VIB - UG - departement Planten Systeembiologie (PSB), UGent
The main characters in this story are the Nictaba lectin orthologues from Arabidopsis thaliana. Lectins are proteins that can selectively and reversibly bind to sugar structures. This class of proteins is widely represented in the plant kingdom but is also present in animals and fungi.
Some plant lectin families are constitutively expressed, while other families show an inducible expression mainly upon stress signals. A plant can suffer from abiotic and biotic stresses. Abiotic stress includes heat, drought, heavy metals, cold and salt stress while biotic stresses consist of insect infestations and pathogen infections.
One of these inducible plant lectins is Nictaba or Nicotiana tabacum agglutinin. It was firstly discovered in the leaves of tobacco, and was identified as a jasmonate inducible protein. In the genome of Arabidopsis thaliana, 31 orthologues of the Nictaba lectin gene have been identified. The Nictaba domain is often linked to another protein domain or a C- or N-terminal region. The most common domain is the F-box domain, other domains include a TIR domain (Toll/interleukin-1 receptor) and a AIG1 (avirulence induced gene 1)-type G domain.
To analyse the evolutionary relationships between the Nictaba-related genes in Arabidopsis and in other species, a phylogenetic tree was made. To have a clear view on the phylogeny of the Nictaba domain, the trees were generated using a multiple sequence alignment (Mega software) for only the Nictaba domain sequences, this to avoid interference of the other (non-lectin) domains with the multiple sequence alignment and phylogenetic tree (RAxML). In the obtained Maximum Likelihood tree, we can observe three main clades, designated as clades A, B and C. The phylogenetic relationship is related to the corresponding protein domain architecture. Clades B and C contain the F-box Nictaba-related genes. Clade A contains separate branches for TIR-Nictaba-related lectins and proteins with only a Nictaba lectin domain.
To investigate the potential involvement of Arabidopsis Nictaba-related lectins in the plant stress response, this project aims to determine the gene expression profiles for different Nictaba orthologues in Arabidopsis thaliana and analyse the conservation of regulatory sequences in the promoter regions of these genes. Therefore a workflow was designed in which we started from the expression profiles from the Nictaba-related genes and defined a set of co-expressed genes. From this set of genes, the gene-of-interest and its co-expressed genes, a gene ontology (GO) enrichment and motif enrichment were produced. The resulting data was visualized in a graph.
To define the expression profile, the project started with a genevestigator search for the 31 Nictaba orthologues in Arabidopsis. We selected microarray experiments from wild type plants for diverse biotic and abiotic stresses (including salicylic acid, methyl jasmonate, abscisic acid, Indole-3-acetic acid (auxin), salt, heat, cold, drought, Pseudomonas, Myzus) and different plant parts. The selection cut-off was set to FC=1.5 and p-value=0.05. Based on these results as well as the organisation of the phylogenetic tree 6 genes with different expression profiles were selected: AT1G80110 (PP2- B11), AT1G31200 (PP2-A9), AT2G02350 (PP2-B9), AT4G19840 (PP2-A1), AT5G52120 (PP2-A14) and AT1G65390 (PP2-A5). The genes PP2- B11, PP2-A14 and PP2-A5 were selected because they show an interesting stress responsive expression profile. For the other 3 genes (PP2-A9, PP2-B9 and PP2-A1) research has been done in our research facility. The 3 different clades of the phylogenetic tree have representatives among the selected genes. PP2-A9 has almost no N- or C-terminal domain and is located in the A-clade together with PP2-A1 (N-terminal domain) and PP2-A5 with a TIR domain. PP2-B11 (F-box) and PP2-B9 (small C-terminal domain) belong to clade B. One gene was selected from clade C: PP2-A14 with an F-box domain linked to a Nictaba domain.
Using their transcription profile, 100 and 200 co-expressed genes for each of the selected Nictaba-related genes were identified (positive and negative correlation). Since it is assumed that co-expressed genes might be involved in the same processes, a GO enrichment analysis was performed, this reveals if a GO term (hierarchical grouping gene descriptions) is significantly more present in the co-expressed genes than in the pool of Arabidopsis genes. In addition, we isolated the sequence 5 kb upstream and 1 kb downstream of the Nictaba-related genes to perform a motif enrichment to identify cis-regulatory elements (and linked transcription factors) enriched in the promoters of these genes.
To give a clear presentation of the data obtained from the GO and motif enrichment, we manipulated the data with Cytoscape, filtering on the q-value value (p-value corrected for the false discovery rate) and the number of hits for the enriched term in the set of the co-expressed genes.
The results of this analysis provide support to the hypothesis that the Arabidopsis Nictaba orthologues are involved in the stress response pathways of the plant. The expression profiles for most Nictaba-related genes show a stress regulated profile. In addition, the in-depth analysis of the selected genes retrieved similar stress related GO terms and cis-regulatory domains. We have shown that this type of analysis can provide information about the function of the gene of interest.
Deze bachelorproef handelt over het integreren van transcriptiefactor – target data van de modelplant Arabidopsis thaliana en het ontwikkelen van een tool voor het visualiseren van deze data in de reeds bestaande CORNET webtool. CORNET is een webtool die netwerken bouwt en visueel weergeeft door de integratie van verschillende data types. Dit gebeurt door twee tools op de CORNET site, de proteïne-proteïne interactie en de co-expressie tool.
Dr. Klaas Vandepoele (BIT)
Dr. Lieven Sterck
+32 (0)9 3313821
Kris Morreel (FBT)
Tom Viaene (FBT)
Sofie Goormachtig (FBT)
Michiel Van Bel (BIT)
Patrick Willems (BIT)