Secondary vascular tissue regeneration after bark girdling in Populus
The bark-girdled trees regenerated new SVT within 12 d and formed new bark within 1 month in P. tomentosa (Fig. 1). In the growing season, the vascular cambium produces secondary phloem outwards and secondary xylem inwards. The surface of the exposed trunk was moist and smooth when the bark of P. tomentosa was removed (Fig. 1a). The cambium had been removed with the bark, leaving only differentiating xylem cells on the surface of the trunk (Fig. 2a,b). From 2 to 6 DAG, the girdled area of trunk became protuberant with a soft surface (Fig. 1b). Cross section revealed the formation of callus, resulting from the division of ray cells of differentiating xylem (diX) at 2 DAG (Fig. 2c). Axial diX cells under callus underwent periclinal and transverse divisions, indicating xylem cell dedifferentiation at 4 DAG (Fig. 2d,e,f). From 6 to 9 DAG, the surface of the girdled area turned green (Fig. 1c). Sieve elements were detected by aniline blue staining under fluorescence microscopy at 6 DAG (Fig. 2i), while cambium was not observed at this time (Fig. 2g,h). The sieve elements were present several cell layers under the isodiametric callus cells, arranged between ray cells (Fig. S1a,d) and connected longitudinally to each other (Fig. S1b), which is similar to what was observed in E. ulmoides (Pang et al., 2008). Moreover, typical phloem patterning was also observed tangentially (Fig. S1c). From 9 to 12 DAG, the girdled trunk became greener, dry and hard (Fig. 1d). Continuous and flat regenerated cambial cells were observed in several cell layers under the regenerated phloem at 12 DAG (Fig. 2j–l).
Figure 1. Morphology of girdled trunk during bark regeneration in Populus tomentosa. The surface of the exposed trunk was moist and smooth when the bark was removed (a). The surface of the girdled trunk became protuberant and soft at 4 d after girdling (DAG), resulting from callus formation (b). It became green at 6 DAG (c), and then became dark green, dry and hard at 12 DAG (d).
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Figure 2. Anatomical observation of secondary vascular tissue (SVT) regeneration after bark girdling in Populus tomentosa. Cambium has been removed with bark during girdling, leaving only differentiating xylem (diX) cells on the surface of the trunk, as shown in the cross section (a) and radial section (b). At 2 d after girdling (DAG), ray cells of diX started to divide and form callus cells (Cal) as shown in the cross section (c). At 4 DAG, more callus cells covered the wounded surface, and axial diX cells under callus underwent periclinal and transverse divisions; dedifferentiating xylem cells (deX) were shown in the cross section (d) and radial section (e). No sieve elements can be detected in deX at this time by aniline blue staining under fluorescence microscopy (f). At 6 DAG, sieve elements were detected by aniline blue staining (i), while cambium was not observed at this time in both cross section (g) and radial section (h). Some of deX under regenerated phloem (rPh) will differentiate into regenerated cambium (diC). At 12 DAG, continuous and flat regenerated cambium (rC) was observed in several cell layers in both cross section (j) and radial section (k) under rPh; it was also shown in an aniline blue-stained radial section (l). Arrowheads show sieve elements. Xy, xylem cells. Bars, 100 μm.
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Based upon our morphological observation of twice-performed girdling experiments, we divided the regeneration process into four stages: initially (Stage 0), differentiating xylem cells are left on the surface of the trunk after bark girdling (Figs 1a, 2a, b); Stage I, from 2 to 6 DAG, xylem cells dedifferentiate (Figs 1b, 2c–f); stage II, from 6 to 9 DAG, sieve element formation (Figs 1c, 2g–i); stage III, from 9 to 12 DAG, wound cambium formation (Figs 1d, 2j–l).
Transcriptome profiling indicates global changes of gene expression during SVT regeneration
For transcriptome profiling analysis, a series of 20-μm-thick tangential cryosections were collected at each stage of regeneration, as illustrated in Fig. 3(a). Five tissues, differentiating xylem (diX, stage 0), dedifferentiating xylem (deX, stage I), regenerated phloem (rPh, stage II), differentiating regenerated cambium (diC, stage II) and regenerated cambium (rC, stage III) were sampled for Affymetrix poplar whole-genome array hybridization (Fig. 3a).
Figure 3. Illustration of sampling procedure and differentially expressed gene (DEG) distribution and quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation of microarray data. (a) Schematic illustration of sampling procedure for microarray and qRT-PCR analysis. A series of 20-μm-thick tangential cryosections was taken for each sample: c. 60- to 80-μm-thick differentiating xylem cells (diX) at stage 0, 40- to 60-μm-thick dedifferentiating xylem (deX) at stage I, regenerated phloem (rPh) and differentiating cambium (diC) at stage II and regenerated cambium (rC) at stage III, as indicated by arrows. Bars with different patterning indicate various tissues (mx, mature xylem). (b) Illustration of the regeneration of phloem and cambium and four pairwise sample comparisons. (c) Numbers of DEGs identified to be up-regulated (striped bars) or down-regulated (filled bars) across distinct regeneration stages. Transcripts with lower confidence bounds (LCBs) ≥ 1.5 in at least one of the four comparisons were flagged as differentially expressed. (d) Comparison between the 90% LCB of fold change reported by microarray and fold change (FC) obtained by qRT-PCR. Genes with LCB > 2, close to 2, 1.5, 1 were chosen for validation. Data were obtained from 28 probe-sets across four comparisons (as shown in Fig. 1b). Log2FC for each comparison is plotted against log2LCB. The intercept of the linear regression line was set to pass through the origin.
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Four pairwise comparisons, (a)deX/diX, (b)rPh/deX, (c)diC/deX and (d)rC/diC, were performed (Fig. 3b). Transcripts with 90% LCBs ≥ 1.5 across at least one of the four comparisons were chosen as differentially expressed genes (DEGs). Extensive changes in gene expression were observed across all stages of regeneration (Fig. 3c). SYBR qRT-PCR of selected transcripts with different LCB values suggested a good correlation between qRT-PCR and microarray results (Fig. 3d). The large numbers of up- and down-regulated transcripts from diX to deX indicated dramatic transcriptome changes during the process of xylem dedifferentiation. On the other hand, much smaller changes in gene expression were observed from diC to rC, implying similar molecular features of the two samples (Fig. 3c). DEGs were then classified based on gene ontology (GO); the numbers of up- or down-regulated genes in each GO category are shown in Table 1 and Fig. S2. Genes associated with transcription, transport, stress response and response to hormone stimulus were strongly regulated during regeneration (Fig. S2). Based on the GO annotation, it can be seen that genes related to the chloroplast, plasma membrane, nucleus and endomembrane system were enriched (Fig. S2). Most of the GO biological process and cellular component categories had similar numbers of genes that were up- or down-regulated (Table 1 and Fig. S2). However, in early regeneration (dedifferentiation, deX/diX), genes related to cell cycle, DNA methylation and chromatin regulation were dominantly up-regulated while genes related to cytoskeleton and cell wall biogenesis and Golgi apparatus were strongly down-regulated (Fig. S2). During phloem and cambium regeneration (rPh/deX and diC/deX), there was a preponderance of up-regulated genes related to transcription (Table 1 and Fig. S2). In addition, genes involved in transport, stress and hormone response were dramatically altered during phloem reformation (Fig. S2).
Table 1. Gene ontology (GO) classification of differentially expressed genes during second vascular tissue regeneration
|GO category||Function category||(a) deX/diX||(b) rPh/deX||(c) diC/deX||(d) rC/diC|
|Response to stress||160||84||109||51||74||42||9||3|
|Response to hormone stimulus||110||102||67||44||45||31||10||2|
|DNA methylation and chromatin assembly or disassembly||37||6||6||22||7||1||0||1|
|Amino acid metabolic process||27||2||9||7||8||5||2||0|
|Programmed cell death and apoptosis||15||18||33||3||21||1||1||2|
|Cell wall biogenesis||3||34||5||4||2||0||1||0|
|Other intracellular components||81||74||26||21||32||6||1||1|
|Other cellular components||1283||1884||1022||551||892||375||142||77|
Xylem cells may acquire regenerative competence through epigenetic regulation and cell cycle re-entry
Investigation of the expression patterns of the related genes indicated that epigenetic regulation and cell cycle re-entry were involved in early SVT regeneration. Three DNA methyltransferases, METHYLTRANSFERASE 1 (MET1), DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), and CHROMOMETHYLASE3 (CMT3) (Finnegan & Kovac, 2000), and DEMETER (DME), a DNA glycosylase with DNA demethylation activity (Gehring et al., 2006; Morales-Ruiz et al., 2006), are involved in DNA methylation regulation in plants. Our data showed that one putative CMT3 and two MET transcripts were up-regulated, whereas two candidate DME genes were down-regulated in early regeneration in deX (Fig. 4a). The opposite expression patterns of putative DNA methyltransferases and DME suggested that DNA methylation regulation is involved during early SVT regeneration.
Figure 4. Transcript expression patterns of epigenetic regulation and cell cycle-related genes during Populus tomentosa secondary vascular tissue (SVT) regeneration. (a) Candidate genes involved in DNA methylation, histone methylation, histone acetylation, chromatin remodeling and polycomb group (PcG) proteins were selected and clustered. (b) Cyclins and cyclin-dependent kinase (CDK) genes with differential expression levels across all samples were identified and clustered. Expression values for each probe-set across all samples were standardized (linearly scaled) to have mean 0 and standard deviation 1 as indicated by red/green-colored squares. The coefficient of variation (CV) for each transcript across samples was used to indicate fluctuation in expression. Complete information for each probe-set in the gene lists can be found in the Supporting Information, Table S1.
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In addition to DNA methylation regulation, we found that the expression of histone modification enzymes was changed in SVT regeneration. Two histone demethylase families, amine oxidase (AOD) and Jumonji C (JmjC) domain-containing histone demethylase families, have been identified in the Populus genome (Zhou & Ma, 2008). Among these putative histone demethylases, six AOD domain-containing transcripts (PtAOD1B, PtAOD2B, PtAOD7A, PtAOD8B, and another two AOD family genes) and five JmjC domain-containing transcripts (PtKDM3D, PtPKDM11, PtPKDM8A, PtPKDM8B and PtPDKM7E) showed diverse expression patterns during regeneration (Fig. 4a and Table S1).
Two putative histone methyltransferases, HMT1 and HMT3, were also found to have expression peaks at diX and rC, respectively (Fig. 4a). In addition, probe-sets representing putative Populus histone acetyltransferase (HAT) and histone deacetylase (HDAC) genes (Pandey et al., 2002) were identified in our dataset. Four HAT (HAG2, HAG3, HAF2 and HAC4) and five HDAC (HDA6, 8, 14, 19 and one plant-specific HDAC, HDT1) transcripts were differentially expressed. However, they exhibited different expression patterns at early regeneration stages (Fig. 4a).
Among the homologous genes of chromatin remodeling-related proteins (Boyko & Kovalchuk, 2008) identified in our dataset, some of them showed significant changes in transcriptional level. In early regeneration, one group of genes consisting of DDM1 and LHP1 were up-regulated while another group of genes, including putative MBD2 and MBD8, MOM1 and one ISW2-like gene, were down-regulated (Fig. 4a).
Polycomb group (PcG) proteins and the Polycomb repressive complexes PRC1 and PRC2 play important roles during cell fate switch and regeneration (Costa & Shaw, 2007; Birnbaum & Alvarado, 2008). All putative homologs of Arabidopsis PcG genes (Pien & Grossniklaus, 2007) were identified in our dataset, including several PRC2 subunits, such as SWINGER (SWN), FIE and MSI1 (Fig. 4a). SWN was down-regulated in deX and then increased at a later stage in rPh and rC. Conversely, MSI1, MSI2, ICU2 and FIE were up-regulated in deX and diC (Fig. 4a). In our earlier proteomic study, a protein homolog of SWN was found at 6 DAG and then disappeared at 10 DAG (Du et al., 2006). Our present transcript data together with previous proteomic data suggest a role for PcG in xylem cell dedifferentiation during SVT regeneration.
During SVT regeneration, almost all differentially expressed type-A cyclins (CYCAs) and type-B cyclins (CYCBs) exhibited highly similar profiles with a significant peak in expression at early regeneration in deX (Fig. 4b). However, exceptions, such as CYCA2 and type-D cyclins (CYCDs), were observed. Transcripts for CYCA2;2 and CYCA2;4 showed the highest expression in diX and down-regulation during regeneration (Fig. 4b). In Populus, PttCYCA2 expression remains high until very late in xylem development (Schrader et al., 2004). Expression of AtCYCA2 in Arabidopsis is considered to be associated with competence for cell division (Burssens et al., 2000). These data suggest that diX cells retained their developmental plasticity and have the capacity to change into other cell types in certain conditions, such as wounding. Several probe-sets representing Cyclin D3:1 exhibited high expression in diC or rC (Fig. 4b). Cyclin-dependent kinases (CDKs) and CDK-like (CKL) displayed different expression profiles; of these, two plant-specific CDKB genes (CDKB1;2 and CDKB2;1) followed the general expression trend of most CYCA and CYCB genes (Fig. 4b). Similarly, most histone transcripts also exhibited an expression peak in deX (Table S1). In cell cycle re-entry experiments conducted in Arabidopsis, most CYCAs and CYCBs exhibit a distinct peak in early mitosis and are both considered as mitotic cyclins, while CDKB genes exhibit their expression peak at either the G2 phase or the G2/M transition (Menges et al., 2005). Comparing the observed specific expression pattern of mitotic cyclins and CDKB genes during SVT regeneration with the regulatory mechanism found in cell re-entry experiments suggests the activation of cell cycle re-entry of differentiating xylem cells in their early cell fate switching process. This is also consistent with our anatomical findings during early SVT regeneration in both P. tomentosa (Fig. 2) and Eucommia, in which different cell division phases of immature xylem cells have been observed (Pang et al., 2008).
Down-regulation of the xylem-specific program and activation of phloem and cambium developmental programs during SVT regeneration
To address the identity change of the diX cells, we examined reported xylem marker genes, such as cellulose synthase (CesA) genes, xyloglucan endotransglycosylase genes PttXET16C/PtXTH35, programmed cell death (PCD)-associated XYLEM CYSTEINE PEPTIDASE (XCP), lignin biosynthesis-related genes and xylem specific transcription factors (TFs) (Schrader et al., 2004; Zhao et al., 2005; Geisler-Lee et al., 2006; Suzuki et al., 2006). Eight probe-sets representing xylem-specific Populus CesA and CesA-like (Csl) genes (PtCesA18/3-2, PtrCesA3, PtCesA3, PtCesA7, PtCesA4/5, PtCslA1/2) were identified from our dataset and all eight CesA transcripts exhibited high expression in diX but were absent or had very low expression in regenerated phloem and cambium tissues (Fig. 5a and Table S2). The transcript of PtXTH35 and homolog of Arabidopsis XCP2 also show preferential expression in diX (Fig. 5a and Table S2). Populus lignin biosynthesis-related enzymes (Hamberger et al., 2007; Shi et al., 2010), including PAL, C4H, C3H, 4CL (Poptr4CL3L), HCT, CCoAOMT (PttCCoAOMT1), COMT (PttCCoAOMT1 and 2) and CAD (PoptrCAD1 and PoptrCAD4/PtrCAD2/PtCAD) genes, exhibited a similar expression profile with enrichment in diX and a dramatic decline in regenerating tissues, implying the loss of xylem characteristics in cells during regeneration (Fig. 5a and Table S2).
Figure 5. Down-regulation of xylem development-related genes during Populus tomentosa secondary vascular tissue (SVT) regeneration. (a) Xylem-specific marker genes and lignification-related enzyme genes. (b) Xylem specification-related transcription factors. Complete information for each probe-set can be found in Supporting Information, Table S2.
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HD-ZIP III genes have been demonstrated to promote xylem specification (Emery et al., 2003; Carlsbecker & Helariutta, 2005). In Populus, homologs of ATHB-8 (PttHB8), ATHB-9/PHV (PttHB9) and ATHB-15 (PttHB15) exhibit a steep increase in expression on the xylem side of the cambial zone (Schrader et al., 2004). In our study, PttHB8, PttHB9 and PttHB15 were specifically expressed in diX and distinctly down-regulated in regenerating tissues (Fig. 5b and Table S2). In addition, examination of xylem-biased NAC domain TFs and MYB TFs (Wilkins et al., 2009; Zhong & Ye, 2009; Yamaguchi & Demura, 2010) showed six NAC TFs and a group of Populus R2R3-MYB TFs were diX-enriched with dramatic down-regulation during regeneration, as shown in Fig. 5b and Table S2. Taken together, the enrichment of these genes in diX validated the enrichment of these genes in secondary xylem cells; furthermore, the significant decrease in expression of these genes in regenerating tissues indicates the shutting-off of the xylem specification program and thus the loss of xylem cell identity during regeneration.
On the other hand, our data suggested that phloem and cambium gene expression programs were activated during SVT regeneration. Transcription factors ALTERED PHLOEM DEVELOPMENT (APL) and KANADI (KAN) are required for phloem specification and differentiation (Kerstetter et al., 2001; Bonke et al., 2003; Emery et al., 2003). During SVT regeneration, transcripts of Populus APL and PttKAN1, PttKAN2 were accumulated in the phloem formation stage (stage II) and their expression level remained high until the appearance of cambium at stage III (Fig. 6a). Besides, several other groups of TFs were also found highly expressed in rPh, including G2-like MYB, Dof and NAM (NO APICAL MERISTEM) (Fig. 6a and Table S3). MYB-RELATED PROTEIN 1 (MYR1) and Dof genes have been found to be phloem-abundant in various species (Zhao et al., 2005; Le Hir et al., 2008). A recent report also shows that Dof5.6/HCA2 is expressed in phloem and cambium of Arabidopsis inflorescence stems (Guo et al., 2009). For all Dof genes identified in the Populus genome (Yang et al., 2006), six transcripts showed clear enrichment in rPh or/and rC (Fig. 6a). Among them, PtrDOF03/33 was the closest homolog of Arabidopsis Dof5.6. Collectively, our results provided molecular evidence that groups of TFs such as MYB, Dof and NAM contributed to phloem and cambium regeneration.
Figure 6. Up-regulation of transcription factors involved in phloem specification (a) and cambium regulation (b) during Populus tomentosa secondary vascular tissue (SVT) regeneration. Complete information for each probe-set in the gene lists can be found in Supporting Information, Table S3.
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Furthermore, to assess the structure and function of the newly formed sieve elements as transport phloem cells, potential phloem structure and function-associated genes were examined. Genes encoding sieve elements/companion cells specific jacalin-related lectins, sieve element specifically synthesized serpins, sucrose synthase and phloem-specific metabolic enzymes could highlight common features of phloem functions in various plant species (Le Hir et al., 2008; Lin et al., 2009). Clustering analysis showed that these related genes were abundant in rPh (Table S3). As shown in Figs 3, S1, callose, the marker for sieve elements, was detected in stages II and III by aniline blue. Accordingly, transcripts of callose synthase were up-regulated in rPh (Table S3). Transcripts of hormone biosynthesis-related genes, such as GA4/gibberellin 3 beta-hydroxylase and ACC synthase, and stress response genes were also present in rPh, (Table S3). In our earlier in vitro experiments, isotope tracing of twigs with [14C]sucrose showed that newly formed sieve elements are capable of transporting sugar during the bark regeneration process in Eucommia (Pang et al., 2008). Genes encoding sugar transporters, amino acid carriers, H+-transporting ATPase (AHA10), potassium channels as well as RNA-binding proteins showed an increase in expression in rPh (Table S3) and thus provided molecular evidence of transport function recovery of phloem. Together, our data indicate the activation of the phloem program during SVT regeneration.
Current evidence indicates that a WUSCHEL-CLAVATA-like mediated regulatory model is active in cambial meristems, as in the apical meristems (Elo et al., 2009; Zhang et al., 2011). In Arabidopsis, the PXY-CLE41/CLE44 loop maintains the undifferentiated pluripotent status in the cambium (Etchells & Turner, 2010). WOX4, a WUSCHEL-related HOMEOBOX gene, may function in the TDIF/CLE41/CLE44-TDR/PXY signaling pathway to promote procambial/cambial cell proliferation (Hirakawa et al., 2010). In Populus, Schrader et al. (2004) have also proposed the existence of a similar feedback loop in the cambium. PttCLV1, a putative ortholog of CLAVATA1, showed very low expression in diX and deX and displayed significant up-regulation in regenerated cambial and phloem cells (Fig. 6b). Transcripts of CLV2 and several other leucine-rich repeat kinases also exhibited similar expression patterns to PttCLV1 (Table S3). Conversely, the receptor-like kinase PttRLK3 and a WUSCHEL-related homeobox family gene PttHB2 showed preferential expression in diX and down-regulated across regeneration. This was similar to what has been found in the normal cambial region, where PttHB2 shows a steep increase in expression toward the xylem side (Schrader et al., 2004).
PttANT and PttPNH, Populus homologs of AINTEGUMENTA and PINHEAD/ZWILLE, show a distinct expression peak in the cambium zone, which implies their involvement in regulation of cambial cell proliferation (Schrader et al., 2004). Probe-sets for PttANT;1 and PttPNH were identified and they all exhibited an expression peak in regenerated cambium (Fig. 6b, Table S3). Interestingly, the accumulation of PttANT transcripts was also detected as early as in deX. The up-regulation of one PINHEAD gene during cambium regeneration was reported and predicted to function in cambium initiation rather than maintenance (Wang et al., 2009). However, our data revealed that the expression of PttPNH remained high until cambium formation, indicating that PINHEAD might be involved in cambium maintenance as well (Fig. 6b). Populus orthologs of Arabidopsis Class I KNOX genes were highly expressed in the cambial zone and functional analysis suggested that they are likely involved in the cambial cell identity maintenance (Schrader et al., 2004; Groover et al., 2006; Du et al., 2009). During regeneration, KNOX1 and KNOX6 showed an increasing trend in regenerated phloem and cambium (Fig. 6b). In addition, a class II KNOX gene, KNAT3/KNOX3, showed high expression in rPh and diC but lower signals in rC (Fig. 6b). The GRAS family TFs SHORTROOT (SHR) and SCARECROW (SCR) contribute to vascular cell patterning in Arabidopsis roots (Helariutta et al., 2000; Nakajima & Benfey, 2002). During cambium regeneration, two Populus homologs of SHR (PttSHR1 and PttSHR2) exhibited maximum expression in rC, while four putative SCR-like 6 (SCL6) revealed different expression patterns toward rC (Fig. 6b and Table S3). The expression profiling of these TFs indicates their potential contribution in cambium regeneration.
Further expression profile analysis revealed that members of additional TF families, such as R2R3-MYB, WRKY, bHLH, bZIP, Whirly and CONSTANS, were enriched in regenerated phloem and cambium (Table S3).
Phytohormone regulation during SVT regeneration
Phytohormones play crucial roles in regulating tissue or organ regeneration in plants (Xu et al., 2006; Grieneisen et al., 2007; Birnbaum & Alvarado, 2008; Sena et al., 2009). An auxin gradient provides a positional signal in pattern specification during SVT development (Uggla et al., 1996, 1998; Mwange et al., 2005). Redistribution of auxin gradients may induce root regeneration (Xu et al., 2006) and roots failed to regenerate when auxin transport was blocked (Sena et al., 2009). To look into the molecular evidence for auxin redistribution during SVT regeneration, we checked the expression patterns of polar auxin transport (PAT) genes including auxin influx carriers (PttLAX, AUX1-like) and efflux carriers (PttPIN, PIN1-lke) identified in Populus (Schrader et al., 2003). The results showed that most AUX1-like genes were highly expressed in diX; however, PttLAX2 and another AUX1-like genes were also enriched in rC (Fig. 7a). PttLAX3, on the other hand, showed a similar expression pattern to PttPIN1 and a PIN1-like gene with high expression in rC. A very different result was found for PttPIN3, which was enriched in deX (Fig. 7a). In the wood-forming region, PttLAX1 and PttLAX2 are both expressed in secondary cell wall-forming xylem but PttLAX2 is also expressed in the cambial zone. PttLAX3, PttPIN1 and PttPIN2 are expressed in the cambium and dividing xylem mother cells, while PttPIN3 shows highest expression in the cortex layer (Schrader et al., 2003). The correlation between the dynamic expression patterns of PAT genes across SVT regeneration and their spatial expression patterns in the normal wood-forming region implied that the expression changes of PAT genes might contribute to the resetting up of auxin gradients during phloem and cambium recovery. By using high-performance liquid chromatography and immunolocalization techniques, we have previously shown the significant change of auxin concentration and distribution within the girdled areas during bark reconstitution (Mwange et al., 2003).
Figure 7. Transcript expression patterns of auxin carriers and auxin receptors genes during secondary vascular tissue (SVT) regeneration. Auxin carrier AUX and PIN genes, auxin receptor TIR and auxin-binding protein1 (ABP1) genes were identified and clustered. The complete information for each probe-set in the gene lists can be found in Supporting Information, Table S4. diX, differentiating xylem; deX, dedifferentiating xylem; rPh, regenerated phloem; diC, differentiating regenerated cambium; rC, regenerated cambium; PAT, polar auxin transport; CV, coefficient of variation.
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Alongside polar auxin transport, auxin signaling also plays an essential role in tissue regeneration. A list of auxin-responsive genes is involved in Arabidopsis root-tip regeneration (Sena et al., 2009) and one IAA-induced TF is up-regulated during poplar secondary vascular system regeneration (Wang et al., 2009). Homologs of auxin receptors (TIR1) and genes involved in auxin response, including Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF), were identified (Table S4). Analysis of TIR1 and auxin-binding protein1 (ABP1) genes demonstrated that putative TIR1 transcripts were up-regulated during SVT regeneration, whereas ABP1 transcript was down-regulated (Fig. 7b). Recent reports indicate that ABP1 is required for the regulation of early auxin-regulated genes and PAT (Robert et al., 2010; Effendi et al., 2011). Our observation might imply different modes of action for the two potential auxin receptors in SVT regeneration. A total of 35 Aux/IAA genes denoted as PoptrIAA and 39 ARF denoted as PoptrARF were discovered in the Populus genome (Kalluri et al., 2007). Twenty-five probe-sets representing 15 PoptrIAA and 18 probe-sets representing 14 PoptrARF were identified from our dataset and they showed differential expression profiles and tissue preference during regeneration (Table S4). The presence of PoptrARF8 in rPh (Table S4) confirmed previous detection of this gene transcript in phloem (Zhao et al., 2005; Le Hir et al., 2008). Several auxin-responsive genes (examined by Sena et al. (2009)) and enzymes putatively involved in Aux/IAA proteolysis were also up-regulated in rPh and rC (Table S4). The regulatory role of auxin during SVT regeneration has also been supported in our experiments carried out in Eucommia, in which application of exogenous IAA on the wound surface accelerates sieve element differentiation (Pang et al., 2008).
In addition to auxin, genes involved in other phytohormone signalling pathways, such as those of cytokinin, GA and ethylene, were also differentially expressed during SVT regeneration (Table S4). Recent studies demonstrate that cytokinins are central hormonal regulators of cambium development (Matsumoto-Kitano et al., 2008; Nieminen et al., 2008; Dettmer et al., 2009). Cytokinins can accelerate sieve tube regeneration and promote callose production around the wound of Coleus internodes (Aloni et al., 1990). Analysis of the expression patterns of cytokinin receptors, type-A and type-B response regulators (RRs) identified in Populus (Ramirez-Carvajal et al., 2008) revealed that transcripts of putative Populus CRE1B and histidine kinase-like (PtHK3a and PtHK3b), four type-A RRs (PtRR1, 2, 7 and 10) and three type-B RRs (PtRR19, 21 and 22) were up-regulated in rPh and/or rC (Table S4). The GA signal transduction protein (spindly), signal response protein GIBBERELLIC ACID INSENSITIVE (GAI) and GA-regulated protein GASA were differentially expressed during regeneration. However, multiple expression patterns for these genes were observed (Table S4). Homologs of ethylene receptor members EIN4, ERT2 and ERS were differentially expressed during regeneration. A number of Populus ERFs (ethylene response factors) and DREBs (dehydration-responsive element binding proteins) were enriched in regenerating tissues (Table S4).