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CLAVATA1-LIKE, a leucine-rich-repeat protein receptor kinase gene differentially expressed during adventitious caulogenesis in Pinus pinaster and Pinus pinea

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Abstract

Molecular cloning and characterization of a CLAVATA1-LIKE gene in Pinus pinaster and Pinus pinea is reported. CLAVATA1 is a well-characterized gene in Arabidopsis integral to the balance between primordial differentiation and meristem proliferation. Currently, it is not known if the Arabidopsis model of in vitro caulogenesis is applicable to conifers. In this work, we study the putative role of the CLAVATA1-LIKE gene during caulogenic induction in cotyledons of P. pinea and P. pinaster after exposure to benzyladenine. Expression analysis showed that the gene was differentially expressed during the first phases of adventitious caulogenesis in cotyledons from both species, suggesting that CLAVATA1-LIKE may play a role in caulogenesis in conifers. A binary vector carrying CLAVATA1-LIKE promoter driven GFP:GUS expression was constructed. Results of genetic transformation showed GUS activity during somatic embryogenic mass proliferation and embryo maturation.

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Acknowledgments

We thank Dr. Kevin Dalton for proofreading the manuscript and Dr. Ruben Alvarez from University of Essex (UK) for the helpful comments. This work was supported by ‘Ministerio de Educación y Ciencia de España’ (AGL2009-12139-C02-01); ‘Plan de Ciencia Tecnología e Innovación del Principado de Asturias’ (IB08-054 and FC10-COF10-07); predoctoral grant from the ‘Ministerio de Educación y Ciencia de España’ (FPU AP2005-0140) to J.M. Álvarez and from ‘Plan de Ciencia Tecnología e Innovación del Principado de Asturias’ (BP10-098) to N. Bueno.

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Author notes

  1. Millán Cortizo

    Present address: INRA, UMR1318, Institut Jean-Pierre Bourgin, RD10, 78000, Versailles, France

Authors and Affiliations

  1. Laboratorio de Biotecnología Agroforestal, Escuela Politécnica de Mieres, Universidad de Oviedo, C/Gonzalo Gutiérrez Quirós, 33600, Mieres, Spain

    José M. Alvarez, Millán Cortizo, Natalia Bueno & Ricardo J. Ordás

  2. Área de Fisiología Vegetal, Departamento BOS, Universidad de Oviedo, C/Catedrático Rodrigo Uría s/n, 33071, Oviedo, Spain

    Ana Rodríguez & Ricardo J. Ordás

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  1. José M. Alvarez
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  2. Millán Cortizo
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  3. Natalia Bueno
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Correspondence to Ricardo J. Ordás.

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José M. Alvarez and Millán Cortizo contributed equally.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Electronic Supplementary Material 1

Primer sequences. Primers used for PCR and Quantitative RT-PCR (PDF 144 kb)

Electronic Supplementary Material 2

Map of the binary vector pPipsCLV1L-GFP:GUS. A promoter fragment of 1,502 bp length was cloned into a Gateway® pENTR/D TOPO vector and introduced by att site LR recombination into the pKGWFS7,0. Sequence annotations were performed with Geneious software (PNG 51 kb)

Electronic Supplementary Material 3

P. pinaster and P. pinea CLUSTALW CDS alignment (PNG 197 kb)

Electronic Supplementary Material 4

Genomic sequence of PipsCLV1L (HQ377527) of 5,373 bp including a 1,513 bp region of the promoter, 79 bp 5’UTR, 119 bp of one intron, 3,045 bp CDS and 617 bp 3’UTR. The 1,502 bp fragment cloned in pPipsCLV1L-GFP:GUS is also indicated. Sequence annotations were performed with the Geneious software (PNG 29 kb)

Electronic Supplementary Material 5

Unrooted phylogenetic tree of the protein kinase domains of the CLV1-like proteins from various species generated using the Geneious software by the UPGMA method and the Jukes-Cantor genetic distance model. (PNG 45 kb)

Electronic Supplementary Material 6

Expression of CLAVATA1-Like in somatic embryos of P. pinaster as determined by Quantitative RT-PCR. Embryogenic masses were harvested after 0, 45 and 90 days of culture on medium supplemented with ABA. (PNG 13 kb)

Electronic Supplementary Material 7

PCR analysis from kanamycin-resistant lines using primers for a 355 bp GFP gene fragment and a 200 bp VirG gene fragment. M: 100 bp DNA ladder (Nippon Genetics). C+: AGL1 pPipsCLV1L-GFP:GUS. C-: P5LV41 non-transformed line. 1–24: kanamycin-resistant lines. (PNG 573 kb)

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Alvarez, J.M., Cortizo, M., Bueno, N. et al. CLAVATA1-LIKE, a leucine-rich-repeat protein receptor kinase gene differentially expressed during adventitious caulogenesis in Pinus pinaster and Pinus pinea . Plant Cell Tiss Organ Cult 112, 331–342 (2013). https://doi.org/10.1007/s11240-012-0240-8

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