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Eucalyptus urograndis stem proteome is responsive to short-term cold stress

Abstract

Eucalyptus urograndis is a hybrid eucalyptus of major economic importance to the Brazilian pulp and paper industry. Although widely used in forest nurseries around the country, little is known about the biochemical changes imposed by environmental stress in this species. In this study, we evaluated the changes in the stem proteome after short-term stimulation by exposure to low temperature. Using two-dimensional gel electrophoresis coupled to high-resolution mass spectrometry-based protein identification, 12 proteins were found to be differentially regulated and successfully identified after stringent database searches against a protein database from a closely related species (Eucalyptus grandis). The identification of these proteins indicated that the E. urograndis stem proteome responded quickly to low temperature, mostly by down-regulating specific proteins involved in energy metabolism, protein synthesis and signaling. The results of this study represent the first step in understanding the molecular and biochemical responses of E. urograndis to thermal stress.

abiotic stress; mass spectrometry; omics; proteomics


Introduction

Eucalyptus is one of the most important plant genera used in the pulp and paper industry. This genus contains ∼700 species that originated in Australia and Indonesia, in addition to several hybrids developed to exploit different plant traits. Currently, Eucalyptus plants are cultivated worldwide because of their rapid adaptability to different climatic conditions and easy use in plant breeding programs. The most widely used species in forest plantations and breeding programs are Eucalyptus grandis, Eucalyptus globulus, Eucalyptus urophylla and Eucalyptus camaldulensis. In addition to these species, several intra- and interspecific hybrids that combine important traits from both parental lines have been successfully bred. Eucalyptus urograndis is one of the most important interspecific hybrids because it combines the rapid growth of E. grandis and the disease/climate tolerance of E. urophylla (Kullan et al., 2012Kullan ARK, Dyk MM, Hefer CA, Jones N, Kanzler A and Myburg AA (2012) Genetic dissection of growth, wood basic density and gene expression in interspecific backcrosses of Eucalyptus grandis and E. urophylla. BMC Genet 13:e60.). This species is currently the hybrid preferred by the Brazilian pulp industry and is consequently the mostly propagated species in commercial forest nurseries in this country.

Although E. urograndis has been successfully adapted to the Brazilian climate, adult trees and young plants are subject to metabolic alterations induced by fluctuations in the growth conditions. The projected increase in the worlds average temperature may result in a new global scenario in which plants may have to cope with erratic temperature patterns of unseasonable warm/cold spells; untimely deacclimation may also lead to freeze damage (Gu et al., 2008Gu L, Hanson PJ, Post M, Kaiser DP, Yang B, Nemani R, Pallardy SG and Meyers T (2008) The 2007 Eastern US spring freeze: Increased cold damage in a warming world? BioScience 58:253–262.; Pagter and Williams, 2013Pagter M and Williams M (2013) Frost dehardening and rehardening of Hydrangea macrophylla stems and buds. HortScience 46:1121–1126.).

Large-scale gene expression profiling provides a robust, high-throughput means of detecting important genes and of guiding plant breeding programs to improve specific plant traits, such as thermotolerance. Proteomics, the large-scale study of the proteins present in a particular biological system, is a powerful multi-disciplinary approach that focuses on the characterization of biological molecules that are synthesized as the final product of gene expression. Information regarding the differential regulation of protein(s) may therefore be useful for monitoring the metabolic responses of plants to environmental disturbances and assisting in the genetic transformation of target plant species. Despite their importance for the pulp industry, few studies have examined the proteome of Eucalyptus species (Celedon et al., 2007Celedon PAF, Andrade A, Meireles KGX, Carvalho MCCG, Caldas DGG, Moon DH, Carneiro RT, Franceschini LM, Oda S and Labate CA (2007) Proteomic analysis of the cambial region in juvenile Eucalyptus grandis at three ages. Proteomics 7:2258–2274.; Bedon et al., 2011Bedon F, Majada J, Feito I, Chaumeil P, Dupuy JW, Lomenech AM, Barre A, Gion JM and Plomion C (2011) Interaction between environmental factors affects the accumulation of root proteins in hydroponically grown Eucalyptus globulus (Labill.). Plant Physiol Bioch 49:69–76., 2012Bedon F, Villar E, Vincent D, Dupuy JW, Lomenech AM, Mabialangoma A, Chaumeil P, Barré A, Plomion C and Gion JM (2012) Proteomic plasticity of two Eucalyptus genotypes under contrasted water regimes in the field. Plant Cell Environ 35:790–805.; Britto et al., 2012Britto DS, Pirovani CP, Gonzalez ER, Silva JF, Gesteira AS and Cascardo JCM (2012) Oxidative stress proteins as an indicator of a low quality of eucalyptus clones for the pulp and paper industry. Genet Mol Res 11:3798–3813.; Valdés et al., 2013Valdés AE, Irar S, Majada JP, Rodriguez A, Fernandez B and Pagès M (2013) Drought tolerance acquisition in Eucalyptus globulus (Labill.): A research on plant morphology, physiology and proteomics. J Proteomics 79:263–276.), with most of them investigating the changes induced by drought stress (Bedon et al., 2011Bedon F, Majada J, Feito I, Chaumeil P, Dupuy JW, Lomenech AM, Barre A, Gion JM and Plomion C (2011) Interaction between environmental factors affects the accumulation of root proteins in hydroponically grown Eucalyptus globulus (Labill.). Plant Physiol Bioch 49:69–76., 2012Bedon F, Villar E, Vincent D, Dupuy JW, Lomenech AM, Mabialangoma A, Chaumeil P, Barré A, Plomion C and Gion JM (2012) Proteomic plasticity of two Eucalyptus genotypes under contrasted water regimes in the field. Plant Cell Environ 35:790–805.; Valdés et al., 2013Valdés AE, Irar S, Majada JP, Rodriguez A, Fernandez B and Pagès M (2013) Drought tolerance acquisition in Eucalyptus globulus (Labill.): A research on plant morphology, physiology and proteomics. J Proteomics 79:263–276.). In this work, we examined the response of young E. urograndis plants to low temperature and identified stem proteins that are potentially related to cold stress responses in this hybrid plant.

Materials and Methods

Plant material

Young E. urograndis S. T. Blake plants (clone PL-3335) were obtained from a local nursery and cultivated in the greenhouse of the Department of Technology at the State University of São Paulo (Jaboticabal, SP, Brazil). After one month of growth, the plants were transferred to growth chambers with a 12 h photoperiod and a temperature of 15 °C for exposure to cold and 30 °C for normal conditions.

Protein extraction

After 24 h of cultivation, E. urograndis stems were isolated and stored at −80 °C until analysis. Protein extracts were obtained by mixing 1.5 g of tissues with 4 mL of extraction media containing 500 mM Tris, 50 mM EDTA, 700 mM sucrose and 100 mM KCl (pH 8.0). After 5 min of incubation at 4 °C, 4 mL of equilibrated phenol (Sigma-Aldrich) was added to the solution and the mixture was stirred for 15 min prior to centrifugation (4000 × g, 10 min, 4 °C). After centrifugation, the phenol fraction was collected, transferred to a new tube and five volumes of cold methanol containing 100 mM ammonium acetate was added. Protein precipitation was done for 12 h and the pellet was resuspended in 300 μL of sample buffer (125 mM Tris-HCl, 1% SDS, 1% dithiothreitol and 20% glycerol) after centrifugation (4000 × g, 15 min; 4 °C). Protein quantitation was done with the Bradford assay using bovine serum albumin as the standard.

Two-dimensional electrophoresis

For two-dimensional (2D) electrophoresis, 100 μg aliquots of proteins were precipitated from the protein extract by adding five volumes of cold acetone followed by incubation for 2 h at −20°C. After centrifugation (4000 × g, 10 min, 4 °C) the protein pellets were resuspended in 150 μL of rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% IPG buffer, 0.002% bromophenol blue) followed by passive sample loading onto IPG-gel strips (7 cm, 4–7 pH gradient) for 12 h. Isoelectric focusing was done using an Ettan IPGPhor 3 system (GE Healthcare) for 20 h and terminated after accumulating 37 kVh. Prior to separation by SDS-PAGE, proteins were reduced (6 M urea, 75 mM Tris-HCl, 30% glycerol, 2% SDS, 0.002% bromophenol blue and 125 mM dithiothreitol) and alkylated (6 M urea, 75 mM Tris-HCl, 30% glycerol, 2% SDS, 0.002% bromophenol blue and 125 mM iodoacetamide) for 30 min at room temperature. Vertical electrophoresis was done in a discontinuous buffer system for 2 h at 100 V. After SDS-PAGE, protein spots were visualized by staining with Coomassie colloidal G-250 for 12 h followed by gel destaining for 3 h using a 30% methanol solution.

Detection of differentially regulated proteins

The 2D gels were analyzed using the program Image Master Platinum v.7 (GE Healthcare). Gel images were acquired in transparent mode using a green color filter. Spots were detected automatically using the parameters smooth, minimum area and saliency. Spot matching was done by using only one landmark with manual correction if necessary. Differential regulation was detected based on the normalized spot volume (%V) parameter. Spot matches between control and treatment gels with a two-fold difference in the %V that was significant (p < 0.0.5) in pair-wise analyses using Students t-test were considered to be differentially stained and, consequently, selected for mass spectrometry analysis. Supplementary data from the 2D gel image analyses is provided in Table S1.

Identification of proteins by tandem mass spectrometry

Prior to the mass spectrometric analyses, proteins were digested in gel according to Shevchenko et al. (2007)Shevchenko A, Tomas H, Havlis J, Olsen JV and Mann M (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860.. Peptides were introduced in the mass spectrometer after separation by reverse phase chromatography using an EASY-nLC 1000 system (Thermo Scientific). After loading onto the analytical column, separation of the tryptic peptides was done using a C18 nano-column (15 cm, 2 μm, 100 Å) for 60 min at a flow rate of 300 nL/min. Mass spectrometric analyses were done in a Q-Exactive instrument (Thermo Scientific) using the data-dependent acquisition method for the ten most abundant peptide ions. Fragment spectra were acquired after HCD (high-energy collision dissociation) fragmentation of the isolated peptide ions under a stepped normalized collision energy equal to 35% (±5%). Spectral correlation was done using the Sequest search tool contained in the Proteome Discoverer software (Thermo Scientific) and run against the E. grandis protein database available for download from the Phytozome portal (Myburg et al., 2014Myburg AA, Grattapaglia D, Tuskan GA, Hellsten U, Hayes RD, Grimwood J, Jenkins J, Lindquist E, Tice H, Bauer D, et al. (2014) The genome of Eucalyptus grandis. Nature 510:356–362.). The search parameters were adjusted for an error tolerance equal to 0.02 Da for the fragment ions and 10 ppm for the parent ions. Oxidation of methionines and carbamidomethylation of cysteines were selected as dynamic and static modifications, respectively, during database searches. Peptide-spectrum matches were considered significant if they had a cross-correlation score ≥1.5, 2.0 or 2.5 for singly, doubly or triply-charged ions, respectively.

Results and Discussion

In this study, we used proteomics to obtain information about the E. urograndis biochemical responses to short-term (24 h) cold stimulus. For this, we initially compared the total protein content between plants grown at a low temperature (15 °C) and those grown at 30 °C. As expected, there were no major changes in protein accumulation after 24 h of exposure to low temperature. Using the extraction procedure described here, protein yields of 0.36 ± 0.09 mg/g (mean ± SD) and 0.45 ± 0.06 mg/g of fresh tissue were obtained for stems of plants grown at 30 °C and 15 °C, respectively.

To gain insight into the fine biochemical alterations that could affect growth and wood biosynthesis, the stem proteome was analyzed using 2D gel electrophoresis applied to young E. urograndis plants grown under both conditions. A subsequent comparative analysis detected 12 protein spots with significant densitometric variation (Figure 1, Table 1). Tandem mass spectrometric analyses followed by database searches using a closely related Eucalyptus species resulted in the successful identification of all proteins from the selected gel spots. The differential regulation of proteins from a wide variety of cellular functions indicates a complex regulatory network in Eucalyptus cells in response to cold stress. Down-regulation of the proteins fructose bisphosphate aldolase (Eucgr.K02073.1, spot 16), glyceraldehyde 3-phosphate dehydrogenase (Eucgr.I01564.1, spot 93) and NADH ubiquinone oxidoreductase (Eucgr.A02717.1, spot 98) suggested a reduction in the energy-related metabolism of E. urograndis stems shortly after cold stress induction.

Figure 1
Representative 2D protein gel obtained for E. urograndis stems. Protein spots selected for mass spectrometry-based identification are marked with an arrow and number, as in Table 1. Molecular mass markers are indicated to the left of the gel.
Table 1
List of differentially regulated proteins identified in the stems of young E. urograndis plants after growth at low temperature for 24 h.

Low temperature stress also decreased the expression of proteins involved in gene transcription, such as the protein Eucgr.K02072.1, described as a transcription factor GT-2 containing a trihelix DNA-binding/SANT domain (spot 87). InterPro and Prosite prediction analyses revealed the presence of a myb-like domain from residue 98 through residue 163 of this differentially regulated protein (Figure 2A). A novel myb transcription factor was recently reported to enhance cold tolerance in Arabidopsis (Su et al., 2014Su LT, Li JW, Liu DQ, Zhai Y, Zhang HJ, Li XW, Zhang QL, Wang Y and Wang QY (2014) A novel MYB transcription factor, GmMYBJ1, from soybean confers drought and cold tolerance in Arabidopsis thaliana. Genes 538:46–55.). Up-regulation of myb-related transcripts/proteins upon cold stress has also been identified in other plants, such as Anthurium sp. (Tian et al., 2013Tian DQ, Pan XY, Yu YM, Wang WY, Zhang F, Ge YY, Shen XL, Shen FQ and Liu XJ (2013) De novo characterization of the Anthurium transcriptome and analysis of its digital gene expression under cold stress. BMC Genomics 14:e827.) and Lilium lancifolium (Wang et al., 2014Wang J, Yang Y, Liu X, Huang J, Wang Q, Gu J and Lu Y (2014) Transcriptome profiling of the cold response and signalling pathways in Lilium lancifolium. BMC Genomics 15:e203.). Although not extensively described, a correlation between myb proteins and cold stress has already been reported for Eucalyptus. Keller et al. (2009)Keller G, Marchal T, San Clemente H, Navarro M, Ladouce N, Wincker P, Couloux A, Teulières C and Marque C (2009) Development and functional annotation of an 11,303-EST collection from Eucalyptus for studies of cold tolerance. Tree Genet Genomes 5:317–327. identified myb-like proteins as the most abundant transcription factors in E. gunnii leaves exposed to cold stress. Large-scale transcript analysis of E. grandis, E. globulus and E. urophylla xylem tissue indicated a differential expression of myb-like proteins across these Eucalyptus species (Salazar et al., 2013Salazar MM, Nascimento LC, Camargo ELO, Gonçalves DC, Neto JL, Marques WL, Teixeira PJPL, Mieczkowski P, Mondego JMC, Carazzolle MF, et al. (2013) Xylem transcription profiles indicate potential metabolic responses for economically relevant characteristics of Eucalyptus species. BMC Genomics 14:e201.). In the present work, the protein Eucgr.K02072.1 identified in young E. urograndis stems was down-regulated after short-term cold exposure.

Figure 2
Structure and molecular phylogeny analysis for the E. urograndis protein Eucgr.K02072.1 (A) ScanProsite output. Predicted myb-like domain profile extends from amino acid residue 98 through residue 163. (B) Neighbor-joining tree branch containing the E. urograndis protein Eucgr.K02072.1 and the closely-related E. grandis sequences.

In addition to the suggested role in plant responses to cold stress, over-expression experiments indicate that myb transcription factors may also regulate lignin genes, such as cinnamoyl-coenzyme A reductase and cinnamyl alcohol dehydrogenase (Goicoechea et al., 2005Goicoechea M, Lacombe E, Legay S, Mihaljevic S, Rech P, Jauneau A, Lapierre C, Pollet B, Verhaegen D, Chaubet-Gigot N, et al. (2005) EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant J 43:553–567.; Barakat et al., 2009Barakat A, Bagniewska-Zadworna A, Choi A, Plakkat U, DiLoreto DS, Yellanki P and Carlson JE (2009) The cinnamyl alcohol dehydrogenase gene family in Populus: Phylogeny, organization, and expression. BMC Plant Biol 9:e26.; Legay et al., 2010Legay S, Sivadon P, Blervacq AS, Pavy N, Baghdady A, Tremblay L, Levasseur C, Ladouce N, Lapierre C, Séguin A, et al. (2010) EgMYB1 and R2R3 transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar. New Phytol 188:774–786.), thereby affecting cell wall thickness and plant growth. Multiple sequence alignments of the E. urograndis protein Eucgr.K02072.1 against the 287 E. grandis genes reported to contain myb-like motifs revealed high sequence similarity between this protein and the E. grandis transcribed genes containing an alcohol dehydrogenase transcription factor motif (Eucgr.H04383.1, Eucgr.A01571.1, Eucgr.J02994.1, Eucgr.A00307.1 and Eucgr.C01064.1 in Figure 2B). This result suggests that if the down-regulated protein Eucgr.K02072.1 is capable of binding to a cinnamyl alcohol dehydrogenase DNA sequence then it may directly affect lignin biosynthesis in E. urograndis stems or could play a role in the NADH/NAD+ balance, and thus in the cell redox status, in order to counteract the imposed temperature stress (Hashida et al., 2009Hashida SN, Takahashi H and Uchimiya H (2009) The role of NAD biosynthesis in plant development and stress responses. Ann Bot 103:819–824.). Currently, one of the best known plant regulatory systems active during cold sensing and acclimation is the C-repeat binding factor (CBF) pathway. In Eucalyptus, two CBF genes have recently been isolated from Eucalyptus gunnii, with both containing myb-like recognition motifs and ABA-responsive elements (Navarro et al., 2009Navarro M, Marque G, Ayax C, Borges JP, Marque C and Teullières C (2009) Complementary regulation of four Eucalyptus CBF genes under various cold condition. J Exp Bot 60:2713–2724.).

Apart from the differential regulation of a myb-like transcription factor, a protein identified as a receptor for activated C kinase (RACK) was also down-regulated in E. urograndis stems during cold stress (spot 92). In Arabidopsis, RACK1 genes seem to negatively regulate ABA responses by direct interaction with the eukaryotic initiation factor 6, a key regulator of the 80S ribosome assembly (Guo et al., 2009aGuo J, Wang S, Wang J, Huang WD, Liang J and Chen JG (2009a) Dissection of the relationship between RACK1 and heterotrimeric G-proteins in Arabidopsis. Plant Cell Physiol 50:1681–1694.,bGuo J, Wang J, Xi L, Huang WD, Liang J and Chen JG (2009b) RACK1 is a negative regulator of ABA responses in Arabidopsis. J Exp Bot 60:3819–3833., 2011Guo J, Wang S, Valerius O, Hall H, Zeng Q, Li JF, Waeston DJ, Ellis BE and Chen JG (2011) Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid. Plant Physiol 155:370–383.). Although the mechanisms that mediate ABA/RACK1 stress responses are still unclear, Speth et al. (2013)Speth C, Willing EM, Rausch S, Schneeberger K and Laubinger S (2013) RACK1 scaffold proteins influence miRNA abundance in Arabidopsis. Plant J 76:433–445. suggested that RACK1 could interfere in ABA related responses by repressing or stimulating the synthesis of microRNAs. A direct correlation between RACK1 and temperature adaptation has been proposed by Ullah et al. (2008)Ullah H, Scappini EL, Moon AF, Williams LV, Armstrong DL and Pedersen LC (2008) Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana. Protein Sci 17:1771–1780. who reported that Arabidopsis plants exposed to elevated temperature showed a reduction in RACK1 protein expression. In addition, the status of sumoylation, a post-translational protein modification (PTM) of RACK1, could play an important role in the heat stress responses (Ullah et al., 2008Ullah H, Scappini EL, Moon AF, Williams LV, Armstrong DL and Pedersen LC (2008) Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana. Protein Sci 17:1771–1780.). As shown here, exposure to low temperature for only 24 h reduced the expression of RACK1 in E. urograndis stems, thereby corroborating a possible role for this protein in temperature-sensing signaling pathways.

Another E. urograndis stem protein that was down-regulated upon short-term cold stress was Eucgr.F02130.2, described as a 14-3-3 protein. Using a data mining approach, Furtado et al. (2007)Furtado EL, Rosa DD, Zamprogno KC, Marino CL, Wilcken CF, Velini ED, Mori ES and Guerrini IA (2007) 14-3-3: Defense and regulatory proteins coding by Eucalyptus genome. Sci Florestalis 73:9–18. identified four 14-3-3-like genes in the E. grandis genome. Large-scale transcript analysis detected 46 14-3-3 transcripts in juvenile xylem tissues of E. grandis (Carvalho et al., 2008Carvalho MCCG, Caldas DGG, Carneiro RT, Moon DH, Salvatierra GR, Franceshini LM, Andrade A, Celedon PAF, Oda S and Labate CA (2008) SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis. Tree Physiol 28:905–919.) and, using a 2D electrophoresis approach, Celedon et al. (2007)Celedon PAF, Andrade A, Meireles KGX, Carvalho MCCG, Caldas DGG, Moon DH, Carneiro RT, Franceschini LM, Oda S and Labate CA (2007) Proteomic analysis of the cambial region in juvenile Eucalyptus grandis at three ages. Proteomics 7:2258–2274. detected the up-regulation of five 14-3-3 protein spots in juvenile xylem in relation to three- and six-year old tissues. Interestingly, in E. urograndis stems, this protein was detected as down-regulated with the same expression profile in two distinct spots (101 and 103). The identification of the same protein in different spots may be explained by the existence of proteolytic cleavage products in the protein extract or by the presence of PTMs in the identified protein.

To examine the possible covalent attachment of protein Eucgr.F02130.2 to a phosphate group, we remined the tandem mass spectra acquired for spots 101 and 103 and compared them against the same protein database with a phosphorylation mass shift (+79.966 Da) set as a dynamic modification. Using this strategy, the peptide AAQDIAQADLASTHPIR was detected in both protein spots and possibly phosphorylated in spot 101 while not phosphorylated in spot 103 (Figure 3). Although the detection of this unique phosphopeptide could not explain the isoelectric mass shift observed between these spots (Figure 1), it was still strong evidence that Eucgr.F02130.2, described as a 14-3-3 protein, is a potential phosphorylation substrate in young E. urograndis stems exposed to cold stress. Despite the existence of transcript and protein evidence for the involvement of 14-3-3 proteins in xylem development, there have been no reports on the involvement of 14-3-3 proteins, or of their differential PTM status, in the Eucalyptus cold response.

Figure 3
Tandem mass spectra of the peptide AAQDIAQADLASTHPIR. The upper panel shows the nearly complete fragmentation pattern (with the acquisition of almost all y-ion series) of the non-phosphorylated precursor ion with m/z equal to 593.3096 (+3). The lower panel shows the fragmentation pattern of the same precursor ion with m/z equal to 619.9706 (+3).

Serine carboxylase-like proteins have traditionally been associated with protein turnover, although recent studies have shown that serine carboxylases share extensive sequence similarity with acyltransferases (Vogt 2010Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20.; Sasaki et al., 2013Sasaki N, Matsuba Y, Abe Y, Okamura M, Momose M, Umemoto N, Nakayama M, Itoh Y and Ozeki Y (2013) Recent advances in understanding the anthocyanin modification steps in carnation flowers. Sci Horti 163:37–45.). The E. urograndis down-regulated protein described as a serine carboxypeptidase1 (accession Eucgr.I01037.1) shared 49% similarity with the protein glucose acyltransferase from Solanum pennellii and S. berthaultii and 47% with a putative acyl-glucose-dependent anthocyanin acyltransferase from Delphinium grandiflorum. Acyl-glucose dependent anthocyanin acyltransferase catalyzes the acylation of anthocyanin pigments, one of the final steps in anthocyanin biosynthesis (Matsuba et al., 2008Matsuba Y, Okuda Y, Abe Y, Kitamura Y, Terasaka K, Mizukami H, Kamakura H, Kawahara N, Goda Y, Sasaki N, et al. (2008) Enzymatic preparation of 1-O-hydroxycinnamoyl-beta-glucoses and their application to the study of 1-O-hydroxycinnamoyl-beta-D-glucose-dependent acyltransferase in anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis. Plant Biotechnol 25:369–375.). Acylation of anthocyanin is thought to assist in the stabilization of this pigment (Cheynier et al., 2006Cheynier V, Duenas-Paton M, Salas E, Maury C, Souquet JM, Sarni-Manchado P and Fulcrand H (2006) Structure and properties of wine pigments and tannins. Am J Enol Vit 57:298–305.) and play a major role in tonoplast transportation (Gomez et al., 2009Gomez C, Terrier N, Torregrosa L, Vialet S, Fournier-Level A, Verriès C, Souquet JM, Mazauric JP, Klein M, Chenyier V, et al. (2009) Grapevine MATE-type proteins act as vacuolar H+-dependent acylated anthocianin transporters. Plant Physiol 150:402–425.). The potential down-regulation of an anthocyanin acyltransferase suggests a reduction in anthocyanin vacuole intake and is likely related to a decrease in the cytoplasmic biosynthesis of this secondary metabolite. This hypothesis agrees well with the proposed reduction in E. urograndis energy-related metabolism, as also indicated by the down-regulation of fructose bisphosphate aldolase (spot 16), glyceraldehyde 3-phosphate dehydrogenase (spot 93) and NADH-ubiquinone oxidoreductase (spot 98) noted above, and the down-regulation of the myb-related protein spot (spot 87). In some plant species, myb transcription factors have been implicated in anthocyanin biosynthesis and storage (Cutanda-Perez et al., 2009Cutanda-Perez MC, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C and Torregrosa L (2009) Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport. Plant Mol Biol 69:633–648.; Li et al., 2012Li L, Ban ZJ, Li XH, Wu MY, Wang AL, Jiang YQ and Jiang YH (2012) Differential expression of anthocyanin biosynthetic genes and transcription factor PcMYB10 in pears (Pyrus communis L.). Plos One 7:e46070.; Singh et al., 2014Singh R, Low ETL, Ooi LCL, Ong-Abdullah M, Nookiah R, Ting NC, Marjuni M, Chan PL, Ithnin M, Manaf MAA, et al. (2014) The oil palm VIRESCENS gene controls fruit colour and encodes a R2R3-MYB. Nat Commun 5:4106.).

Eucalyptus urograndis stems also showed inhibition of the expression of proteins related to cell division and elongation. Gel spot 91, which contained the protein identified as cytochrome b5-like heme/steroid binding domain protein (Eucgr.C04037.1), showed down-regulation upon short-term cold treatment. In Arabidopsis thaliana, over-expression of the membrane steroid binding protein 1 resulted in shortened hypocotyls, suggesting a close relationship between cell elongation and steroid binding proteins (Yang et al., 2005Yang XH, Xu ZH and Xue HW (2005) Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation. Plant Cell 17:116–131.). In addition, the Arabidopsis rlf-1 (reduced lateral root formation) mutant showed reduced cotyledon growth and cell division inhibition, indicating that the RLF gene, which encodes a protein with a cytochrome b5-like heme/steroid binding domain (At5g09680), positively modulates cell division (Ikeyama et al., 2010Ikeyama Y, Tasaka M and Fukaki H (2010) RLF, a cytochrome b5-like/steroid binding domain protein, controls lateral root formation independently of ARF7/19-mediated auxin signaling in Arabidopsis thaliana. Plant J 62:865–875.). In accordance with these studies, the protein endo-1,3;1,4-β-D-glucanase, reported to be involved in cell elongation (Zhu et al., 2006Zhu J, Chen S, Alvarez S, Asirvatham VS, Schachtman DP, Wu Y and Sharp RE (2006) Cell wall proteome in the maize primary root elongation zone. I. Extraction and identification of water-soluble and lightly ionically bound proteins. Plant Physiol 140:311–325.; Guillaumie et al., 2008Guillaumie S, Goffner D, Barbier O, Martinant JP, Pichon M and Barrière Y (2008) Expression of cell wall related genes in basal and ear internodes of silting brown-midrib-3, caffein acid O-methyltransferase (COMT) down-regulated, and normal maize plants. BMC Plant Biol 8:e71.; Komatsu and Yanagawa, 2013Komatsu S and Yanagawa Y (2013) Cell wall proteomics of crops. Front Plant Sci 4:17.), was found to be down-regulated in E. urograndis stems after short-term exposure to low temperature stress; this finding corroborated the hypothesis of a negative relationship between cold stress and cell elongation in E. urograndis stems.

Conclusions

In this work, we used a proteomics approach to identify proteins potentially involved in the E. urograndis cold stress response. Despite using a low-throughput/low sensitivity proteomics strategy it was possible to detect the differential regulation of 12 proteins potentially involved in a quick metabolic response of E. urograndis stems after short-term exposure to low temperature. The proteins identified here could be useful markers for monitoring cold stress in this species or as potential targets in molecular-based programs aimed at expanding the use of this species in forest plantations.

  • Supplementary Material
    The following online material is available for this article:
    • Table S1 Experimental data for E. urograndis stem protein isolation and 2D gel analysis.

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

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant nos. 2011/11650-0 and 2011/51949-5). G.A.L., N.A.C. and T.S.B. were supported by research fellowships from FAPESP (grant nos. 2013/06370-4, 2013/06352-6, 2011/23582-0).

  • Associate Editor: Dario Grattapaglia

References

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Internet Resources

Publication Dates

  • Publication in this collection
    Apr-Jun 2015

History

  • Received
    04 Aug 2014
  • Accepted
    07 Nov 2014
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