Abstract
Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20-80 fold more than in anther but similar to that of non-embryogenic callus, which was 20-50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity.
Highlights
Somatic embryogenesis (SE) is the process whereby somatic cells can develop into plants via characteristic morphological stages (Shah et al, 2002) and is a notable example of plant totipotency
AcSERK3 is a member of a small family of receptor-like kinases (RLKs), all of which have a predicted signal peptide, five leucine-rich regions, a typical serine-proline rich juxtamembrane region, and a C-terminal kinase domain that is conserved in all of the 11 subdomains, as described for serine/threonine protein kinases (Shah et al, 2001a)
The similarity of the deduced amino acid sequence of AcSERK3 with AtSERK1 (85%) suggested that the corresponding regions in AcSERK3 may play a similar role to AtSERK1
Summary
Somatic embryogenesis (SE) is the process whereby somatic cells can develop into plants via characteristic morphological stages (Shah et al, 2002) and is a notable example of plant totipotency. Somatic embryogenesis can be used for rapid propagation of valuable clones and for gene transfer and regeneration of genetically modified plants (Schellenbaum et al, 2008). Pineapple is a vegetative propagated crop that depends on suckers because it is self-sterile. Somatic embryogenesis is a highly efficient means of propagating pineapples and can be induced in this species by exposure to 2,4-dichlorophenoxyacetic acid (2,4-D), with the frequency of somatic embryo induction reaching 95%; the number of somatic embryos per callus (3x3x3 mm) after 40 days (d) of culture is ~48 (He et al, 2007). The origin of SE in pineapple is unicellular (He et al, 2010; Ma et al, 2012a,b). Somatic embryogenesis provides a good way to genetically transform pineapple
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