Inferring Hypotheses on Functional Relationships of Genes: Analysis of the Arabidopsis thaliana Subtilase Gene Family

Abstract

The gene family of subtilisin-like serine proteases (subtilases) in Arabidopsis thaliana comprises 56 members, divided into six distinct subfamilies. Whereas the members of five subfamilies are similar to pyrolysins, two genes share stronger similarity to animal kexins. Mutant screens confirmed 144 T-DNA insertion lines with knockouts for 55 out of the 56 subtilases. Apart from SDD1, none of the confirmed homozygous mutants revealed any obvious visible phenotypic alteration during growth under standard conditions. Apart from this specific case, forward genetics gave us no hints about the function of the individual 54 non-characterized subtilase genes. Therefore, the main objective of our work was to overcome the shortcomings of the forward genetic approach and to infer alternative experimental approaches by using an integrative bioinformatics and biological approach. Computational analyses based on transcriptional co-expression and co-response pattern revealed at least two expression networks, suggesting that functional redundancy may exist among subtilases with limited similarity. Furthermore, two hubs were identified, which may be involved in signalling or may represent higher-order regulatory factors involved in responses to environmental cues. A particular enrichment of co-regulated genes with metabolic functions was observed for four subtilases possibly representing late responsive elements of environmental stress. The kexin homologs show stronger associations with genes of transcriptional regulation context. Based on the analyses presented here and in accordance with previously characterized subtilases, we propose three main functions of subtilases: involvement in (i) control of development, (ii) protein turnover, and (iii) action as downstream components of signalling cascades. Supplemental material is available in the Plant Subtilase Database (PSDB) (http://csbdb.mpimp-golm.mpg.de/psdb.html) , as well as from the CSB.DB (http://csbdb.mpimp-golm.mpg.de).

Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis

“…Assuming that a set of genes coexpressed under a given experimental regimen is involved in the same or related metabolic pathway, candidate genes involved in the regulation or synthesis steps of a particular metabolic pathway can be comprehensively identified, or at least predicted with some confidence, by using publicly available transcriptome databases (9)(10)(11)(12)(13). Although the strategy of coexpression analysis has great potential for versatility, its application has thus far been limited in the actual discovery of useful genes in important pathways in Arabidopsis (11).…”

A robust omics-based approach for the identification of glucosinolate biosynthetic genes

“…Apparently, however, coexpression analysis does work for this purpose only when a complete biological process is coordinately regulated at the level of mRNA accumulation. (3) Even if the knockout of genes belonging to a gene family, the members of which have unknown biological function, fails to reveal any apparent phenotype, the function of these genes can be predicted on the basis of their coexpression relationship with other genes (Rautengarten et al 2005). (4) Coexpression analysis can even be conducted using a non-targeted approach without any preexisting hypothesis.…”

Molecular cloning and characterization of a subtilisin-like protease from Arabidopsis thaliana

“…Eukaryotic subtilases are encoded by a variety of organisms, including animals, plants, and fungi, and form the SB subfamily within the S8 serine protease family (Rautengarten et al, 2005).…”