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Modulation of Arabidopsis and monocot root architecture by CLAVATA3/EMBRYO SURROUNDING REGION 26 peptide.

Czyzewicz N, Shi CL, Vu LD, Van De Cotte B, Hodgman C, Butenko MA, Smet ID - J. Exp. Bot. (2015)

Bottom Line: Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling.In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed.Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Leicestershire LE12 5RD, UK.

No MeSH data available.


CLE26p alanine scanning on A. thaliana. (A) Sequence of synthetic CLE peptides used. (B) Representative pictures of mCLE26p-treated wild-type seedlings at 12 d after germination. (C, D) Quantification of primary root length (C) and emerged lateral root density (D) for mCLE26p-treated wild-type seedlings. The bar graphs indicate the mean ±SE. Statistical significance (Student’s t-test) compared with no peptide (*) and to CLEp treatment (♦) is indicated: ***/♦♦♦P<0.001, */♦P<0.05. Scale bar=1cm. (This figure is available in colour at JXB online.)
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Figure 4: CLE26p alanine scanning on A. thaliana. (A) Sequence of synthetic CLE peptides used. (B) Representative pictures of mCLE26p-treated wild-type seedlings at 12 d after germination. (C, D) Quantification of primary root length (C) and emerged lateral root density (D) for mCLE26p-treated wild-type seedlings. The bar graphs indicate the mean ±SE. Statistical significance (Student’s t-test) compared with no peptide (*) and to CLEp treatment (♦) is indicated: ***/♦♦♦P<0.001, */♦P<0.05. Scale bar=1cm. (This figure is available in colour at JXB online.)

Mentions: Given that application of low—possibly physiologically more relevant—concentrations of CLE26p gave a reduction in primary root length and an increase in lateral root density, it was decided to focus on CLE26p for a more in-depth analysis of the sequence–activity relationship. To identify amino acids critical for CLE26p function, an alanine scan was performed using primary root length and lateral root density as biological assays (Fig. 4A–D; Supplementary Fig. S5 at JXB online). Seedlings grown on media containing 10 μM mCLE26pR1A, mCLE26pP4A, mCLE26pR5A, mCLE26pG6A, mCLE26pD8A, mCLE26pP9A, mCLE26pI10A, mCLE26pH11A, and mCLE26pN12A showed no significant decrease in primary root length compared with untreated A. thaliana, and were significantly different from non-mutated CLE26p treatment (Fig. 4C). The total number of emerged lateral roots was similarly not decreased for these mCLE26p variants compared with the CLE26p control treatment (Supplementary Fig. S5). This suggested that these amino acid residues are critical for CLE26 function. In contrast, amino acid residues 2 and 3 do not appear to be critical for CLE function, since mCLE26pK2A and mCLE26pV3A displayed the same, but a less strong effect on primary root length as the non-mutated CLE26p variant (a decrease of between 52% and 63%) (Fig. 4C). Interestingly, mCLE26pP7A displayed increased activity, with respect to both primary root length (a further decrease of 11%) and lateral root density (a further increase of 32%), compared with wild-type CLE26p (Fig. 4C, D). Finally, mCLE26pD8A resulted in a slightly longer root than untreated seedling roots, but did not have an effect on lateral root density (Fig. 4C, D). Taken together, the series of alanine-substituted CLE26 peptides revealed several amino acids which are critical for bioactivity of CLE26, and pinpointed mCLE26pP7A and mCLE26pD8A as a hyperactive and a possible antagonistic peptide, respectively. Previous analyses of critical amino acid residues in, for example, CLE41/CLE44 [also referred to as TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)] (Ito et al., 2006) and CLV3 (Kondo et al, 2008) identified similar amino acids that are important for the peptide to have the required bioactivity.


Modulation of Arabidopsis and monocot root architecture by CLAVATA3/EMBRYO SURROUNDING REGION 26 peptide.

Czyzewicz N, Shi CL, Vu LD, Van De Cotte B, Hodgman C, Butenko MA, Smet ID - J. Exp. Bot. (2015)

CLE26p alanine scanning on A. thaliana. (A) Sequence of synthetic CLE peptides used. (B) Representative pictures of mCLE26p-treated wild-type seedlings at 12 d after germination. (C, D) Quantification of primary root length (C) and emerged lateral root density (D) for mCLE26p-treated wild-type seedlings. The bar graphs indicate the mean ±SE. Statistical significance (Student’s t-test) compared with no peptide (*) and to CLEp treatment (♦) is indicated: ***/♦♦♦P<0.001, */♦P<0.05. Scale bar=1cm. (This figure is available in colour at JXB online.)
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Related In: Results  -  Collection

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Figure 4: CLE26p alanine scanning on A. thaliana. (A) Sequence of synthetic CLE peptides used. (B) Representative pictures of mCLE26p-treated wild-type seedlings at 12 d after germination. (C, D) Quantification of primary root length (C) and emerged lateral root density (D) for mCLE26p-treated wild-type seedlings. The bar graphs indicate the mean ±SE. Statistical significance (Student’s t-test) compared with no peptide (*) and to CLEp treatment (♦) is indicated: ***/♦♦♦P<0.001, */♦P<0.05. Scale bar=1cm. (This figure is available in colour at JXB online.)
Mentions: Given that application of low—possibly physiologically more relevant—concentrations of CLE26p gave a reduction in primary root length and an increase in lateral root density, it was decided to focus on CLE26p for a more in-depth analysis of the sequence–activity relationship. To identify amino acids critical for CLE26p function, an alanine scan was performed using primary root length and lateral root density as biological assays (Fig. 4A–D; Supplementary Fig. S5 at JXB online). Seedlings grown on media containing 10 μM mCLE26pR1A, mCLE26pP4A, mCLE26pR5A, mCLE26pG6A, mCLE26pD8A, mCLE26pP9A, mCLE26pI10A, mCLE26pH11A, and mCLE26pN12A showed no significant decrease in primary root length compared with untreated A. thaliana, and were significantly different from non-mutated CLE26p treatment (Fig. 4C). The total number of emerged lateral roots was similarly not decreased for these mCLE26p variants compared with the CLE26p control treatment (Supplementary Fig. S5). This suggested that these amino acid residues are critical for CLE26 function. In contrast, amino acid residues 2 and 3 do not appear to be critical for CLE function, since mCLE26pK2A and mCLE26pV3A displayed the same, but a less strong effect on primary root length as the non-mutated CLE26p variant (a decrease of between 52% and 63%) (Fig. 4C). Interestingly, mCLE26pP7A displayed increased activity, with respect to both primary root length (a further decrease of 11%) and lateral root density (a further increase of 32%), compared with wild-type CLE26p (Fig. 4C, D). Finally, mCLE26pD8A resulted in a slightly longer root than untreated seedling roots, but did not have an effect on lateral root density (Fig. 4C, D). Taken together, the series of alanine-substituted CLE26 peptides revealed several amino acids which are critical for bioactivity of CLE26, and pinpointed mCLE26pP7A and mCLE26pD8A as a hyperactive and a possible antagonistic peptide, respectively. Previous analyses of critical amino acid residues in, for example, CLE41/CLE44 [also referred to as TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)] (Ito et al., 2006) and CLV3 (Kondo et al, 2008) identified similar amino acids that are important for the peptide to have the required bioactivity.

Bottom Line: Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling.In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed.Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Leicestershire LE12 5RD, UK.

No MeSH data available.