To identify essential genes that play important roles in wood formation, an efficient approach is to characterize genes that are expressed at key switches of SVS development. We used cDNA microarrays and our previously established SVS regeneration system to identify genes that are preferentially expressed at the key switches of the SVS regeneration process. Similar to the results of our previous proteomics study [13
], this work found that the transcript level also changed dramatically at these key switch points, at which the anatomy, structures, and functions of the cells change with the appearance of new types of cells and tissues. More than 67% of the genes we identified had the same function description as in a previous work by Hertzberg and coworkers [10
], verifying the results and further confirming their involvement in wood formation. However, a large number of the genes (9.7%) were only identified in our study. This could have occurred because the previous study [10
] only analyzed the transcriptional profiles in the cambium zone and immature and mature xylem, whereas we analyzed the transcriptional profiles following the dynamic process of SVS regeneration, allowing us to obtain genes with more dynamic changes in transcript levels or those that are only strongly transcribed during the transitions. For instance, the gene encoding the Ag13 protein precursor (S050) was only transcribed strongly at 18 days AG, when the differentiation of cambium into xylem was beginning, and was transcribed at low levels during the other stages.
In the early stages of SVS regeneration, meristem cells appeared at 6 days AG, possibly from dedifferentiated immature xylem cells; the cells then increased in number by cell division and finally had formed continuous cambium-like layers at 10 days AG. At 14 days AG, a vascular cambium had formed as a continuous and regular layer. During this process, several genes encoding transcription regulators (including PINHEAD and AUX/IAA), primary cell wall-associated proteins, and proteins of unknown function were up- or downregulated, indicating their functions in cell differentiation and meristem formation. PINHEAD was upregulated only at the stage of initiation of meristem cells, and was downregulated after 10 days AG (S003, additional file 3
). In Arabidopsis
, PINHEAD is expressed in the vasculature, and the development of shoot apical meristems is abnormal in the pinhead mutant. The PINHEAD gene should encode a component of a hypothetical meristem-forming competence factor and may play a role in primary meristem initiation [14
]. We hypothesize that the strong expression of PINHEAD triggers certain cells to dedifferentiate into meristem cells at 6 days AG, and is no longer needed when the nearly continuous cambium-like layers have formed. Therefore, PINHEAD may be involved in cambium initiation rather than cambium maintenance. Recent advances have shown that several plant growth regulators, including auxin [16
], cytokinin [19
], brassinosteroid [21
], and proteoglycan [23
], regulate the development of SVS as signaling substances. A radial concentration gradient of auxin forms across the cambium region in the aspen stem and is related to secondary xylem development [17
]. Mutations in several auxin response factors (ARFs) or auxin (IAA)-induced proteins disrupt the normal body organization along the apical-basal axis and result in discontinuous and reduced vascular formation [24
]. An IAA-induced gene (S069, additional file 3
) is expressed at 10 days AG, suggesting that the auxin-activated expression of IAAs regulates the transcription of genes involved in cambium formation.
The genes expressed in the cambium zone were revealed by a cDNA array analysis employing the microsectioning technique, and marker genes for cambial initials (Peakset 2), phloem (Peakset 1), and xylem mother cells (Peakset 3) were found, with totals of 19, 103, and 36 genes, respectively [26
]. In contrast, in our study, the initiation of cambial cells, rather than maintenance of the stem layer in the normal cambial zone sampled in the previous study, could be traced, allowing us to obtain a large number (71, additional file 2
) of up- and downregulated genes involved in the transition from callus cells to stem cells. In addition, considerable numbers (41%) of these genes were of unknown function, indicating they might be novel genes that play roles in this process. Nevertheless, nine genes in our data were found that had the same (6) or a similar (3) function description as the nine counterparts (47%) in Peakset 2 for cambial initials, suggesting that these genes are important for both the formation and maintenance of the stem layer; therefore, further investigation into their roles in cambial activity is warranted. Similarly, our SVS regeneration system identified genes expressed during the differentiation of stem cells (increased cell layers in the cambium zone) into these mother cells and the maintenance of their identity, and 56 such genes (42% of the total genes of known function) in our data had the same or similar function description as markers in Peaksets 1 and 3 in the previous study [26
]. However, we could not partition these dynamically transcribed genes into phloem and xylem mother cells as in the previous study due to the limitation of our regeneration system in which these two types of cells appeared at nearly the same time. In summary, we studied the temporal expression of genes involved in cambial activity, whereas the previous work [26
] emphasized genes that are spatially expressed across the cambium zone. In addition, both studies found common genes of interest that play roles in this process. Combining these two experimental systems to comprehensively profile the genes that are spatially and temporally involved in SVS development by employing whole-genome poplar chips should provide valuable results.
In the later stages of SVS regeneration (from 16 to 22 days AG), the newly formed cambium zone began to differentiate, and finally a normal wood-forming structure was completely regenerated. Genes encoding MYB-family proteins (S002, S111, S112, S129, additional file 3
) and several small heat shock proteins (HSPs) (S174, S184, S188, additional file 3
) were either up- or downregulated. A promoter analysis of genes that encode enzymes associated with lignin biosynthesis revealed that AC elements are necessary for xylem-localized gene expression [27
]. In Eucalyptus
, EgMYB2, a member of the R2R3-MYB family, can bind to the promoters of the EgCCR
genes, which contain AC elements, and regulate their transcription [28
]. The thickness of the secondary cell wall of transgenic tobacco plants overexpressing EgMYB2
was dramatically increased and the lignin profile was altered, suggesting that EgMYB2 is a positive regulator of both lignin biosynthesis and secondary wall formation in xylem [28
undergoes secondary growth in roots, hypocotyls, and stems under certain conditions [29
]. The transcriptional profiling of induced secondary growth in Arabidopsis
showed that several MYB transcription factors regulate the activities and differentiation of xylem and phloem [34
]. Our results showed MYB-family genes transcribed at high levels at 18 and 22 days AG (S002, S111, S112, S129, additional file 3
), revealing that certain MYB proteins are involved in the regulation of xylem development.
Interestingly, during the regeneration process, rare genes encoding enzymes involved in lignin biosynthesis were found to differ at the transcript level, except for genes encoding 4-coumarate:CoA ligase (4CL) (S141, S155, additional file 3
) and cinnamate 4-hydroxylase (C4H) (S036, additional file 3
). However, the expression of genes associated with cell wall biosynthesis and assembly, such as extensins and expansins, changed dramatically, reflecting the transitions of cell types during this regeneration process but without large changes in the chemical composition of the cell walls. Since our cDNA microarray was prepared from a cambium zone-specific subtractive library, and the regenerated tissues mainly included cambium, immature xylem, and a small amount of phloem, lacking secondary cell wall deposition, the expression of most of the genes associated with lignin biosynthesis was not extensively observed.
Small HSPs, induced by gibberellins or methyl jasmonate, play roles in plant growth and development [35
], including wood formation [36
]. The transcripts of several small HSPs (S174, S184, S188, additional file 3
) changed dramatically at 16, 18, and 22 days AG in our study, indicating that small HSPs are highly involved in cambium differentiation and xylem development. In addition, several genes of unknown function were strongly transcribed at key switches of the regeneration process, suggesting their potential roles in SVS regeneration. The transcript levels of genes encoding the P0510C12.9 protein at 10 days AG were 12- to 37-fold higher than at 6 days AG, and the high transcript levels persisted through the regeneration process. At 16 days AG, the transcription of the P0510C12.9 gene (five such genes were found, but only S061 is listed in additional file 3
) was significantly stronger than at 14 days AG. These proteins of unknown function might be important players in wood formation. The transcription of numerous genes encoding these HSPs and unknown proteins was found to be highly dynamic during this process, and it would be interesting to further elucidate their roles in wood formation.