Expression profiling of CTR1-like and EIN2-like genes in buds and leaves of Populus tremula, and in vitro study of the interaction between their polypeptides

Abstract

In Arabidopsis, the serine/threonine protein kinase Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 2 polypeptide (EIN2) functions are key negative and positive components, respectively, in the ethylene signalling route. Here, we report on an in silico study of members of the CTR1-like and EIN2-like polypeptide families from poplars. The expression of CTR1-like and EIN2-like genes such as Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a was studied in Populus tremula buds and leaves in response to dehydration, various light conditions and under senescence. In buds under dehydration, the maximal fold-change of the Ptre-CTR1, Ptre-CTR3 and Ptre-EIN2a expression level recorded almost identical values. This suggests that maintenance of a constant ratio between the transcript levels of genes encoding positive and negative ethylene signalling components is required under stress. The expression of the studied genes was 1.4-to 3-fold higher in response to darkness, but 4.5- to 51.2-fold and 21.6- to 51.2-fold higher under the early and moderate leaf senescence, respectively. It is worth noting that the senescence-related Ptre-EIN2a and Ptre-CTR3a expression profiles were very similar. Using in vitro assays, we demonstrated the ability of the catalytic domain of Ptre-CTR1 to phosphorylate the Ptre-EIN2a-like polypeptide, which is similar to that in Arabidopsis. The target substrate, the Ptre-CEND2a polypeptide (C-terminal part of Ptre-EIN2a), was only phosphorylated by the protein kinase Ptre-CTR1 and not by Ptre-CTR3. Moreover, the addition of Ptre-CTR3 polypeptides (-CTR3a or -CTR3b forms) to the reaction mixture had an inhibitory effect on Ptre-CTR1 auto- and trans-phosphorylation. In contrast to Ptre-CTR1, Ptre-CTR3 may act as a positive regulator in ethylene signalling in poplar; however, this hypothesis requires in vivo confirmation. Thus, the ethylene signalling route in poplar seems to be under the control of certain additional mechanisms which have not been reported in Arabidopsis.

Keywords: 1-Aminocyclopropane-1-Carboxylate oxidase; Constitutive triple response 1; Ethylene; Ethylene insensitive 2; Poplar.