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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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Characterization of RUL1, MOL1, and PXY expression.(A–C) Detection of RUL1 (A), MOL1 (B) and PXY (C) transcripts by RISH on cross sections of vegetative shoot tips. Asterisks label the apical meristem, arrowheads the cambium-specific signals in leaf bundles. Size bar in (A): 100 µm, same magnification in (A–C). (D) qRT-PCR-based analysis of transcript accumulation in the bottom-most centimeter of the stem and a fragment 3 cm further apically (compare Figure 1C). (E) qRT-PCR-based analysis of RUL1, MOL1, PXY, and WOX4 mRNA abundance in the first internode above the rosette of the corresponding mutants. (F) Genetic interaction between MOL1 and RUL1. Lateral extension of the ICD immediately above the uppermost rosette leaf is shown. (G) Unrooted tree for RUL1, MOL1, and PXY protein sequences, and their closest homologs from Populus trichocarpa based on full length protein sequences. There are two homologs each for MOL1 and PXY with a similar degree of sequence similarity. For RUL1, the situation is less straightforward as another Arabidopsis protein (AT5G58300) belongs to the same sub-clade partly displaying low bootstrap values. The scale bar represents 0.1 amino acid substitutions per position. Bootstrap values are given in percentage.
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pgen-1001312-g006: Characterization of RUL1, MOL1, and PXY expression.(A–C) Detection of RUL1 (A), MOL1 (B) and PXY (C) transcripts by RISH on cross sections of vegetative shoot tips. Asterisks label the apical meristem, arrowheads the cambium-specific signals in leaf bundles. Size bar in (A): 100 µm, same magnification in (A–C). (D) qRT-PCR-based analysis of transcript accumulation in the bottom-most centimeter of the stem and a fragment 3 cm further apically (compare Figure 1C). (E) qRT-PCR-based analysis of RUL1, MOL1, PXY, and WOX4 mRNA abundance in the first internode above the rosette of the corresponding mutants. (F) Genetic interaction between MOL1 and RUL1. Lateral extension of the ICD immediately above the uppermost rosette leaf is shown. (G) Unrooted tree for RUL1, MOL1, and PXY protein sequences, and their closest homologs from Populus trichocarpa based on full length protein sequences. There are two homologs each for MOL1 and PXY with a similar degree of sequence similarity. For RUL1, the situation is less straightforward as another Arabidopsis protein (AT5G58300) belongs to the same sub-clade partly displaying low bootstrap values. The scale bar represents 0.1 amino acid substitutions per position. Bootstrap values are given in percentage.

Mentions: To analyze the interaction of both signaling components, we first determined whether they are generally co-expressed by performing RISH in vegetative shoot tips. This revealed that RUL1 mRNA localizes to the (pro)cambium mainly in more mature leaf primordia (Figure 6A). In contrast, no MOL1 mRNA accumulation was detected in the apex, suggesting that it is specific to the cambium in the stem (Figure 6B). Both expression domains are unlike the PXY expression domain, which is found in procambium strands starting in very young leaf primordia (Figure 6C). However, qRT-PCR analyses showed that the mRNA abundance of all three genes correlates positively with the progression of secondary growth along the stem (Figure 6D). Analysis of transcript accumulation in the respective mutant backgrounds revealed an increase of RUL1 and PXY mRNA abundance in mol1-1 (Figure 6E). As a possible downstream target of the identified receptors, we also analyzed WOX4 transcript abundance in rul1-2, mol1-1, and pxy-4 backgrounds. Consistent with a role of PXY as an activator of WOX4[29], the analysis revealed decreased WOX4 transcript levels in pxy-4 mutants (Figure 6E). In contrast, as for RUL1 and PXY mRNAs, an increase of WOX4 mRNA levels in mol1-1 backgrounds was detected, whereas the level was almost unchanged in rul1-2 (Figure 6E). The up-regulation of this selection of cambium-expressed genes in mol1 confirms a role of MOL1 as a negative regulator of cambium activity, at least partly, upstream of RUL1 and PXY. To test whether MOL1 and RUL1 function in a linear mode of action or rather in parallel pathways, we analyzed the genetic interaction between MOL1 and RUL1. The analysis of the mol1-1 rul1-1 double mutant revealed a wild-type-like rate of vascular tissue production in interfascicular regions in comparison to the respective single mutants (Figure 6F). Taken together, we conclude that there is no direct and exclusive dependence between MOL1 and RUL1 activity, but that both genes function in parallel in the cambium to balance the production of secondary vascular tissues.


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)

Characterization of RUL1, MOL1, and PXY expression.(A–C) Detection of RUL1 (A), MOL1 (B) and PXY (C) transcripts by RISH on cross sections of vegetative shoot tips. Asterisks label the apical meristem, arrowheads the cambium-specific signals in leaf bundles. Size bar in (A): 100 µm, same magnification in (A–C). (D) qRT-PCR-based analysis of transcript accumulation in the bottom-most centimeter of the stem and a fragment 3 cm further apically (compare Figure 1C). (E) qRT-PCR-based analysis of RUL1, MOL1, PXY, and WOX4 mRNA abundance in the first internode above the rosette of the corresponding mutants. (F) Genetic interaction between MOL1 and RUL1. Lateral extension of the ICD immediately above the uppermost rosette leaf is shown. (G) Unrooted tree for RUL1, MOL1, and PXY protein sequences, and their closest homologs from Populus trichocarpa based on full length protein sequences. There are two homologs each for MOL1 and PXY with a similar degree of sequence similarity. For RUL1, the situation is less straightforward as another Arabidopsis protein (AT5G58300) belongs to the same sub-clade partly displaying low bootstrap values. The scale bar represents 0.1 amino acid substitutions per position. Bootstrap values are given in percentage.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001312-g006: Characterization of RUL1, MOL1, and PXY expression.(A–C) Detection of RUL1 (A), MOL1 (B) and PXY (C) transcripts by RISH on cross sections of vegetative shoot tips. Asterisks label the apical meristem, arrowheads the cambium-specific signals in leaf bundles. Size bar in (A): 100 µm, same magnification in (A–C). (D) qRT-PCR-based analysis of transcript accumulation in the bottom-most centimeter of the stem and a fragment 3 cm further apically (compare Figure 1C). (E) qRT-PCR-based analysis of RUL1, MOL1, PXY, and WOX4 mRNA abundance in the first internode above the rosette of the corresponding mutants. (F) Genetic interaction between MOL1 and RUL1. Lateral extension of the ICD immediately above the uppermost rosette leaf is shown. (G) Unrooted tree for RUL1, MOL1, and PXY protein sequences, and their closest homologs from Populus trichocarpa based on full length protein sequences. There are two homologs each for MOL1 and PXY with a similar degree of sequence similarity. For RUL1, the situation is less straightforward as another Arabidopsis protein (AT5G58300) belongs to the same sub-clade partly displaying low bootstrap values. The scale bar represents 0.1 amino acid substitutions per position. Bootstrap values are given in percentage.
Mentions: To analyze the interaction of both signaling components, we first determined whether they are generally co-expressed by performing RISH in vegetative shoot tips. This revealed that RUL1 mRNA localizes to the (pro)cambium mainly in more mature leaf primordia (Figure 6A). In contrast, no MOL1 mRNA accumulation was detected in the apex, suggesting that it is specific to the cambium in the stem (Figure 6B). Both expression domains are unlike the PXY expression domain, which is found in procambium strands starting in very young leaf primordia (Figure 6C). However, qRT-PCR analyses showed that the mRNA abundance of all three genes correlates positively with the progression of secondary growth along the stem (Figure 6D). Analysis of transcript accumulation in the respective mutant backgrounds revealed an increase of RUL1 and PXY mRNA abundance in mol1-1 (Figure 6E). As a possible downstream target of the identified receptors, we also analyzed WOX4 transcript abundance in rul1-2, mol1-1, and pxy-4 backgrounds. Consistent with a role of PXY as an activator of WOX4[29], the analysis revealed decreased WOX4 transcript levels in pxy-4 mutants (Figure 6E). In contrast, as for RUL1 and PXY mRNAs, an increase of WOX4 mRNA levels in mol1-1 backgrounds was detected, whereas the level was almost unchanged in rul1-2 (Figure 6E). The up-regulation of this selection of cambium-expressed genes in mol1 confirms a role of MOL1 as a negative regulator of cambium activity, at least partly, upstream of RUL1 and PXY. To test whether MOL1 and RUL1 function in a linear mode of action or rather in parallel pathways, we analyzed the genetic interaction between MOL1 and RUL1. The analysis of the mol1-1 rul1-1 double mutant revealed a wild-type-like rate of vascular tissue production in interfascicular regions in comparison to the respective single mutants (Figure 6F). Taken together, we conclude that there is no direct and exclusive dependence between MOL1 and RUL1 activity, but that both genes function in parallel in the cambium to balance the production of secondary vascular tissues.

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