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Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and the CLE-LIKE signal peptide genes in the Pinophyta.

Strabala TJ, Phillips L, West M, Stanbra L - BMC Plant Biol. (2014)

Bottom Line: The CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species.The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants.Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scion, 49 Sala St, PO Box 3020, Rotorua 3010, New Zealand. strabala@clear.net.nz.

ABSTRACT

Background: There is a rapidly growing awareness that plant peptide signalling molecules are numerous and varied and they are known to play fundamental roles in angiosperm plant growth and development. Two closely related peptide signalling molecule families are the CLAVATA3-EMBRYO-SURROUNDING REGION (CLE) and CLE-LIKE (CLEL) genes, which encode precursors of secreted peptide ligands that have roles in meristem maintenance and root gravitropism. Progress in peptide signalling molecule research in gymnosperms has lagged behind that of angiosperms. We therefore sought to identify CLE and CLEL genes in gymnosperms and conduct a comparative analysis of these gene families with angiosperms.

Results: We undertook a meta-analysis of the GenBank/EMBL/DDBJ gymnosperm EST database and the Picea abies and P. glauca genomes and identified 93 putative CLE genes and 11 CLEL genes among eight Pinophyta species, in the genera Cryptomeria, Pinus and Picea. The predicted conifer CLE and CLEL protein sequences had close phylogenetic relationships with their homologues in Arabidopsis. Notably, perfect conservation of the active CLE dodecapeptide in presumed orthologues of the Arabidopsis CLE41/44-TRACHEARY ELEMENT DIFFERENTIATION (TDIF) protein, an inhibitor of tracheary element (xylem) differentiation, was seen in all eight conifer species. We cloned the Pinus radiata CLE41/44-TDIF orthologues. These genes were preferentially expressed in phloem in planta as expected, but unexpectedly, also in differentiating tracheary element (TE) cultures. Surprisingly, transcript abundances of these TE differentiation-inhibitors sharply increased during early TE differentiation, suggesting that some cells differentiate into phloem cells in addition to TEs in these cultures. Applied CLE13 and CLE41/44 peptides inhibited root elongation in Pinus radiata seedlings. We show evidence that two CLEL genes are alternatively spliced via 3'-terminal acceptor exons encoding separate CLEL peptides.

Conclusions: The CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species. Only one CLE peptide sequence has been 100% conserved between gymnosperms and angiosperms over 300 million years of evolutionary history, the CLE41/44-TDIF peptide and its likely conifer orthologues. The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants. Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.

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Multiple alignment of Arabidopsis and predicted Pinophyta CLEL protein amino acid sequences. A schematic diagram of a generic CLEL protein representing the main features of CLEL proteins is shown above the alignment. The amino (H2N) terminus of the schematic protein is followed by the signal peptide (SP), the first non-conserved sequence (NCS1), the CLEL domain (CLD) and the second non-conserved sequence (NCS2) found at the COOH terminus of some CLEL proteins. Presumed cleavage sites of the SP and the mature CLEL peptide sequence are indicated by large arrowheads. The multiple alignment depicts the individual SPs of each putative full-length protein sequence with grey highlighting. The SignalP 4.1-predicted cleavage sites are indicated by the small arrowheads. The CLEL motif, including the two conserved asp-tyr amino acids at the amino termini of the predicted CLEL peptides, is indicated by white lettering. The predicted CLEL peptides are indicated by black highlighting except for the asp-tyr sequence, which is indicated by brick red highlighting.
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Figure 2: Multiple alignment of Arabidopsis and predicted Pinophyta CLEL protein amino acid sequences. A schematic diagram of a generic CLEL protein representing the main features of CLEL proteins is shown above the alignment. The amino (H2N) terminus of the schematic protein is followed by the signal peptide (SP), the first non-conserved sequence (NCS1), the CLEL domain (CLD) and the second non-conserved sequence (NCS2) found at the COOH terminus of some CLEL proteins. Presumed cleavage sites of the SP and the mature CLEL peptide sequence are indicated by large arrowheads. The multiple alignment depicts the individual SPs of each putative full-length protein sequence with grey highlighting. The SignalP 4.1-predicted cleavage sites are indicated by the small arrowheads. The CLEL motif, including the two conserved asp-tyr amino acids at the amino termini of the predicted CLEL peptides, is indicated by white lettering. The predicted CLEL peptides are indicated by black highlighting except for the asp-tyr sequence, which is indicated by brick red highlighting.

Mentions: In contrast to the CLE family, the ROOT GROWTH FACTOR/CLE-LIKE/GOLVEN (RGF/CLEL/GLV) gene family has only recently been identified and described[19-21]. Like the CLE genes, they encode short, secreted peptides that affect aspects of plant development. Structurally, the RGF/CLEL/GLV genes are similar to the CLE genes in that they encode precursor proteins with a signal peptide, followed by an NCS1 region with a C-terminally oriented 12-15 aa peptide that is post-translationally processed to the active form (Figure 2). Also like the CLE genes, some CLEL genes encode proteins with C-terminal NCS2 regions of varying lengths (Figure 2). The CLEL peptides, as their name suggests, have very similar sequences to the CLE peptides. A key difference between the CLE and CLEL peptides is that the CLEL peptides are variable in length at 13-16 amino acids, as compared to the 12 amino acids of the CLE peptides. Perhaps the most salient distinguishing feature between CLE and CLEL peptides is the aspartic acid-tyrosine pair at the N-termini of all but one the RGF/CLEL/GLV active peptides. The sole exception to this rule is found in the GLV9 peptide, which contains a functionally conserved glutamic acid residue at its N-terminus in place of aspartic acid[21]. At least some of the CLEL peptides are post-translationally tyrosine sulphated, which is essential for aspects of their activity in vivo, including RAM homeostasis[19]. Interestingly, the conserved amino-terminal asp-tyr pair of the CLEL peptides is a characteristic shared with the sulphotyrosine peptide ligands PLANT PEPTIDE CONTAINING SULFATED TYROSINE 1 (PSY1) and PHYTOSULFOKINE (PSK)[22,23]. However, PSK and PSY1 are not otherwise similar to the CLEL peptides.


Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and the CLE-LIKE signal peptide genes in the Pinophyta.

Strabala TJ, Phillips L, West M, Stanbra L - BMC Plant Biol. (2014)

Multiple alignment of Arabidopsis and predicted Pinophyta CLEL protein amino acid sequences. A schematic diagram of a generic CLEL protein representing the main features of CLEL proteins is shown above the alignment. The amino (H2N) terminus of the schematic protein is followed by the signal peptide (SP), the first non-conserved sequence (NCS1), the CLEL domain (CLD) and the second non-conserved sequence (NCS2) found at the COOH terminus of some CLEL proteins. Presumed cleavage sites of the SP and the mature CLEL peptide sequence are indicated by large arrowheads. The multiple alignment depicts the individual SPs of each putative full-length protein sequence with grey highlighting. The SignalP 4.1-predicted cleavage sites are indicated by the small arrowheads. The CLEL motif, including the two conserved asp-tyr amino acids at the amino termini of the predicted CLEL peptides, is indicated by white lettering. The predicted CLEL peptides are indicated by black highlighting except for the asp-tyr sequence, which is indicated by brick red highlighting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016512&req=5

Figure 2: Multiple alignment of Arabidopsis and predicted Pinophyta CLEL protein amino acid sequences. A schematic diagram of a generic CLEL protein representing the main features of CLEL proteins is shown above the alignment. The amino (H2N) terminus of the schematic protein is followed by the signal peptide (SP), the first non-conserved sequence (NCS1), the CLEL domain (CLD) and the second non-conserved sequence (NCS2) found at the COOH terminus of some CLEL proteins. Presumed cleavage sites of the SP and the mature CLEL peptide sequence are indicated by large arrowheads. The multiple alignment depicts the individual SPs of each putative full-length protein sequence with grey highlighting. The SignalP 4.1-predicted cleavage sites are indicated by the small arrowheads. The CLEL motif, including the two conserved asp-tyr amino acids at the amino termini of the predicted CLEL peptides, is indicated by white lettering. The predicted CLEL peptides are indicated by black highlighting except for the asp-tyr sequence, which is indicated by brick red highlighting.
Mentions: In contrast to the CLE family, the ROOT GROWTH FACTOR/CLE-LIKE/GOLVEN (RGF/CLEL/GLV) gene family has only recently been identified and described[19-21]. Like the CLE genes, they encode short, secreted peptides that affect aspects of plant development. Structurally, the RGF/CLEL/GLV genes are similar to the CLE genes in that they encode precursor proteins with a signal peptide, followed by an NCS1 region with a C-terminally oriented 12-15 aa peptide that is post-translationally processed to the active form (Figure 2). Also like the CLE genes, some CLEL genes encode proteins with C-terminal NCS2 regions of varying lengths (Figure 2). The CLEL peptides, as their name suggests, have very similar sequences to the CLE peptides. A key difference between the CLE and CLEL peptides is that the CLEL peptides are variable in length at 13-16 amino acids, as compared to the 12 amino acids of the CLE peptides. Perhaps the most salient distinguishing feature between CLE and CLEL peptides is the aspartic acid-tyrosine pair at the N-termini of all but one the RGF/CLEL/GLV active peptides. The sole exception to this rule is found in the GLV9 peptide, which contains a functionally conserved glutamic acid residue at its N-terminus in place of aspartic acid[21]. At least some of the CLEL peptides are post-translationally tyrosine sulphated, which is essential for aspects of their activity in vivo, including RAM homeostasis[19]. Interestingly, the conserved amino-terminal asp-tyr pair of the CLEL peptides is a characteristic shared with the sulphotyrosine peptide ligands PLANT PEPTIDE CONTAINING SULFATED TYROSINE 1 (PSY1) and PHYTOSULFOKINE (PSK)[22,23]. However, PSK and PSY1 are not otherwise similar to the CLEL peptides.

Bottom Line: The CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species.The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants.Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.

View Article: PubMed Central - HTML - PubMed

Affiliation: Scion, 49 Sala St, PO Box 3020, Rotorua 3010, New Zealand. strabala@clear.net.nz.

ABSTRACT

Background: There is a rapidly growing awareness that plant peptide signalling molecules are numerous and varied and they are known to play fundamental roles in angiosperm plant growth and development. Two closely related peptide signalling molecule families are the CLAVATA3-EMBRYO-SURROUNDING REGION (CLE) and CLE-LIKE (CLEL) genes, which encode precursors of secreted peptide ligands that have roles in meristem maintenance and root gravitropism. Progress in peptide signalling molecule research in gymnosperms has lagged behind that of angiosperms. We therefore sought to identify CLE and CLEL genes in gymnosperms and conduct a comparative analysis of these gene families with angiosperms.

Results: We undertook a meta-analysis of the GenBank/EMBL/DDBJ gymnosperm EST database and the Picea abies and P. glauca genomes and identified 93 putative CLE genes and 11 CLEL genes among eight Pinophyta species, in the genera Cryptomeria, Pinus and Picea. The predicted conifer CLE and CLEL protein sequences had close phylogenetic relationships with their homologues in Arabidopsis. Notably, perfect conservation of the active CLE dodecapeptide in presumed orthologues of the Arabidopsis CLE41/44-TRACHEARY ELEMENT DIFFERENTIATION (TDIF) protein, an inhibitor of tracheary element (xylem) differentiation, was seen in all eight conifer species. We cloned the Pinus radiata CLE41/44-TDIF orthologues. These genes were preferentially expressed in phloem in planta as expected, but unexpectedly, also in differentiating tracheary element (TE) cultures. Surprisingly, transcript abundances of these TE differentiation-inhibitors sharply increased during early TE differentiation, suggesting that some cells differentiate into phloem cells in addition to TEs in these cultures. Applied CLE13 and CLE41/44 peptides inhibited root elongation in Pinus radiata seedlings. We show evidence that two CLEL genes are alternatively spliced via 3'-terminal acceptor exons encoding separate CLEL peptides.

Conclusions: The CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species. Only one CLE peptide sequence has been 100% conserved between gymnosperms and angiosperms over 300 million years of evolutionary history, the CLE41/44-TDIF peptide and its likely conifer orthologues. The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants. Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.

Show MeSH