Variability of tracheid dimension and cell wall chemical composition in response to variations in edapho-climatic conditions
Anatomical, chemical and physiological studies have shown that the consequences of environmental fluctuations such as drought stress can be tracked within the secondary xylem of forest tree species (Zahner, 1968; Liphschitz & Waisel, 1970; Barber et al., 2000). Wood can therefore be considered as a bio-marker of environmental changes. In this report, the analysis of wood properties of developing xylem (cell wall chemical composition, fibre morphology and metabolites) sampled during the 2003 growing season gave us a unique opportunity to study the phenotypic plasticity of differentiating xylem in a single P. pinaster genotype in response to changes in edapho-climatic conditions.
PCA (Fig. 7) suggested that differences in climatic conditions strongly influenced the observed annual pattern of cell wall chemistry and fibre morphology. Higher relative atmospheric humidity and precipitation promoted the accumulation of proteins and hemicelluloses, while less water promoted lignin and resinic acid biosynthesis. Moreover, higher SMD and temperature induced an increase in cellulose (cH7) accumulation.
Tracheid dimension also responded to variation in edapho-climatic conditions (see Fig. 2). A negative relationship was found between fibre length and SMD. The shorter tracheids of sample T5 also had higher cellulose content, probably reflecting a thicker cell wall, characteristic of LW-forming fibre. Pittermann et al. (2006a) reported that an increase in cell wall reinforcement was associated with a decrease in tracheid length, implying that stronger tracheids tended to be shorter. Pittermann et al. (2006b) also found that tracheid diameter was nearly optimized to achieve the greatest hydraulic efficiency for a given tracheid length. In contrast, the positive relationship that was found between lignin content and fibre width is probably the result of a higher proportion of primary cell wall (more lignin-rich) in EW-forming tracheids (i.e. in samples T1 and T2), rather than a causal correlation.
Resinic acids accumulated early in the season (T1 and T2 samples) and then dropped to very low levels for samples T3 and T4, and finally increased at T5. We attributed the reduction at T3 and T4 to the heavy rainfall before sampling date T3. This suggestion is supported by the finding that climatic conditions affect the accumulation of secondary metabolites and particularly resinic acids in developing xylem. In conifers, it is well known that water deficit causes the accumulation of constitutive terpenes and flow of constitutive resin (Lombardero et al., 2000; Turtola et al., 2003), as well as an increase in the number of specialized terpene secretory structures (Lewinsohn et al., 1991).
Molecular plasticity during the growing season
Using our stringent statistical criteria, we found that 19% of genes (667 genes) were differentially expressed during the 2003 growing season. A similar proportion was found by Egertsdotter et al. (2004) in Pinus taeda. Among these genes, 21.8% displayed a maximum:minimum ratio > 10, showing that the transcriptome of differentiating xylem was greatly affected during the annual course of wood formation.
Based on their expression profiles, differentially expressed genes were clustered into five groups. The two most important clusters (1 and 2) contained 83% of the genes, corresponding to genes differentially regulated in EW- and LW-forming tissues. Three minor clusters (3, 4 and 5) included fewer genes whose expression profiles could be interpreted as responsive to particular edapho-climatic conditions encountered during the study period.
In the following sections we will discuss some of the genes found in the contrasting clusters, referred to as ‘EW’ and ‘LW’ responsive genes for clusters 1 and 2, respectively, as well as genes belonging to clusters 3, 4 and 5, which we refer to as ‘specific edapho-climatic condition’ responsive genes.
EW responsive genes A total of 95 genes were found to be over-expressed early in the season (cluster 1) with a fold-change ratio > 10, of which six (whose expression profiles were analyzed by qPCR; Supporting Information Fig. S2) presented ratios > 100. These included the following.
Three genes encoding metallothionein-like proteins (MTs; BX249412 (302-fold, validated by qPCR; Supporting Information Fig. S2b), BX249603 (32.9-fold) and BX252580 (14.7-fold)) were over-expressed in EW-forming tissues. MTs play a role in detoxification of heavy metals and in homeostasis of intracellular metal ions (Cobbett & Goldsbrough, 2002). Although their exact function is still not completely understood, it seems that MTs may be expressed as part of a general stress response. Bhalerao et al. (2003) and Andersson et al. (2004) reported that MTs are induced during leaf senescence. Xylogenesis is characterized by the genetically programmed loss of cell structure and metabolic function, leading to cell death (Fukuda, 1996). We suggest that MTs could play a crucial role as metal chelators, in protecting differentiating xylem cells from the toxic effects of metal ions released during the lignification and programmed cell death (PCD) steps.
A glutamate-ammonia ligase (also glutamine synthetase (GS); EC 220.127.116.11; BX253698; 202-fold) was also validated by qPCR (Supporting Information Fig. S2c). This enzyme is implicated in the nitrogen metabolism cycle. In actively lignifying cells the phenylpropanoid-N cycle involves the enzymes phenylalanine ammonia-lyase (PAL) (EC18.104.22.168), GS and possibly glutamine-oxyglutarate aminotransferase (GOGAT) (EC 22.214.171.124, EC 126.96.36.199), which has a major role in rapidly recycling the ammonium liberated by the PAL reaction. The high level of accumulation of GS at the beginning of the growing season is also consistent with the higher lignin content of samples T1 and T2 probably resulting from increased PAL activity. Indeed, one PAL (BX248906; 3.5-fold) was also found to be over-expressed during ‘EW’ formation.
An arabinogalactan/proline-rich protein (AGP; BX249981; 100-fold, validated by qPCR; Supporting Information Fig. S2d) belongs to a family of proteins in P. pinaster that have been found to be abundantly expressed in wood-forming tissues (Paiva, 2006). Proteome analysis of P. pinaster wood-forming tissues also showed that one spot corresponding to an AGP was identified as an ‘EW’ protein (Gion et al., 2005). Numerous potential roles of AGP during xylogenesis have been proposed, including roles in cell division and expansion (reviewed by Schultz et al., 2000), secondary cell wall initiation (Kieliszewski & Lamport, 1994), and PCD (Schindler et al., 1995, Greenberg, 1996). The over-expression of this putative AGP-like protein could be related to the higher rate of fusiform initials differentiating in spring.
A gene encoding a putative protein (BX249775; 153-fold, validated by qPCR; Supporting Information Fig. S2e) was also highly expressed in EW-forming tissues. It should be noted here that the length of this EST was very small (132 bp) which may explain its lack of homology with a gene of known function.
Two other genes, encoding a glycine-rich RNA-binding protein 7 (GR-RBP; BX252406; 101-fold) and an iron transport multicopper oxidase FET5 (MCO-FET5; BX252150; 126-fold), could not be validated by qPCR with the tested primer pair, because of low efficiency.
• Cell division-related genes
Genes encoding a cyclin A/CDK2-associated protein (BX255795) and cyclin-dependent kinase regulatory subunit 1 (CKS-1; BX251584) were over-expressed in EW-forming tissue and showed fold-change ratios of 10.8 and 5.2, respectively. These genes are known to be involved in the control of cell cycle progression (Horvath et al., 2003). This result indicates that cell division was probably highly activated at the beginning of the season, in agreement with Uggla et al. (2001).
• Energy-related genes
Among the genes up-regulated in EW-forming tissues, and related to energy production, we found a fructose-bisphosphate aldolase (EC188.8.131.52; BX249425; 58-fold, also validated by qPCR; Supporting Information Fig. S2f), a cytochrome c1 (BX254511; 32.3-fold), a naphthoate synthase (EC 184.108.40.206; BX250373; 11.5-fold), and a glyceraldehyde 3-phosphate dehydrogenase (G3PDH; EC 220.127.116.11; BX249100; 11-fold).
Fructose-bisphosphate aldolase (BX249425) was also found to be highly expressed in xylem compared with seven other tissues (Paiva, 2006). However, it should be noted that another transcript of fructose-bisphosphate aldolase (BX249029; 22.9-fold) was also found in the LW cluster (cluster 2), suggesting the presence of members of the same gene family with different levels of regulation in different types of wood-forming tissues.
G3PDH is implicated in primary metabolism, namely in energy production. Le Provost et al. (2003) previously showed that a transcript of this enzyme was up-regulated in EW-forming tissues.
• Genes involved in sugar transport and cell wall biogenesis
Sucrose has been shown to be the major carbohydrate of cambial metabolism (Krabel, 2000). In the 2003 growing season, we found that the polysaccharide content in the apolar fraction reached its maximum in sample T3. We also found that fructose and hexapyranose types increased from T1 to T3 in the polar metabolic fraction, followed by a decrease of sucrose (Fig. 6 and Supporting Information Table S4). At the molecular level, four transcripts showing carbohydrate transporter activity were found in cluster 1, including a D-xylose-proton symporter (D-xylose transporter; BX251928; 26.9-fold), a putative sugar transporter (BX250728; 24.3-fold), and two plasma membrane H+-ATPases (PM H+-ATPases; BX249881, 9.6-fold and BX253719, 7-fold). The PM H+-ATPase is a key enzyme that generates the proton-motive force that drives the uptake of nutrients such as sugars and ions across the plasma membrane of growing plant cells. It seems to be especially important in the uptake of potassium ions (Hoth et al., 1997). This uptake is essential for osmotic regulation and cell enlargement in differentiating tissues (MacRobbie, 1977; Hsiao & Läuchli, 1986). Arend et al. (2002) reported increased abundance of PM H+-ATPase in spring in cambial cells and expanding xylem. Paiva (2006) also reported that a gene coding for PM H+-ATPase (TC51926) was abundantly represented in the differentiating xylem library of P. pinaster. He also showed that the corresponding tentative consensus (TC51926) of the Pine Gene Index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=pine) was significantly over-expressed in xylem libraries when compared with other tissue libraries. A seasonal variation of PM H+-ATPase transcripts was also observed in the bud tissue of the peach tree (Prunus persica; Gévaudant et al., 2001) and in roots of Pinus sylvestris (Iivonen & Vapaavuori, 2002).
Intracellular traffic-related genes were found to be highly expressed in EW-forming tissues. These included an exportin 1 (BX249137; 94.6-fold), a GTP-binding protein SAR 1 (BX249833; 41.3-fold), and an ADP-ribosylation factor (BX253253; 26.8-fold).
We also found several genes implicated in the carbohydrate/polysaccharide metabolism of the cell wall to be strongly up-regulated in EW-forming tissues, namely, a dTDP-glucose 4,6-dehydratase (EC 18.104.22.168; BX252145; 75.3-fold), a cellulose synthase (PpinCESA3; BX250234; 38.5-fold), a glucan endo-1,3-beta-D-glucosidase (EC 22.214.171.124; BX249285; 35.5-fold), and an alpha-1,4-glucan-protein synthase (UDP-forming; EC 126.96.36.199; BX250805; 34.2-fold).
To conclude, the over-expression in EW of genes involved in cell wall carbohydrate metabolism, sugar transport and intracellular trafficking suggests that a substantial mobilization of carbohydrates occurred at the beginning of the growing season. Indeed, during cambial growth, the cambial region acts as a strong axial sink, which probably competes for minerals and assimilates with other sinks such as young leaves and roots (Dünisch & Bauch, 1994; Krabel, 2000).
LW responsive genes Fold-change ratios for LW-related genes were much lower compared with EW-related genes. In addition, for > 72.5% of the LW differentially expressed genes, we could not assign a known function, a proportion that was much higher than the average of the P. pinaster xylem ESTs, which was 53% (Paiva, 2006). A total of 37 genes were found to be over-expressed late in the season (cluster 2) with fold-change ratios > 10. Among these, 29 corresponded to putative proteins or proteins of unknown function.
Only three genes showed a maximum:minimum ratio > 100. These included two putative proteins (BX251778, 666 bp, 112-fold and BX253111, 122 bp, 165-fold) and one β-tubulin (BX249177; 129-fold). LW tracheids are characterized by a thicker secondary cell wall, which mainly consists of highly ordered cellulose deposits. Microtubules mainly consist of α-tubulin and β-tubulin, and are implicated, among other functions, in the orientation of cellulose microfibrils during the differentiation of tracheary elements (Chaffey et al., 1998; Spokevicius et al., 2007; Oakley et al., 2007). Interestingly, one F-actin capping protein alpha subunit (BX250456; 3.1-fold), which is involved in actin assembly, was found to be co-regulated with this β-tubulin. Together these results highlight the importance of cytoskeletal proteins in LW formation. Genes of the ‘transcription’ category were also well represented in cluster 2, which contained six genes from this category, including a gene encoding a zinc finger protein 216 (BX248938; 49.6-fold).
Effect of particular edapho-climatic conditions on gene expression Our sampling strategy also made it possible to detect modifications of the transcriptome (identified from peaks of transcript accumulation) in response to particular changes in edapho-climatic conditions.
Sampling date T2 was preceded by a long period (30 d) without effective precipitation (daily mean 0.5 mm and 18 d without rainfall), and with high temperatures for the time of year and continuous loss of SWC. LUE was maximal at T2 and we also observed an increase in transpiration rate, probably as a result of leaf area expansion. Additionally, a pronounced decline in stomatal conductance was predicted by the GRAECO model, suggesting that the trees were under drought stress. We therefore suggest that genes of cluster 3 (Fig. 8), which showed a peak of expression at T2, are involved in a physiological response to increasing loss of SWC. This cluster comprised genes coding for stress-responsive proteins, that is, three heat-shock proteins (BX255667, 5.4-fold; BX249170, 9.2-fold and BX251102, 42.5-fold), one protease (aspartic proteinase; BX249116; 15.8-fold), and an abscisic stress ripening protein (ASR; BX249387; 6.5-fold). A peptidylprolyl isomerase (BX2499248) showed the highest fold-change ratio of all differentially expressed genes of this study (469-fold). Peptidylprolyl isomerase is implicated in the ubiquitin-proteasome pathway. It should be noted that another peptidylprolyl isomerase (BX249687; 27.3-fold) and a ubiquitin-protein ligase (EC 188.8.131.52; BX248908; 11.1-fold) were also found in cluster 1, and were thus considered as EW-related genes. Together, these results suggest the direct recruitment of stress-responsive genes, as well as genes involved in the modification of the metabolic machinery, via the ubiquitin-proteasome pathway.
A cellulose synthase (PpinCesa1; 97% identical to PtCesa2; BX249248; 6.7-fold), a caffeate O-methyltransferase (COMT; BX254093; 3.0-fold), and a proline-rich protein (PRP; ortholog of PtaPRP1; 2.9-fold) were also found to be over-expressed in sample T2. PpinCesa1 is involved in cellulose biosynthesis in secondary cell walls, while COMT is implicated in lignification. PRPs have also been reported to be involved in lignification (Cassab, 1998). Indeed, PRPs may provide sites for selective complex formation with phenolic precursors, for example for initiation or polymerization of the lignin polymer (Whitmore, 1978; Zhang et al., 2000). The up-regulation of these cell wall biosynthesis-related genes could be related to the reinforcement and reduced permeability of the cell wall in response to the rapid decrease of SWC.
Heavy rainfall preceded T3 sampling, temporarily increasing SWC. This physiological state was reflected by genes of cluster 4, which showed a peak of expression at T3 (Fig. 8). This cluster was characterized by a high number of transcripts related to protein synthesis, consistent with the peak of protein content measured by FTIR spectroscopy and analytical pyrolysis. The fold-change ratios of six ribosomal genes were found to vary between 2.3 and 39. Another interesting gene encoded a GTP-binding nuclear protein RAN (BX249426; 61.0-fold), which is known to regulate the karyopherins, which are involved in nucleocytoplasmic transport, also indicative of high transcriptional activity occurring at T3.
Finally, the genes included in cluster 5 showed a peak of expression at T4 (Fig. 8). Their expression profile could be related to the sudden increase of SMD and abnormally high temperatures in late spring. Ten genes with known functions were found to have a fold-change ratio > 2, including a gibberellin (GA)-stimulated 5 (GASA5)-like protein (BX249894; 3.5-fold), NBS/LRR (BX254260; 3.1-fold), an arabinogalactan protein (AGP; BX255488; 2.9-fold), and a putative plasma membrane-associated protein (BX255221; 2.6-fold). Arabidopsis GASA is homologous to the original GA-regulated tomato (Lycopersicum esculentum) gene GA-stimulated 1 (GAST1) (Aubert et al., 1998), with GAST1 homologs implicated in cell division (Aubert et al., 1998), cell elongation (Taylor & Scheuring, 1994) and radial cell expansion (Kotilainen et al., 1999). Israelsson et al. (2005) reported a dramatic increase in expansion zones of wood-forming tissues where concentrations of bioactive GA were highest. The coincidence between a peak of this P. pinaster GASA5-like expression and an increase in fibre width also suggests a role for this gene in radial expansion of maritime pine wood-forming tissues. AGPs are cell wall proteins that have been implicated in many processes of plant growth, development and adaptation, including, cell proliferation, expansion and differentiation, and plant defence (reviewed by Majewska-Sawka & Nothnagel, 2000). AGPs have been reported to be among the most expressed genes in poplar (Populus spp.) and pine stems (Sterky et al., 1998, 2004; Paiva, 2006).
Concluding remarks and perspectives
In this paper, the metabolic profiling and cell wall chemical composition of wood-forming tissues and fibre morphology were assessed during a growing season. We found that differences in edapho-climatic conditions (mainly air temperature and soil water availability) strongly influenced the observed annual patterns of metabolite and chemical composition. Our results show that, in favourable eco-physiological conditions, trees channel carbon and energy towards growth and cell division, and increase protein and hemicellulose contents. Conversely, water deficit induced an increase in the allocation of carbon to cellulose and lignin biosynthesis, probably reflecting an increase in cell wall thickening for hydraulic conductance protection. In addition, transcript profiling provided us with new hints about the molecular players involved in wood formation, and their plasticity during the growing season. A step forward is to ask whether this plastic response has an adaptive value. A recent study in P. pinaster (Eveno et al., 2008) identified, among differentially expressed candidate genes for the drought stress response, genes that showed a departure from neutrality and demographic equilibrium, which is a first indication of the adaptive value of these genes.