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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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Transcript detection by RISH using antisense and sense probes.(A and B) AT5G05160/RUL1, (C and D) PXY, (E and F) ATHB8, (G and H) AT5G51350/MOL1, (I and J) AGO4, (K and L) AT5G57130, (M and N) WOX4, (O) SCM (GUS probe), (P) ACA8 (GUS probe), (Q) GUS sense probe on ACA8:GUS line. (A, C, E, G, I, K, M, O, P) show results using antisense probes, (B, D, F, H, J, L, N, Q) using sense probes. Arrowheads indicate cambium-specific mRNA accumulation and asterisks label the position of primary vascular bundles. Stem sections come from immediately above the uppermost rosette leaf of 15 cm tall plants. Size bar in (A): 100 µm, same magnification in (A–Q).
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pgen-1001312-g004: Transcript detection by RISH using antisense and sense probes.(A and B) AT5G05160/RUL1, (C and D) PXY, (E and F) ATHB8, (G and H) AT5G51350/MOL1, (I and J) AGO4, (K and L) AT5G57130, (M and N) WOX4, (O) SCM (GUS probe), (P) ACA8 (GUS probe), (Q) GUS sense probe on ACA8:GUS line. (A, C, E, G, I, K, M, O, P) show results using antisense probes, (B, D, F, H, J, L, N, Q) using sense probes. Arrowheads indicate cambium-specific mRNA accumulation and asterisks label the position of primary vascular bundles. Stem sections come from immediately above the uppermost rosette leaf of 15 cm tall plants. Size bar in (A): 100 µm, same magnification in (A–Q).

Mentions: To validate the above conclusion, we concentrated on ‘early’ genes from Group 2, categorized as being involved in developmental processes, signal transduction, and transcription (see GO annotations, http://www.arabidopsis.org/tools/bulk/go/index.jsp, for a more detailed analysis). As we were primarily interested in genes specifically regulating secondary growth, we eliminated those genes for which severe embryo defects in corresponding mutants had been described [44], leaving us with 13 genes (Table 1). Expression of these genes and WOX4 was analyzed by RNA in situ hybridization (RISH) with the exception of AHP3 and ATSEN1, for which no specific RNA probes could be designed due to extended sequence similarity to close homologues. For the remaining genes, sense and antisense probes were hybridized to cross-sections taken from immediately above the uppermost rosette leaf of 30 cm-tall inflorescence stems. For those genes for which GUS reporter lines were available [45]–[48], corresponding reporter lines were analyzed by using a GUS-specific RNA probe. As a result, for all but three cases in which no mRNA accumulation could be detected (Table 1), mRNA was found to accumulate in a continuous domain consisting of one to three cells in a radial orientation located between xylem and phloem tissue (Figure 4). The domain of RNA accumulation detected for all positive genes was similar to that of the cambium-specific genes PXY and WOX4, suggesting that all genes are similarly cambium-specific or, at least, transcribed in close proximity. This verification of the cambium-specific mRNA accumulation of a large proportion of the genes identified suggests that we had indeed elucidated the mRNA profile established during cambium formation in planta, and that CIS incubation can be used for the identification and analysis of cambium regulators.


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)

Transcript detection by RISH using antisense and sense probes.(A and B) AT5G05160/RUL1, (C and D) PXY, (E and F) ATHB8, (G and H) AT5G51350/MOL1, (I and J) AGO4, (K and L) AT5G57130, (M and N) WOX4, (O) SCM (GUS probe), (P) ACA8 (GUS probe), (Q) GUS sense probe on ACA8:GUS line. (A, C, E, G, I, K, M, O, P) show results using antisense probes, (B, D, F, H, J, L, N, Q) using sense probes. Arrowheads indicate cambium-specific mRNA accumulation and asterisks label the position of primary vascular bundles. Stem sections come from immediately above the uppermost rosette leaf of 15 cm tall plants. Size bar in (A): 100 µm, same magnification in (A–Q).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040665&req=5

pgen-1001312-g004: Transcript detection by RISH using antisense and sense probes.(A and B) AT5G05160/RUL1, (C and D) PXY, (E and F) ATHB8, (G and H) AT5G51350/MOL1, (I and J) AGO4, (K and L) AT5G57130, (M and N) WOX4, (O) SCM (GUS probe), (P) ACA8 (GUS probe), (Q) GUS sense probe on ACA8:GUS line. (A, C, E, G, I, K, M, O, P) show results using antisense probes, (B, D, F, H, J, L, N, Q) using sense probes. Arrowheads indicate cambium-specific mRNA accumulation and asterisks label the position of primary vascular bundles. Stem sections come from immediately above the uppermost rosette leaf of 15 cm tall plants. Size bar in (A): 100 µm, same magnification in (A–Q).
Mentions: To validate the above conclusion, we concentrated on ‘early’ genes from Group 2, categorized as being involved in developmental processes, signal transduction, and transcription (see GO annotations, http://www.arabidopsis.org/tools/bulk/go/index.jsp, for a more detailed analysis). As we were primarily interested in genes specifically regulating secondary growth, we eliminated those genes for which severe embryo defects in corresponding mutants had been described [44], leaving us with 13 genes (Table 1). Expression of these genes and WOX4 was analyzed by RNA in situ hybridization (RISH) with the exception of AHP3 and ATSEN1, for which no specific RNA probes could be designed due to extended sequence similarity to close homologues. For the remaining genes, sense and antisense probes were hybridized to cross-sections taken from immediately above the uppermost rosette leaf of 30 cm-tall inflorescence stems. For those genes for which GUS reporter lines were available [45]–[48], corresponding reporter lines were analyzed by using a GUS-specific RNA probe. As a result, for all but three cases in which no mRNA accumulation could be detected (Table 1), mRNA was found to accumulate in a continuous domain consisting of one to three cells in a radial orientation located between xylem and phloem tissue (Figure 4). The domain of RNA accumulation detected for all positive genes was similar to that of the cambium-specific genes PXY and WOX4, suggesting that all genes are similarly cambium-specific or, at least, transcribed in close proximity. This verification of the cambium-specific mRNA accumulation of a large proportion of the genes identified suggests that we had indeed elucidated the mRNA profile established during cambium formation in planta, and that CIS incubation can be used for the identification and analysis of cambium regulators.

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