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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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Synthetic CLE peptide treatments of Pinus radiata seedlings. Seedlings were either mock-inoculated (water, no peptide) (A), CLE13 (B,F), CLE41 (C,G), or both CLE13 and CLE41 combined (D,H). Peptide inoculations were with 10 μM (1X) peptide(s) (B,C,D), or 100 μM (10X) (F,G,H) peptide(s). E. Plot of average root lengths observed in A-D, F-H, with corresponding lettering. Error bars represent the standard deviations.
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Figure 6: Synthetic CLE peptide treatments of Pinus radiata seedlings. Seedlings were either mock-inoculated (water, no peptide) (A), CLE13 (B,F), CLE41 (C,G), or both CLE13 and CLE41 combined (D,H). Peptide inoculations were with 10 μM (1X) peptide(s) (B,C,D), or 100 μM (10X) (F,G,H) peptide(s). E. Plot of average root lengths observed in A-D, F-H, with corresponding lettering. Error bars represent the standard deviations.

Mentions: To begin to assess whether the close sequence conservation of the predicted CLE peptides between Arabidopsis and the Pinophyta conferred similar phenotypic effects on pine seedlings to those observed in Arabidopsis, we applied two synthetic CLE peptides, CLE13 and CLE41/44-TDIF to in vitro-germinated Pinus radiata zygotic embryos. These two peptides were chosen since they were either identical (CLE41/44), or differing by only one amino acid (CLE13) from predicted conifer CLE peptides (Figure 5). Additionally, these peptides belong to important CLE subfamilies that exert opposite effects on root growth, yet have been demonstrated to exert synergistic effects on the development of vascular tissue in Arabidopsis[28]. As in Arabidopsis seedlings, the CLE13 peptide inhibited root elongation at concentrations as low as 10 μM (Figure 6B,E,F). CLE41/44-TDIF also inhibited root elongation in germinated pine zygotic embryos, and its effect was indistinguishable from CLE13 at 100 μM concentration (Figure 6C,E,G). Combining the CLE13 and CLE41 peptides resulted in essentially the same effect as application of CLE13 alone, although some root elongation was observed in the 100 μM dual application (Figure 6D,E,H). No reproducible effect on vascular tissue either in the root or the shoot was observed in these plants (data not shown).


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)

Synthetic CLE peptide treatments of Pinus radiata seedlings. Seedlings were either mock-inoculated (water, no peptide) (A), CLE13 (B,F), CLE41 (C,G), or both CLE13 and CLE41 combined (D,H). Peptide inoculations were with 10 μM (1X) peptide(s) (B,C,D), or 100 μM (10X) (F,G,H) peptide(s). E. Plot of average root lengths observed in A-D, F-H, with corresponding lettering. Error bars represent the standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Synthetic CLE peptide treatments of Pinus radiata seedlings. Seedlings were either mock-inoculated (water, no peptide) (A), CLE13 (B,F), CLE41 (C,G), or both CLE13 and CLE41 combined (D,H). Peptide inoculations were with 10 μM (1X) peptide(s) (B,C,D), or 100 μM (10X) (F,G,H) peptide(s). E. Plot of average root lengths observed in A-D, F-H, with corresponding lettering. Error bars represent the standard deviations.
Mentions: To begin to assess whether the close sequence conservation of the predicted CLE peptides between Arabidopsis and the Pinophyta conferred similar phenotypic effects on pine seedlings to those observed in Arabidopsis, we applied two synthetic CLE peptides, CLE13 and CLE41/44-TDIF to in vitro-germinated Pinus radiata zygotic embryos. These two peptides were chosen since they were either identical (CLE41/44), or differing by only one amino acid (CLE13) from predicted conifer CLE peptides (Figure 5). Additionally, these peptides belong to important CLE subfamilies that exert opposite effects on root growth, yet have been demonstrated to exert synergistic effects on the development of vascular tissue in Arabidopsis[28]. As in Arabidopsis seedlings, the CLE13 peptide inhibited root elongation at concentrations as low as 10 μM (Figure 6B,E,F). CLE41/44-TDIF also inhibited root elongation in germinated pine zygotic embryos, and its effect was indistinguishable from CLE13 at 100 μM concentration (Figure 6C,E,G). Combining the CLE13 and CLE41 peptides resulted in essentially the same effect as application of CLE13 alone, although some root elongation was observed in the 100 μM dual application (Figure 6D,E,H). No reproducible effect on vascular tissue either in the root or the shoot was observed in these plants (data not shown).

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