriculture (Araus et al., 2002). However, stress tolerance by genetic modification is difficult to achieve due to the involvement of complex traits in plants. The manipulation of certain classes of proteins, especially those having a direct protective role, could reveal good candidates to improve plant stress tolerance (Wang et al., 2003).

Various genes are known to be stress-induced (Seki et al., 2002). Expression profiling has become an important tool to investigate how an organism responds to environmental changes and, subsequently how these transcriptional changes may thus define both tolerant and sensitive responses (Hazen et al., 2003). The difference between tolerance and susceptibility may arise from the regulation of a basic set of genes (Xiong and Zhu, 2002; Taji et al., 2004; Kant et al., 2006). In the Citrus transcriptome survey, we have identified 305 genes implicated in different functions known to be intrinsically related to plant water stress tolerance (Figure 8).

Apart from the complex regulatory network between drought and other environmental stresses, a tight correlation amongst distinct classes of proteins has been demonstrated. For example, the overexpression of BiP (a scarce transcript in citrus libraries) was apparently associated to a decreased responsiveness of antioxidative enzymes under water deficit, notably in relation to SOD, identified in a large number of CitEST libraries (Alvim et al., 2001). New LEA proteins hypothesized to function as radical scavengers under oxidative stress conditions imposed by the dehydration have recently been described (Hara et al., 2004). In contrast, mitochondria-localized LEAs could exert a complementary role to osmolytes, which mainly participate in cytosol stabilization (Grelet et al., 2005). Another aspect that should be considered is the capacity of the plant to recover from an imposed constraint. The regulation of certain transcripts could be an indication of this capacity, as it is the case for aquaporins in salt-stressed rice roots (Kawasaki et al., 2001).

Finally, it has been demonstrated that the capacity to cope with adverse conditions is strongly dependent on a stress-anticipatory preparedness in tolerant species (Gong et al., 2005). Therefore, the approach adopted in this study will bring about trends to be pursued in the functional characterization and transgenic analyses of the identified components.

Internet Resources

Supplementary Online Material

Table S1 - Citrus ESTs with homology to functionally characterized genes involved in ion transporters from Arabidopsis thaliana and other model species.

Table S2 -Citrus ESTs with homology to the major intrinsic protein (MIP) gene family from Arabidopsis thaliana and other model species.

Table S3 -Citrus ESTs with homology to genes involved in osmolyte biosynthesis.

Table S4 -Citrus ESTs with homology to the heat shock proteins (HSP) gene family from Arabidopsis thaliana and other model species.

Table S5 - Citrus ESTs with homology to the late embryogenesis abundant proteins (LEA) gene family from Arabidopsis thaliana and other model species.

Table S6 -Citrus ESTs with homology to the SP1-related coding genes from Arabidopsis thaliana.

Table S7 - Citrus ESTs with homology to superoxide dismutase protein families from Arabidopsis thaliana.

Table S8 - Citrus ESTs with homology to peroxidase protein families from Arabidopsis thaliana.

Table S9 - Citrus ESTs with homology to ascorbate-glutathione cycle-related protein families from Arabidopsis thaliana.

Table S10 - Citrus ESTs with homology to alternative oxidase protein families from Arabidopsis thaliana.

Table S11 - Citrus ESTs with homology to catalase protein families from Arabidopsis thaliana.

Table S12 - Citrus ESTs with homology to thioredoxin protein from Solanum tuberosum.

Are provided with the electronic version of this article.

This material is available as part of the online article from http://www.scielo.br/gmb.

Received: July 24, 2006; Accepted: February 8, 2007.

Associate Editor: Marco Aurélio Takita

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Publication Dates

• Publication in this collection
  26 Nov 2007
• Date of issue
  2007

History

• Accepted
  08 Feb 2007
• Received
  24 July 2006