The Arabidopsis lectin EULS3 is involved in stomatal closure
Introduction
Plants are sessile organisms that cannot escape from adverse conditions. Therefore, they have evolved intricate mechanisms to defend themselves against stresses such as cold, heat, drought, insect herbivory and pathogen attack, resulting in a change of the expression for specific genes implicated in protection against these stresses. During the last decade, it has become evident that plants also react to environmental stress by enhancing the expression of certain lectin genes. These carbohydrate-binding proteins have long been overlooked, since they cannot be detected under normal growth conditions. Only after the plant has been subjected to stress, the expression levels increase considerably. This new class of so-called inducible lectins differs from the classical lectins that are constitutively expressed at high levels and as a consequence have been the subject of most plant lectin research so far. In contrast to the classical lectins which usually reside in the vacuole, most of the inducible lectins locate to the nucleus and the cytoplasmic compartment of the cell [1]. The fact that the expression of these new lectins is induced by stress led to the hypothesis that these proteins play a role in the stress physiology of plants.
The class of inducible, nucleocytoplasmic lectins comprises a wide range of proteins with different carbohydrate-binding motifs [2]. Based on the sequence of the carbohydrate recognition domain involved, a classification was elaborated which allowed to group these individual lectins in six families of carbohydrate-binding proteins, among which the recently discovered Euonymus europaeus (spindle tree) lectin (EUL) family [3]. The EUL family comprises all proteins that show sequence homology to the Euonymus lectin present in the fleshy arils of the spindle tree. An extensive screening of publicly accessible genome databases revealed that the EUL domain is remarkably well conserved throughout the plant kingdom, and is present in virtually all green plants, suggesting an important role for this protein domain [4]. The majority of the EUL proteins consist of a single EUL domain preceded by an unrelated N-terminal domain whereas some proteins are composed of two tandemly arrayed EUL domains. The nomenclature of the EUL proteins is based on the plant species and the type of EUL protein present. This work focuses on the EUL protein from Arabidopsis thaliana, a protein with a single EUL domain of the S3 type which will further be referred to as the ArathEULS3 lectin.
The ArathEULS3 sequence (At2g39050) is the only sequence in the genome of A. thaliana containing an EUL domain [3]. Recently, ArathEULS3 was expressed in the yeast Pichia pastoris and the recombinant protein was characterized. Glycan microarray analyses have proven that ArathEULS3 can be considered a true lectin since it interacts with glycans containing Lewis Y, Lewis X and lactosamine motifs [5]. Based on the sequence homology to the EULS3 lectin from rice (OrysaEULS3) and the results from microarray experiments, it was hypothesized that the expression of the lectin gene is upregulated by environmental cues related to osmotic stress [6]. qRT-PCR analyses confirmed that the ArathEULS3 expression in plants is low but stable under normal growth conditions but is significantly elevated after treatment with salt (NaCl), abscisic acid (ABA) and methyl jasmonate (MeJA) [7]. Until now, however, very little is known about the precise physiological role of proteins with EUL domains.
To study the importance of the EULS3 lectin from Arabidopsis in plant growth and development we identified its binding partners using tandem affinity purification (TAP) analyses. The physiological importance of the interaction between ArathEULS3 and its interacting partners was evaluated in physiological studies involving the stomatal response of Arabidopsis leaves and disease symptom development after Pseudomonas syringae infection experiments using transgenic lines with overexpression or reduced expression of ArathEULS3. These data clearly yield new ideas with respect to the physiological importance of ArathEULS3 for plant development.
Section snippets
Plant materials
Wild type seeds of A. thaliana ecotype Columbia (Col-0) were sown in artificial soil (Jiffy-7, 44 mm Ø) (AS Jiffy Products, Drobak, Norway) in an ARATRAY® (ARASYSTEMS, Ghent, Belgium). The trays were incubated for 3 days at 4 °C in the dark to eliminate any residual dormancy of the seeds, and were then transferred to a growth chamber at 21 °C with a 16/8 light/dark photoperiod. For collection of samples from aerial tissues during development, whole plantlets were collected 6, 15 and 22 days after
Tandem affinity purification of the full length ArathEULS3 protein yields two novel interactors
ArathEULS3 is a chimeric protein with a calculated molecular mass of 35.6 kDa, consisting of an N-terminal domain with unknown function (18.3 kDa) and a C- terminal carbohydrate-binding domain (17.3 kDa). The full length ArathEULS3 protein was tagged N- or C-terminally with the GS tag containing an Immunogoblin G binding peptide and a Streptavidin binding peptide separated by one or two TEV protease cleavage sites resulting in an N-terminal tag of 20.6 kDa and a C-terminal tag of 22.0 kDa (Fig. 1A)
ATS3A and ATS3B interact with the N-terminal domain of ArathEULS3
TAP experiments were performed for the full length ArathEULS3 protein as well as its individual domains, C- or N-terminally tagged with the GS tag. All independent TAP replications performed with the full length protein and the N-terminal domain yielded the same interaction partners ATS3A and ATS3B, both identified with >99% confidence. Taking into account that TAP results only yield interactions which are able to withstand the purification procedure, this result indicates that the interactions
Acknowledgements
This work was funded primarily by the Fund for Scientific Research—Flanders (FWO grant G006114N) and the Research Council of Ghent University (project BOF15/GOA/005).
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