File S4
Extended discussion
Cell-wall synthesis and strengthening may be temporarily inhibited while cell-wall degradation/loosening may be induced
It has been suggested that the above-discussed hormonal changes induce modulations in cell-wall metabolism that allow loosening of the cell wall and cell extension. Indeed, low-oxygen conditions have been found to induce the expression of extensin genes, which are believed to be a major determinant of cell expansion (Lasanthi-Kudahettige et al., 2007; Steffens et al., 2006).
In the current study, significant enrichment of cell-wall functions was evident in the downregulated gene set. Downregulation of genes encoding enzymes for pectin and cellulose biosynthesis suggest that wall synthesis may be inhibited. Three isoforms of cellulose synthase were downregulated as well. In barley, high cellulose synthase transcript levels were recorded in the maturation zone of stems and roots, and lower levels in developing grain, floral tissues and the elongation zone of leaves, suggesting that cellulose synthesis is induced only when the cells stop expanding (Burton et al., 2004).
Two isoforms of endo-1,4-beta-glucanase (EGase) were reduced as well. Interestingly, membrane-anchored EGase (KOR) is involved in cellulose biosynthesis in both primary and secondary cell walls of Arabidopsis (Nicol et al., 1998). Mutations in membrane-anchored EGases in Arabidopsis result in defects in cell-wall formation and reduced cellulose content (Lane et al., 2001; Szyjanowicz et al., 2004). Co-regulation of KOR and three cellulose synthase genes has been recorded in aspen as well (Bhandari et al., 2006).
A reduction was also found in the transcript level of a UDP-D-glucuronate 4-epimerase gene, which catalyzes the conversion of UDP-D-glucuronate to UDP-D-galacturonate, an activated precursor necessary for pectin synthesis (Usadel et al., 2004).
Downregulation was also documented in pectinacetylesterase and proline-rich proteins, which may testify to a reduction in wall-strengtheningactivity. Pectinacetylesterase activity contributes to loss of cell-wall extensibility by enhancing aggregation processes between galacturonan chains inside the apoplasm (Breton et al., 1996). Proline-rich proteins are structural components of the cell wall which play a role in its strengthening (Cassab, 1998).
Cellulose synthesis and deposition begin when the cells stop growing, leading to cell-wall thickening (Milioni et al., 2001). Downregulation of functions related to this process might be necessary during cell enlargement, which involves cell-loosening activities. Accordingly, downregulation of genes involved in cell-wall synthesis and strengthening was accompanied by upregulation of several functions related to cell-wall loosening.
Expansin, which is expressed in expanding tissues and is involved in cell-wall loosening (Cosgrove, 1999; Li et al., 2003), was upregulated by both stimuli. Induction was also evident for pectin methylesterase and polygalacturonase genes, responsible for pectin degradation. Pectin methylesterase not only demethylates pectin to produce substrates for polygalacturonase, but also reduces pH via proton release when methoxyl groups of pectin are converted to carboxyl groups. This change in pH has been proposed to control the activity of other cell-wall-degrading enzymes that are optimally active at low pH, thereby facilitating cell expansion and growth (Nari et al., 1986). In addition, a beta-1,3 glucanase gene, which has been reported to function in the removal of extracellular 1,3-β-D-glucan around plasmodesmata, was induced by the treatments. Interestingly, induction of birch apex meristem dormancy was accompanied by the formation of 1,3-β-D-glucan-containing sphincters on all plasmodesmata (Rinne and van der Schoot, 1998), while stimulation of bud dormancy release by chilling was accompanied by restoration of the symplasmic organization of the meristem, mediated by 1,3-β-D-glucanase (Rinne et al., 2001). The upregulation of 1,3-β-D-glucanase observed in the current study raises the possibility that artificial dormancy-release agents, such as HC and HS, lead to a similar restoration of the symplasmic organization of the grape bud meristem. Interestingly, an ethylene-responsive element binding protein (EREBP) has been reported to be involved in the regulation of glucanase during tobacco seed germination (Leubner-Metzger et al., 1998).
Taken together, our data suggest that cell-wall loosening may be induced by the treatments while cell-wall synthesis and strengthening are inhibited. Reorganization of cell-wall metabolism has also been seen during dormancy release of raspberry buds (Mazzitelli et al., 2008), including upregulation of an extensin gene, which concurs with the induction of loosening activity.
Cell-cycle machinery is probably turned on during the process of dormancy release
Previous studies have suggested that endodormant cells are predominantly arrested in the G1 phase of the cell cycle. Entry of the cells into the S phase may indicate that this arrest has been removed (Rohde et al., 1997). Histone mRNA accumulation is closely correlated with the S phase and is considered a good marker for proliferating cells (Fobert et al., 1994). The observed upregulation of histones H2A and H2B may thus support entry into the S phase in response to HS and HC stimuli. Upregulation of histone genes has also been documented during dormancy release of raspberry (Mazzitelli et al., 2008) and leafy spurge(Horvath et al., 2002) buds.
Chromosome condensation during the mitotic phase is influenced by post-translational modification of histones (Buchanan et al., 2000). Accordingly, we recorded upregulation of HDA2, a subunit of histone deacetylase complex 1 (Wu et al., 2001; Kanta et al., 2006), and downregulation of a SET domain-containing protein, which shares high similarity with the histone-lysine N-methyltransferase that functions in histone methylation (Baumbusch et al., 2001). This may testify to an influence of the treatments on the status of histone acetylation/methylation. In agreement with this, changes in DNA methylation and histone multi-acetylation have been reported during dormancy release of potato tubers (Law and Suttle, 2003, 2004).
The observed upregulation of tousled-like protein kinase further supports cell-cycle activation, since its activity is linked to active DNA replication during the S phase (Mello and Almouzni, 2001; Tyler, 2002; Carrera et al., 2003). Cell-cycle activation could also be inferred from upregulation of a gene encoding a CRN protein. CRN proteins, encoded by CDC16 and CDC23, are required for progression through the G2/M transition of the cell cycle in yeast (Icho and Wickner, 1987; Hirano et al., 1990).
Pin1-type peptidyl-prolyl cis/trans isomerase facilitates protein folding and is required for both normal mitotic progression and re-entry into the cell cycle from quiescence in yeast and animal systems (Joseph et al., 2003; He et al., 2004). In this study, Pin1 was induced by both stimuli, consistent with the hypothesis that the treatments may induce re-entry of the cells into the cell cycle. Some genes which function in chromosome assembly and segregation during the mitotic phase were also induced by both stimuli, indicating that cell division begins within the bud following the treatments. Two chromosome segregation-related genes, encoding a kinesin-like protein and nuclear matrix constituent protein 1 (NMCP1), as well as a phragmoplastin gene, were upregulated. The kinesin-like protein is a homolog of a microtubule motor protein that functions in early spindle assembly during the mitotic phase in Arabidopsis (Ambrose and Cyr, 2007). The NMCP1 gene has been related to chromosome segregation, based on its chromosome-segregation ATPase domain (Masuda et al., 1997), and phragmoplastin is a dynamin-like protein that is associated with cell-plate formation in plants (Gu and Verma, 1996). These data suggest that cell division starts following the treatments.
Interestingly, a Mob1-like protein was downregulated. Mob1-like proteins are localized in the cell-division plate during cytokinesis in alfalfa root tip (Citterio et al., 2005). In Drosophila, some MOB superfamily proteins have growth-inhibitory functions (Lai et al., 2005) and their expression in alfalfa reproductive tissues has been related to a potential growth-inhibitory effect (Citterio et al., 2005). Downregulation of a grape Mob-1-like homolog by both stimuli may be related to the removal of growth inhibition during dormancy release.
Rearrangement of amino acid and protein metabolism in response to dormancy-release stimuli
Rearrangement of amino acid and protein metabolism in the treated grape buds can be clearly inferred from the significant enrichment of the chaperone, ubiquitination and protein-degradation categories (CH, UB, PA, respectively) in the upregulated gene set, and from the regulation mode of selected genes involved in amino acid metabolism.
Protein folding, ubiquitination, and degradation
Under normal conditions, the coordinated machinery of heat-shock 70 chaperones (Hsp70, Dnak) and their co-chaperones (e.g. DnaJ/Hsp40 and GrpE) assists in the folding of de-novo synthesized polypeptides and in the import/translocation of precursor proteins (Hartl, 1996). Under stress conditions, however, the machinery of these heat-shock chaperones and co-chaperones is involved in facilitating the refolding and proteolytic degradation of non-native proteins (Wang et al., 2004). The Hsp90 heterocomplex, which also plays a key role in protein degradation and trafficking, is stabilized by the co-chaperone p23 (Queitsch et al., 2002). The upregulation of two isoforms of DnaJ, an isoform of Hsp70 and an isoform of p23, under the stress conditions developed in the treated buds may testify to post-translational modifications, trafficking and degradation of proteins in response to HS and HC stimuli. This is in agreement with the upregulation of Ca2+-dependent calreticulin/calnexin chaperones (Pang et al., 2007), the significant upregulation of some small GTP-binding proteins (Keilin et al., 2007 and current study) and the over-representation of the UB category in the upregulated gene set, all responsible for regulation of vesicle trafficking and protein degradation.
Within the latter category, several ubiquitin and polyubiquitin genes, as well as a protease that cleaves polyubiquitin, were upregulated, providing the cell with an ample supply of ubiquitin monomers (Callis et al., 1995). Induction was also evident for genes encoding the major proteins in the ubiquitination machinery (ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2 and several isoforms of ubiquitin protein ligase E3). Similarly, induction of genes coding for polyubiquitin and enzymes of the ubiquitination machinery has been recorded during chilling-induced dormancy release of raspberry buds (Mazzitelli et al., 2007).
In yeast, interaction of the Cdc48 complex with an endoplasmic reticulum (ER) membrane-bound ubiquitin ligase is mediated by a UBX-domain protein (Ubx2), and has a central role in ER-associated protein degradation (ERAD) of misfolded polypeptides in the ubiquitin proteasome system (Neuber et al., 2005). Upregulation was recorded in the treated buds of genes encoding Ubx2 and all the components of the Cdc48 complex, including Cdc48, Npl4 and Ufd1 (Hitchcock et al., 2001). Therefore, it is suggested that the Cdc48-dependent ubiquitination pathway may be involved in ERAD in the treated buds. Possible activation of ERAD may be supported by the upregulation of the two Ca2+-binding chaperone genes, calnexin and calreticulin, in the buds. These proteins are located in the ER, where they operate the “quality control” processes of protein modification (Michalak et al., 2002).
The induction of SKP1 and F-box protein genes in the treated buds may indicate that SCF complex-dependent ubiquitination is induced. The latter is involved in cell-cycle regulation, plant-hormone signal transduction, organ development and plant defense (Lechner et al., 2006). Physical association of SGT1 (suppressor of the G2 allele of skp1) with SCF complex is required in yeast for both the G1/S and G2/M transitions in the cell cycle (Kitagawa et al., 1999). Induction of the SGT1 gene in the treated buds supports the possibility that SCF complex-dependent ubiquitination is induced and may be involved in activation of the cell cycle.
Rearrangement of amino acid metabolism may testify to an increase in OAA availability and induction of ethylene production
Downregulation of the phosphoglycerate dehydrogenase gene, involved in the transformation of phosphoglycerate to serine, may concur with a need for the final products of glycolysis for energy production and NAD+ regeneration. However, upregulation was evident for glutamine synthetase, aspartate transaminase and phosphoserine aminotransferase, involved in the biosynthesis of glutamine, aspartate and serine, respectively (Buchanan et al., 2000; Hart et al., 2007). Interestingly, under anoxia there is upregulation of aspartate aminotransferase, which channels amino acids to and from glycolysis via OAA (Buchanan et al., 2000; Lasanthi-Kudahettige et al., 2007). While glutamine synthetase and aspartate transaminase were upregulated in the treated buds, asparagine synthase was downregulated. A similar effect of upregulation of glutamine synthetase and downregulation of asparagines synthase expression has been recorded in cases of high sugar availability (Oliveira et al., 2001; Thum et al., 2003). This information suggests ties between possible upregulation of sucrose degradation and glycolysis and the recorded changes in amino acid metabolism.
Glutamine, glutamate, asparagine and aspartate serve as the nitrogen sources for the synthesis of other amino acids (Buchanan et al., 2000). Accordingly, upregulation was evident for glutamine amidotransferase, which catalyzes the fifth step in histidine biosynthesis (Fujimori and Ohta, 1998), as well as for amino acid acetyltransferase, which is involved in the synthesis of arginine from glutamate (Takahara et al., 2007).
Upregulation was also evident for cystathionine gamma-synthase, catalyzing the first step in the biosynthesis of methionine from cysteine and homoserine, the latter originating from aspartate (Buchanan et al., 2000; Hacham et al., 2006; Katz et al., 2006). On the other hand, adenosyl homocysteinase, which is part of another pathway that recycles S-adenosyl-methionine (SAM) to produce methionine (Buchanan et al., 2000), was downregulated. SAM is a precursor for ethylene biosynthesis (Buchanan et al., 2000). The coordinated upregulation of methionine production from homoserine and cysteine and downregulation of the pathway that recycles SAM to methionine is in agreement with upregulation of ethylene biosynthesis. Interestingly, changes in ethylene metabolism and signaling have been documented under low-oxygen conditions (Benschop et al., 2006; Steffens et al., 2006; Lasanthi-Kudahettige et al., 2007) and seem to occur in the grape bud as well, in response to both stimuli (see below).
Plant growth regulators metabolism and signaling
The roles of plant-growth regulators during bud dormancy release are not yet clear (Saure, 1985; Faust et al., 1997; Horvath et al., 2003; Olsen, 2003). In the present study, most of the genes related to plant-hormone synthesis, signaling and response were downregulated
Interestingly, a homolog of Auxin Independent 1 (AXI1), which is induced by auxin and functions in cell division in tobacco protoplasts (Walden et al., 1994; Harling et al., 1997), was induced. However, there was both up- and downregulation of auxin transport and auxin efflux carriers, and both auxin-repressed and auxin-induced genes were downregulated. It has been previously assumed that auxins may be involved in processes related to bud burst and outgrowth, but not in dormancy release (Wood, 1983; Saure, 1985). However, recent data show an increase in auxin content in potato tuber buds from harvest until the end of the dormancy period, and strong upregulation of an auxin response factor (ARF6) at the end of the dormancy stage, in correlation with the overcoming of dormancy (Faivre-Rampant et al., 2004; Sorce et al., 2005). The current analysis is not definitive regarding the involvement of auxin in dormancy release.
Downregulation of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, which functions in cytokinin biosynthesis (Takahashi et al., 1998), and KNOTTED1-like homeobox protein, which increases de-novo cytokinin biosynthesis (Chen et al., 2004; Sakamoto et al., 2006), suggest that cytokinin biosynthesis is temporarily inhibited.
Downregulation was recorded for brassinosteroid (BR) insensitive 1 (BRIl) and brassinosteroid signaling positive regulator (BES1/BZR1) genes. BRIl is the receptor of BR (Clouse et al., 1996; Li and Chory, 1997) and BES1/BZR1 is a downstream component of BR signaling (Wang et al., 2002).However, increased expression of the BR biosynthesis enzyme DWF4 and greater amounts of BR are observed in the BR-insensitive mutants bri1 and bin2, suggesting that regulation of endogenous levels of BR is linked with the BR signal in a feedback control loop (Choe et al., 2002; Goda et al., 2002). Hence, downregulation of components of BR-signal transduction does not necessarily testify to downregulation of BR biosynthesis and BR-mediated gene expression, and may even reflect its induction. Since BRs induce cell elongation, further analysis of this issue is warranted.
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