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Characterization of transcriptome remodeling during cambium formation identifies MOL1 and RUL1 as opposing regulators of secondary growth.

Agusti J, Lichtenberger R, Schwarz M, Nehlin L, Greb T - PLoS Genet. (2011)

Bottom Line: Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity.Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments.By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation.

View Article: PubMed Central - PubMed

Affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.

ABSTRACT
Cell-to-cell communication is crucial for the development of multicellular organisms, especially during the generation of new tissues and organs. Secondary growth--the lateral expansion of plant growth axes--is a highly dynamic process that depends on the activity of the cambium. The cambium is a stem cell-like tissue whose activity is responsible for wood production and, thus, for the establishment of extended shoot and root systems. Attempts to study cambium regulation at the molecular level have been hampered by the limitations of performing genetic analyses in trees and by the difficulty of accessing this tissue in model systems such as Arabidopsis thaliana. Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity. Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments. By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation. In summary, we provide a means for investigating cambium regulation in unprecedented depth and present two signaling components that control a process responsible for the accumulation of a large proportion of terrestrial biomass.

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Expression of identified genes in various tissues.(A) Average of mean array signal intensity (MSI) relations as described [21] for genes present in Groups 1 and 2 comparing xylem (X), phloem/cambium (PC) and non-vascular (NV) tissues from hypocotyls. (B) Percentage of genes classified as being specifically expressed in WUS, FIL or CLV3 expression domains (Supplementary Table 5 in [37]) comparing ‘all’ genes present in the genome, genes found in Group 1, and in Group 2, respectively. (C and D) Radial (C) and longitudinal (D) distribution of expression levels of genes from Group 1 and 2 in root meristems based on the values for the top 50% of varying probe sets described in Supplementary Table 12 in [36]. Average expression levels of genes listed in Table S2 (Group 1) and listed in Table S3 (Group 2) are shown. In (C), domains are defined by the expression of GFP marker lines. For (D), roots were dissected at different longitudinal positions resulting in samples representing subsequent developmental stages (see [36] for details). Sample 1 contains the quiescent center and tissue-specific stem cells. CC  =  companion cells.
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pgen-1001312-g003: Expression of identified genes in various tissues.(A) Average of mean array signal intensity (MSI) relations as described [21] for genes present in Groups 1 and 2 comparing xylem (X), phloem/cambium (PC) and non-vascular (NV) tissues from hypocotyls. (B) Percentage of genes classified as being specifically expressed in WUS, FIL or CLV3 expression domains (Supplementary Table 5 in [37]) comparing ‘all’ genes present in the genome, genes found in Group 1, and in Group 2, respectively. (C and D) Radial (C) and longitudinal (D) distribution of expression levels of genes from Group 1 and 2 in root meristems based on the values for the top 50% of varying probe sets described in Supplementary Table 12 in [36]. Average expression levels of genes listed in Table S2 (Group 1) and listed in Table S3 (Group 2) are shown. In (C), domains are defined by the expression of GFP marker lines. For (D), roots were dissected at different longitudinal positions resulting in samples representing subsequent developmental stages (see [36] for details). Sample 1 contains the quiescent center and tissue-specific stem cells. CC  =  companion cells.

Mentions: Next, we were interested to see whether there were indications that the identified genes were indeed cambium-specific. Therefore, we analyzed their expression values in different tissues employing data from tissue-specific transcriptional profilings described previously. Characterization of the transcript profile of a mixture of cambium and phloem tissues from hypocotyls represents the closest approximation to a cambium-specific profile described for Arabidopsis to date [21]. Calculating the relationship between mean signal intensities (MSI) in phloem/cambium, xylem, and non-vascular tissues [21] for genes from Group 1, we found a bias toward a phloem/cambium-specific expression (Figure 3A). The same analysis for genes from Group 2 showed an even stronger bias, suggesting that, by applying more stringent selection criteria, we increased tissue specificity within the group of selected genes (Figure 3A). Consistently, analysis of the expression of the selected genes according to high-resolution expression maps of the shoot apical meristem and the root tip [36], [37], revealed a bias towards expression in phloem tissues with a stronger bias for Group 2 (Figure 3C). Interestingly, we did not observe a bias toward a stem cell-specific expression in root or shoot apical meristems (Figure 3B–3D), arguing against a large overlap of gene expression profiles when comparing lateral and apical meristems. Importantly, the search for known cambium markers identified PXY and ATHB8 as members of Group 1 and 2. Both genes belong to the small group of genes described to be specifically expressed in (pro)cambium cells [24], [43], and their presence serves as an indicator for the tissue specificity of the identified group of genes in intact plants. Because WOX4, a cambium regulator identified recently [29], is not present on the ATH1 array, we performed qRT-PCR experiments in order to reveal WOX4 transcript abundance in our LCM-derived samples. This analysis showed that the WOX4 transcript accumulates preferentially in interfascicular regions of 5 day NAA-treated fragments (Figure S4). Taken together, we conclude that cambium identity is established in interfascicular regions of CIS-incubated fragments, and that our approach provides a series of genes expressed in a tissue specific manner at different stages during cambium formation in intact plants.


Characterization of transcriptome remodeling during cambium formation identifies MOL1 and RUL1 as opposing regulators of secondary growth.

Agusti J, Lichtenberger R, Schwarz M, Nehlin L, Greb T - PLoS Genet. (2011)

Expression of identified genes in various tissues.(A) Average of mean array signal intensity (MSI) relations as described [21] for genes present in Groups 1 and 2 comparing xylem (X), phloem/cambium (PC) and non-vascular (NV) tissues from hypocotyls. (B) Percentage of genes classified as being specifically expressed in WUS, FIL or CLV3 expression domains (Supplementary Table 5 in [37]) comparing ‘all’ genes present in the genome, genes found in Group 1, and in Group 2, respectively. (C and D) Radial (C) and longitudinal (D) distribution of expression levels of genes from Group 1 and 2 in root meristems based on the values for the top 50% of varying probe sets described in Supplementary Table 12 in [36]. Average expression levels of genes listed in Table S2 (Group 1) and listed in Table S3 (Group 2) are shown. In (C), domains are defined by the expression of GFP marker lines. For (D), roots were dissected at different longitudinal positions resulting in samples representing subsequent developmental stages (see [36] for details). Sample 1 contains the quiescent center and tissue-specific stem cells. CC  =  companion cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040665&req=5

pgen-1001312-g003: Expression of identified genes in various tissues.(A) Average of mean array signal intensity (MSI) relations as described [21] for genes present in Groups 1 and 2 comparing xylem (X), phloem/cambium (PC) and non-vascular (NV) tissues from hypocotyls. (B) Percentage of genes classified as being specifically expressed in WUS, FIL or CLV3 expression domains (Supplementary Table 5 in [37]) comparing ‘all’ genes present in the genome, genes found in Group 1, and in Group 2, respectively. (C and D) Radial (C) and longitudinal (D) distribution of expression levels of genes from Group 1 and 2 in root meristems based on the values for the top 50% of varying probe sets described in Supplementary Table 12 in [36]. Average expression levels of genes listed in Table S2 (Group 1) and listed in Table S3 (Group 2) are shown. In (C), domains are defined by the expression of GFP marker lines. For (D), roots were dissected at different longitudinal positions resulting in samples representing subsequent developmental stages (see [36] for details). Sample 1 contains the quiescent center and tissue-specific stem cells. CC  =  companion cells.
Mentions: Next, we were interested to see whether there were indications that the identified genes were indeed cambium-specific. Therefore, we analyzed their expression values in different tissues employing data from tissue-specific transcriptional profilings described previously. Characterization of the transcript profile of a mixture of cambium and phloem tissues from hypocotyls represents the closest approximation to a cambium-specific profile described for Arabidopsis to date [21]. Calculating the relationship between mean signal intensities (MSI) in phloem/cambium, xylem, and non-vascular tissues [21] for genes from Group 1, we found a bias toward a phloem/cambium-specific expression (Figure 3A). The same analysis for genes from Group 2 showed an even stronger bias, suggesting that, by applying more stringent selection criteria, we increased tissue specificity within the group of selected genes (Figure 3A). Consistently, analysis of the expression of the selected genes according to high-resolution expression maps of the shoot apical meristem and the root tip [36], [37], revealed a bias towards expression in phloem tissues with a stronger bias for Group 2 (Figure 3C). Interestingly, we did not observe a bias toward a stem cell-specific expression in root or shoot apical meristems (Figure 3B–3D), arguing against a large overlap of gene expression profiles when comparing lateral and apical meristems. Importantly, the search for known cambium markers identified PXY and ATHB8 as members of Group 1 and 2. Both genes belong to the small group of genes described to be specifically expressed in (pro)cambium cells [24], [43], and their presence serves as an indicator for the tissue specificity of the identified group of genes in intact plants. Because WOX4, a cambium regulator identified recently [29], is not present on the ATH1 array, we performed qRT-PCR experiments in order to reveal WOX4 transcript abundance in our LCM-derived samples. This analysis showed that the WOX4 transcript accumulates preferentially in interfascicular regions of 5 day NAA-treated fragments (Figure S4). Taken together, we conclude that cambium identity is established in interfascicular regions of CIS-incubated fragments, and that our approach provides a series of genes expressed in a tissue specific manner at different stages during cambium formation in intact plants.

Bottom Line: Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity.Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments.By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation.

View Article: PubMed Central - PubMed

Affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.

ABSTRACT
Cell-to-cell communication is crucial for the development of multicellular organisms, especially during the generation of new tissues and organs. Secondary growth--the lateral expansion of plant growth axes--is a highly dynamic process that depends on the activity of the cambium. The cambium is a stem cell-like tissue whose activity is responsible for wood production and, thus, for the establishment of extended shoot and root systems. Attempts to study cambium regulation at the molecular level have been hampered by the limitations of performing genetic analyses in trees and by the difficulty of accessing this tissue in model systems such as Arabidopsis thaliana. Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity. Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments. By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation. In summary, we provide a means for investigating cambium regulation in unprecedented depth and present two signaling components that control a process responsible for the accumulation of a large proportion of terrestrial biomass.

Show MeSH
Related in: MedlinePlus