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The Populus Class III HD ZIP transcription factor POPCORONA affects cell differentiation during secondary growth of woody stems.

Du J, Miura E, Robischon M, Martinez C, Groover A - PLoS ONE (2011)

Bottom Line: Synthetic miRNA knock down of POPCORONA results in abnormal lignification in cells of the pith, while overexpression of a miRNA-resistant POPCORONA results in delayed lignification of xylem and phloem fibers during secondary growth.POPCORONA misexpression also results in coordinated changes in expression of genes within a previously described transcriptional network regulating cell differentiation and cell wall biosynthesis, and hormone-related genes associated with fiber differentiation.POPCORONA illustrates another function of Class III HD ZIPs: regulating cell differentiation during secondary growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Forest Genetics, Pacific Southwest Research Station, U.S. Forest Service, Davis, California, United States of America.

ABSTRACT
The developmental mechanisms regulating cell differentiation and patterning during the secondary growth of woody tissues are poorly understood. Class III HD ZIP transcription factors are evolutionarily ancient and play fundamental roles in various aspects of plant development. Here we investigate the role of a Class III HD ZIP transcription factor, POPCORONA, during secondary growth of woody stems. Transgenic Populus (poplar) trees expressing either a miRNA-resistant POPCORONA or a synthetic miRNA targeting POPCORONA were used to infer function of POPCORONA during secondary growth. Whole plant, histological, and gene expression changes were compared for transgenic and wild-type control plants. Synthetic miRNA knock down of POPCORONA results in abnormal lignification in cells of the pith, while overexpression of a miRNA-resistant POPCORONA results in delayed lignification of xylem and phloem fibers during secondary growth. POPCORONA misexpression also results in coordinated changes in expression of genes within a previously described transcriptional network regulating cell differentiation and cell wall biosynthesis, and hormone-related genes associated with fiber differentiation. POPCORONA illustrates another function of Class III HD ZIPs: regulating cell differentiation during secondary growth.

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Expression of PCN during Populus stem development revealed by whole mount in situ hybridization.Antisense PCN (first and second columns), sense negative control (third column), and positive control (fourth column) probes were hybridized to stem sections from two month old tissue culture grown trees. (a) Section from first elongating internode hybridized with antisense PCN probe. PCN is expressed broadly during primary growth, with strongest expression associated with procambium. (b) Higher magnification of first elongating internode hybridized with antisense PCN probe. (c) Section from first elongating internode hybridized with sense PCN probe (negative control), showing minimal background hybridization. (d) Section from first elongating internode hybridized with antisense pop50S probe (positive control). (e) Section from the fourth internode, hybridized with antisense PCN probe. PCN is expressed broadly in the cambial zone, and strongly in differentiating xylem. (f) Higher magnification of (e). (g) Section from the fourth internode hybridized with negative control sense PCN probe. (h) Section from fourth internode hybridized with positive control antisense pop50S probe. (i) Section from seventh internode hybridized with antisense PCN probe. PCN expression is mostly associated with differentiating xylem cells and lightly in cambial zone. (j) Higher magnification of (i). (k) Section from seventh internode hybridized with sense PCN probe (negative control). (l) Section from seventh internode hybridized with positive control antisense pop50S probe. (m) Section from the base internode hybridized with antisense PCN probe. PCN expression is largely limited to the differentiating xylem cells and cambial zone. (n) Higher magnification of (m). (o) Section from the base internode hybridized with sense PCN probe (negative control). (p) Section from the base internode hybridized with positive control antisense pop50S probe. Cambial zone (Ca), Phloem fiber (Pf), Procambium (Pc), Ray (r), Xylem (Xy), Bar = 100 µm.
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pone-0017458-g003: Expression of PCN during Populus stem development revealed by whole mount in situ hybridization.Antisense PCN (first and second columns), sense negative control (third column), and positive control (fourth column) probes were hybridized to stem sections from two month old tissue culture grown trees. (a) Section from first elongating internode hybridized with antisense PCN probe. PCN is expressed broadly during primary growth, with strongest expression associated with procambium. (b) Higher magnification of first elongating internode hybridized with antisense PCN probe. (c) Section from first elongating internode hybridized with sense PCN probe (negative control), showing minimal background hybridization. (d) Section from first elongating internode hybridized with antisense pop50S probe (positive control). (e) Section from the fourth internode, hybridized with antisense PCN probe. PCN is expressed broadly in the cambial zone, and strongly in differentiating xylem. (f) Higher magnification of (e). (g) Section from the fourth internode hybridized with negative control sense PCN probe. (h) Section from fourth internode hybridized with positive control antisense pop50S probe. (i) Section from seventh internode hybridized with antisense PCN probe. PCN expression is mostly associated with differentiating xylem cells and lightly in cambial zone. (j) Higher magnification of (i). (k) Section from seventh internode hybridized with sense PCN probe (negative control). (l) Section from seventh internode hybridized with positive control antisense pop50S probe. (m) Section from the base internode hybridized with antisense PCN probe. PCN expression is largely limited to the differentiating xylem cells and cambial zone. (n) Higher magnification of (m). (o) Section from the base internode hybridized with sense PCN probe (negative control). (p) Section from the base internode hybridized with positive control antisense pop50S probe. Cambial zone (Ca), Phloem fiber (Pf), Procambium (Pc), Ray (r), Xylem (Xy), Bar = 100 µm.

Mentions: PCN is expressed broadly in the cambial zone and xylem of Populus shoots. Whole mount in situ hybridization was used to visualize PCN expression in tangential sections from different developmental stages of Populus stems (Materials and Methods). During primary growth and the transition to secondary growth, PCN is expressed broadly in the cambial zone and in developing xylem (Fig. 3a,b). Later in development, phloem fiber differentiation becomes evident (Fig. 3e,f) and weak signal is seen outside of the cambial zone at this stage in the developing phloem, including the phloem fibers (Fig. 3e,f). Moving further down the stem into developmentally older tissues, PCN expression is maintained within developing xylem, and is most pronounced in rays (Fig. 3 i,j). At the base of the stem, strongest expression is found in the cambial zone, with reduced expression in the secondary xylem (Fig. 3m,n). However, expression is still pronounced within the rays traversing the secondary xylem (Fig. 3n). In comparison to sense-probe negative controls (Fig. 3c,g,k,o), the in situ staining of experimental sections with the PCN antisense probe is specific and has relatively low background. However, based on comparison of PCN antisense and sense negative control sections we cannot exclude the possibility of low PCN expression, cross hybridization to related genes, or diffusion of the stain in epidermis, pith cells, or other tissues/cell types. Differential staining caused by differences in cytoplasmic density of cell types is revealed by an antisense probe for a presumably ubiquitously expressed gene encoding a 50S ribosomal protein (Fig. 3d,h,l,p).


The Populus Class III HD ZIP transcription factor POPCORONA affects cell differentiation during secondary growth of woody stems.

Du J, Miura E, Robischon M, Martinez C, Groover A - PLoS ONE (2011)

Expression of PCN during Populus stem development revealed by whole mount in situ hybridization.Antisense PCN (first and second columns), sense negative control (third column), and positive control (fourth column) probes were hybridized to stem sections from two month old tissue culture grown trees. (a) Section from first elongating internode hybridized with antisense PCN probe. PCN is expressed broadly during primary growth, with strongest expression associated with procambium. (b) Higher magnification of first elongating internode hybridized with antisense PCN probe. (c) Section from first elongating internode hybridized with sense PCN probe (negative control), showing minimal background hybridization. (d) Section from first elongating internode hybridized with antisense pop50S probe (positive control). (e) Section from the fourth internode, hybridized with antisense PCN probe. PCN is expressed broadly in the cambial zone, and strongly in differentiating xylem. (f) Higher magnification of (e). (g) Section from the fourth internode hybridized with negative control sense PCN probe. (h) Section from fourth internode hybridized with positive control antisense pop50S probe. (i) Section from seventh internode hybridized with antisense PCN probe. PCN expression is mostly associated with differentiating xylem cells and lightly in cambial zone. (j) Higher magnification of (i). (k) Section from seventh internode hybridized with sense PCN probe (negative control). (l) Section from seventh internode hybridized with positive control antisense pop50S probe. (m) Section from the base internode hybridized with antisense PCN probe. PCN expression is largely limited to the differentiating xylem cells and cambial zone. (n) Higher magnification of (m). (o) Section from the base internode hybridized with sense PCN probe (negative control). (p) Section from the base internode hybridized with positive control antisense pop50S probe. Cambial zone (Ca), Phloem fiber (Pf), Procambium (Pc), Ray (r), Xylem (Xy), Bar = 100 µm.
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Related In: Results  -  Collection

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pone-0017458-g003: Expression of PCN during Populus stem development revealed by whole mount in situ hybridization.Antisense PCN (first and second columns), sense negative control (third column), and positive control (fourth column) probes were hybridized to stem sections from two month old tissue culture grown trees. (a) Section from first elongating internode hybridized with antisense PCN probe. PCN is expressed broadly during primary growth, with strongest expression associated with procambium. (b) Higher magnification of first elongating internode hybridized with antisense PCN probe. (c) Section from first elongating internode hybridized with sense PCN probe (negative control), showing minimal background hybridization. (d) Section from first elongating internode hybridized with antisense pop50S probe (positive control). (e) Section from the fourth internode, hybridized with antisense PCN probe. PCN is expressed broadly in the cambial zone, and strongly in differentiating xylem. (f) Higher magnification of (e). (g) Section from the fourth internode hybridized with negative control sense PCN probe. (h) Section from fourth internode hybridized with positive control antisense pop50S probe. (i) Section from seventh internode hybridized with antisense PCN probe. PCN expression is mostly associated with differentiating xylem cells and lightly in cambial zone. (j) Higher magnification of (i). (k) Section from seventh internode hybridized with sense PCN probe (negative control). (l) Section from seventh internode hybridized with positive control antisense pop50S probe. (m) Section from the base internode hybridized with antisense PCN probe. PCN expression is largely limited to the differentiating xylem cells and cambial zone. (n) Higher magnification of (m). (o) Section from the base internode hybridized with sense PCN probe (negative control). (p) Section from the base internode hybridized with positive control antisense pop50S probe. Cambial zone (Ca), Phloem fiber (Pf), Procambium (Pc), Ray (r), Xylem (Xy), Bar = 100 µm.
Mentions: PCN is expressed broadly in the cambial zone and xylem of Populus shoots. Whole mount in situ hybridization was used to visualize PCN expression in tangential sections from different developmental stages of Populus stems (Materials and Methods). During primary growth and the transition to secondary growth, PCN is expressed broadly in the cambial zone and in developing xylem (Fig. 3a,b). Later in development, phloem fiber differentiation becomes evident (Fig. 3e,f) and weak signal is seen outside of the cambial zone at this stage in the developing phloem, including the phloem fibers (Fig. 3e,f). Moving further down the stem into developmentally older tissues, PCN expression is maintained within developing xylem, and is most pronounced in rays (Fig. 3 i,j). At the base of the stem, strongest expression is found in the cambial zone, with reduced expression in the secondary xylem (Fig. 3m,n). However, expression is still pronounced within the rays traversing the secondary xylem (Fig. 3n). In comparison to sense-probe negative controls (Fig. 3c,g,k,o), the in situ staining of experimental sections with the PCN antisense probe is specific and has relatively low background. However, based on comparison of PCN antisense and sense negative control sections we cannot exclude the possibility of low PCN expression, cross hybridization to related genes, or diffusion of the stain in epidermis, pith cells, or other tissues/cell types. Differential staining caused by differences in cytoplasmic density of cell types is revealed by an antisense probe for a presumably ubiquitously expressed gene encoding a 50S ribosomal protein (Fig. 3d,h,l,p).

Bottom Line: Synthetic miRNA knock down of POPCORONA results in abnormal lignification in cells of the pith, while overexpression of a miRNA-resistant POPCORONA results in delayed lignification of xylem and phloem fibers during secondary growth.POPCORONA misexpression also results in coordinated changes in expression of genes within a previously described transcriptional network regulating cell differentiation and cell wall biosynthesis, and hormone-related genes associated with fiber differentiation.POPCORONA illustrates another function of Class III HD ZIPs: regulating cell differentiation during secondary growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Forest Genetics, Pacific Southwest Research Station, U.S. Forest Service, Davis, California, United States of America.

ABSTRACT
The developmental mechanisms regulating cell differentiation and patterning during the secondary growth of woody tissues are poorly understood. Class III HD ZIP transcription factors are evolutionarily ancient and play fundamental roles in various aspects of plant development. Here we investigate the role of a Class III HD ZIP transcription factor, POPCORONA, during secondary growth of woody stems. Transgenic Populus (poplar) trees expressing either a miRNA-resistant POPCORONA or a synthetic miRNA targeting POPCORONA were used to infer function of POPCORONA during secondary growth. Whole plant, histological, and gene expression changes were compared for transgenic and wild-type control plants. Synthetic miRNA knock down of POPCORONA results in abnormal lignification in cells of the pith, while overexpression of a miRNA-resistant POPCORONA results in delayed lignification of xylem and phloem fibers during secondary growth. POPCORONA misexpression also results in coordinated changes in expression of genes within a previously described transcriptional network regulating cell differentiation and cell wall biosynthesis, and hormone-related genes associated with fiber differentiation. POPCORONA illustrates another function of Class III HD ZIPs: regulating cell differentiation during secondary growth.

Show MeSH