Combining SSH, HDCA, cDNA macroarrays and real-time RT-PCR to identify genes regulated during bud burst
We used SSH and HDCA to generate and isolate cDNA clones of genes regulated during bud burst in oak. This approach was reported as very efficient to rapidly identify differentially expressed genes (e.g. SSH and cDNA microarray used by Yang et al. (1999), or SSH, SH and differential screening used by Nakata & McConn, 2002)). In our case, the combination of SSH, HDCA and cDNA macroarrays has the obvious advantage of reducing the number of differentially expressed clones allowing for a closer look on the remaining ones. First, SSH was performed to ensure the isolation of specific and rare transcripts. Then, the HDCA permitted to reduce by half the number of clones, by selecting those that showed a significantly different expression between different stages of bud development. Finally, cDNA macroarrays highlighted genes highly regulated during bud burst. By using this stepwise procedure, we combined the advantages of global and more targeted gene discovery approaches. While thousands of clones were retained by the SSH, HDCA screening procedures reduced this number to several hundred (801 clones). Molecular pathways highly regulated during bud burst were thus highlighted.
Real-time RT-PCR experiments validated the results obtained by reverse-Northern blot. Despite problems inherent in quantification using this technique (Bustin, 2002; Vandesompele et al., 2002), real-time RT-PCR has been used as a powerful tool to validate arrays results (Chtanova et al., 2001; Jones et al., 2002; Pfaffl et al., 2003). Although some discrepancies occurred, differences of expressions were of similar magnitude to those obtained using reverse-Northern blot. The use of RT-PCR has been reported in animal physiology, endocrinology, immunology, virology, microbiology (Hopkins, 2002; Pfaffl et al., 2003; Schams et al., 2003; Pinzani et al., 2004; Stram et al., 2004; Thomas et al., 2004) and plant science (Khan & Shih, 2004; Thomas et al., 2004). It has been described as a technique of choice to assess the expression levels of gene transcripts, and preferred to the more traditional macroarray methods (Walker, 2002). In our study, the observed differences of expression patterns between both techniques could be explained by the amplification phase used to synthesize double-stranded cDNA in the case of macroarray radioactive targets. Although PCR reactions on single cDNAs were stopped during the exponential phase, some transcripts could have been artificially favoured or disfavored. Moreover, some differences could be caused by cross-hybridizations between genes from multigene families that probably occur on arrays, contrary to real-time RT-PCR, where primers are gene-specific.
As described in the original paper by Diatchenko et al. (1996), SSH is efficient ‘for generating cDNAs highly enriched for differentially expressed genes of both high and low abundance’. On one hand, six out of the 10 genes analysed by real-time RT-PCR were highly differentially expressed between stages 0 and I, namely LEA5 (Fig. 7a), galactinol synthase (Fig. 7b), DAG2 (Fig. 7c), alpha-amylase/subtilisin inhibitor (RASI) (Fig. 7f), At2g14910 (Fig. 7h) and At4g24120 (T19F6.8) (Fig. 7i). On the other hand, RASI and LEA5 transcripts were sixfold and 1000-fold more abundant than the control gene in the quiescent stage, respectively, whereas DAG2 or Zwh21.1 transcripts were 55-fold less abundant than the 60S ribosomal protein transcript used as control. These results provide candidate genes with potential roles in bud burst. It should be noted that several genes described as regulating seed germination, dormancy or bud burst were identified (e.g. DAG2 and SKP1) among the 10 genes assessed by real-time RT-PCR. This finding constituted the first level of validation for these candidate genes. Indeed, the DAG2-like zinc finger protein isolated in our study, has been hypothesized to act on a maternal switch that controls seed germination in Arabidopsis thaliana (Gualberti et al., 2002), and seed and bud dormancy have been hypothesized to involve similar processes (Rohde et al., 2000). For its part, S-phase kinase-associated protein 1 A (SKP1) is a component of the SCF complex necessary to trigger the G1 to S-phase transition in yeast, and possibly in plants (Horvath et al., 2003). Knowing that Devitt & Stafstrom (1995) found that cells in paradormant buds are arrested in G1 and at the G2/M boundary, SKP1 constitutes a relevant candidate gene for bud burst.
Molecular mechanisms revealed at the onset of bud burst
This work also provided tools to dissect molecular mechanisms regulated during bud burst. Although a majority of isolated transcripts were expressed during the entire process, some were specific for a particular stage or at least significantly up- or down-regulated at a specific stage. Because the monitoring of bud burst extended from the fully quiescent stage (0) to the stage of elongating shoots (V), transcript accumulation along the sequence not only changed due to bud burst but also due to elongating tissues. However, the morphological and cytological variation (Figs 1 and 3) of the bud clearly suggested that the transition between stage 0 and I corresponded to the release from ecodormancy. Potential roles of specific transcripts of stages 0 and I (i.e. the quiescent and the swelling bud) are discussed later. Conversely, genes preferentially expressed at the end of bud burst will not be considered as candidate genes for bud burst, as they appeared to be more related to leaf development than to bud burst.
Cell rescue and defense-related genes were expressed during the quiescent stage and at the onset of bud burst. Desiccation stress obviously occurred at the quiescent stage, as exemplified by LEA5 expression pattern (Fig. 7a). Indeed, although their precise biological function remains obscure (Wise & Tunnacliffe, 2004), transcripts encoding LEA proteins have been isolated in a wide range of vegetative tissues of plants under moisture stress. In addition to LEA proteins, dehydrin-like and heat-shock transcripts were found to accumulate at the onset of flushing, probably acting to protect cells from desiccation and temperature stresses, through accumulation of dehydrins and heat-shock proteins. Transcripts of galactinol synthase also increased at the onset of bud burst (Fig. 7b). Galactinol synthase catalyses the first step in the biosynthesis of raffinose family oligosaccharides (RFO) from UDP-galactose. These RFOs are thought to play a role in the desiccation tolerance of seeds, and galactinol synthase has already been reported to be involved in drought and heat-stress tolerance (Pukacka & Wojkiewicz, 2002; Taji et al., 2002; Zhao et al., 2003). As a result of these different mechanisms, the integrity of both membranes and proteins may be maintained in the bud tissues. It is also well known that water stress enhances the production of reactive oxygen species and increases susceptibility to pathogens (Bray et al., 2000). On one hand, we found that oxidative stress, counteracted by the activation of catalase, acting in free radical removal, was activated during bud break. Moreover, the regulation of the osmotic potential by the accumulation of dehydrins, LEA, or sugars could also contribute to the protection of cells against oxidation (Rhodes & Hanson, 1993). On the other hand, the Endochitinase PR4 precursor gene was upregulated during quiescent and early stages and may contribute to defense against pathogens.
Expression of histones H3 and H4, as well as that of putative transcription factors related to DAG2 (Fig. 7c) and MONOPTEROS from Arabidopsis thaliana, were found to be induced at the onset of bud burst. While elevated expression of histones H3 and H4 appears to reflect increased cell division activity in general, induction of some distinct transcription factor-like genes might provide some clues about developmental processes specifically taking place during bud burst. DAG2, by contrast, was shown to act as a transcription factor specifically involved in the maternal control of seed germination (Gualberti et al., 2002). In buds, as in seeds, it may potentially act on dormancy release, supporting the existence of a common basis for the control of seed and bud dormancy (Rohde et al., 2000), as first hypothesized by Wareing (1956). In line with this model, we also found that expression of a homologue of MONOPTEROS appears to be induced during bud burst. MP is an auxin response factor (ARF) that seems to act as a transcriptional activator, required for the control of axis formation in the embryo and in auxin-dependent cell expansion (Hardtke & Berleth, 1998; Hardtke et al., 2004). A similar role could be attributed to the MP homologue found in oak buds. By analogy to its counterpart in Arabidopsis, this gene might transduce auxin signals, essential for early developmental events occurring during bud burst. Expression of presumptive cell cycle-regulators was also affected by bud burst. Indeed, SKP1 (Fig. 7d) and pollen-specific protein SF21 (Fig. 7e) were upregulated at stage IV. This result may indicate that cell division and differentiation are shifted during bud burst: cell division occurring during the first stages and both cell division and differentiation at stage IV. Conversely, expression of SKP1 remained constant between quiescent and early active stages, as previously observed for the transition to dormancy for cambial meristems (Schrader et al., 2004). There was no gene related to epigenesis/cold requirement found to be regulated in our experiment.
Activity of glycosyl hydrolases, such as glucan endo-1,3-β-glucosidase and cyanogenic β-glucosidase precursor, were also enhanced at the quiescent and early stages. These enzymes are induced by gibberellic acid and are known to play a role in both cell-wall mobilization and cell elongation (Hrmova & Fincher, 2001), through hydrolysis of glycosidic bonds linking cell-wall components. Induction of these enzymes during bud burst could thus reflect the initiation of outgrowth taking place in early stages of bud burst. By contrast, another regulator of carbohydrate modification, namely an α-amylase/subtilisin inhibitor was highly expressed at the quiescent stage only (Fig. 7f), suggesting that hydrolysis of storage starch or glycogen is repressed in the quiescent bud. The observed reduction in the expression of this gene upon bud swelling would indicate the onset of starch mobilization at this developmental stage.
In general, an increase in the expression of genes essential for energy supply could be observed at the end of bud burst, as shown by expression patterns of transcripts encoding for photosystem II reaction center M protein, cytochrome B6-F complex, plastocyanin, oxygen-evolving enhancer protein 2, ATP synthase ɛ -chain, that were all classified into the Group III. In parallel, RubisCo expression was also upregulated, as well as that of glyceraldehyde-3-phosphate dehydrogenase (Fig. 7g), as previously reported by Wang et al. (1991). Indeed, these authors demonstrated that enzyme activity of the glycolytic pathway increased at the release of dormancy. Regarding the developmental stages used in this study (Fig. 1), this increase in the expression of energy-related transcripts is undoubtedly caused by leaf development, which starts at stage III, but mainly develops at stages IV and V.
In addition to these known genes, several unknown transcripts were shown to be differentially expressed using real-time RT-PCR (Fig. 7h–j). Two of these hypothetical proteins, T19F6.8 and Zwh21.1, contain domains specific for oligo-peptide transporters and F-box proteins, respectively. A presumptive function of these genes in the control of bud burst remains to be determined.