Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro.

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.

Characterization and expression analysis of AcSERK2, a somatic embryogenesis and stress resistance related gene in pineapple.

The isolation and characterization of AcSERK2, another somatic embryogenesis receptor-like kinase (SERK) gene homolog, confirmed that there was a SERK gene family in pineapple. AcSERK2 shared high similarity to AcSERK1 and other SERKs, containing all the characteristic domains of the SERK family. The expressed recombinant protein of pET-AcSERK2 was soluble. AcSERK2 expression was effectively induced by 2,4-dichlorophenoxyacetic acid (2,4-D) and maintained in high level at the early stage of somatic embryogenesis (SE). The results of in situ hybridization showed that AcSERK2 expressed at high level in the competent cells under the induction of 2, 4-D and at low level during the development of meristematic center to globular embryo. This indicated that AcSERK2 could be used as a potential marker gene to monitor the acquisition of embryogenic competence. However, AcSERK2 expression was also detected in all the tested organs and the expression level decreased along with the development of calyx, ovule and ovary. These results suggested that AcSERK2 maybe play a broader role in organ development. AcSERK2 transcription could be induced by exogenous hormones and abiotic stress. It was deduced that AcSERK2 may participate in response to exogenous hormones and abiotic stress regulation in pineapple. The expression pattern of AcSERK2 during SE differed from that of AcSERK1, and this indicated that the SERK genes may be non-redundant in somatic embryogenesis.