Abstract
BackgroundThere 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.ResultsWe 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.ConclusionsThe 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.
Highlights
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
We conducted a TBLASTN search in the NCBI/ EMBL/DDBJ gymnosperm Expressed sequence tag (EST) databases for CLEL family members using the A. thaliana CLEL motif sequences
The discovery of genes encoding perfectly conserved CLE41/44-TRACHEARY ELEMENT DIFFERENTIATION (TDIF) peptide orthologues in all the Pinophyta species that are known to have CLAVATA3-EMBRYO-SURROUNDING REGION (CLE) genes is strongly suggestive of a conserved role between conifers and dicots for these peptides in the regulation of vascular cambium homeostasis. This hypothesis is all the more compelling considering that there is essentially no other sequence conservation between these Arabidopsis and conifer gene sequences, suggesting very strong selective pressure for the conservation of the CLE41/44-TDIF peptide sequence among species with a vascular cambium. Consistent with such a hypothesis, all the conifer CLE41/44-TDIF ESTs we identified in our EST database meta-analysis were sourced from RNA isolated from inner bark and/or phloem or root tissues (Additional file 3: Table S1)
Summary
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. The CLE gene protein-coding sequences terminate with the Cterminal amino acid of the mature CLE peptide. Not all CLE genes conform to this paradigm, and C-terminal non-conserved sequences (NCS2) ranging from 3 to 450 nt (1-150 aa) have been observed in CLE genes from various species (Figure 1). These sequences are apparently trimmed from the precursor protein by a carboxypeptidase activity [16,17,18]
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