75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

83 Characterization of Orthologous Subtilases in Arabidopsis and Tomato
Franziska Huttenlocher, Anna Cedzich, Katrin Ullrich, Annick Stintzi, Andreas Schaller
University of Hohenheim, Institute of Plant Physiology and Biotechnology, 70599 Stuttgart, Germany
Subtilases or subtilisin-like serine proteinases are encoded by large gene families in higher plants. In Arabidopsis
thaliana, 56 subtilase genes have been annotated. Within the scope of the AFGN program and in collaboration with four
European and partners, we are aiming at the functional characterization of the Arabidopsis subtilase family using T-DNA
insertion mutants, detailed expression analysis, and computational approaches (Rautengarten et al., 2005). However,
loss-of-function analysis revealed obvious phenotypical differences for only two of the mutants, i.e. sdd1 and ale1. The
vast majority of the T-DNA-insertion lines did not exhibit any defects under standard growth conditions (cf. poster by
Knappenberger et al.). To gain further insight into the function of the gene family, we extend our studies to tomato,
comparing the organization of the subtilase gene family, the expression of orthologous genes and the properties of the
enzymes.
Phylogenetic analysis suggests the tomato genes for subtilases LeSBT1, 2, and 3 to be orthologous to At5g67360,
At5g51170 and At5g67090 , respectively. Functional equivalence of the three pairs of genes is supported by similar
expression patterns: promoter::GUS analysis indicated expression for LeSBT1 and At5g67360 in the vasculature, in sepal
and petal abscission zones and in the style. LeSBT2 and At5g51170 were found to be expressed similarly in guard cells,
while the promoter activity of LeSBT3 and At5g67090 was detected in the vasculature as well as in roots, particularly
at the root tip and the sites where lateral roots protrude. For the analysis of the biochemical properties, LeSBT3 was
overexpressed in a plant cell suspension culture and purified to homogeneity f rom culture supernatants. Using synthetic
oligopeptides as substrates, MALDI-TOF MS analysis of the cleavage products indicated a preference of recombinant
LeSBT3 for glutamine in the P1 position of its substrates. A detailed kinetic analysis of the LeSBT3 enzyme was performed
using a fluorigenic peptide substrate derived from the preliminary analysis of substrate specificity. How the properties of
recombinant LeSBT3 compare to those of the corresponding Arabidopsis subtilase remains to be seen.
Ref.: Rautengarten, C., Steinhauser, D., Büssis, D., Stintzi, A., Schaller, A., Kopka, J., Altmann, T. (2005): Inferring hypotheses on functional
relationships of genes: Analysis of the Arabidopsis thaliana subtilase gene family. PLoS Comput. Biol. 1(4): e40
84 Analysis of the Arabidopsis thaliana Subtilisin-Like Serine Protease Gene Family
Mathias Knappenberger, Annick Stintzi, Andreas Schaller
Institute of Plant Physiology and Biotechnology - University Hohenheim - 70593 Stuttgart - Germany
Subtilases or subtilisin-like serine proteinases are the closest homologs in plants of mammalian proprotein or
prohormone convertases. In Arabidopsis thaliana, they are encoded by a gene family of 56 members.
Thus far, a specific function has been assigned to just two of them: SDD1 specifies stomatal density and distribution
and ALE1 is required for cuticle formation and epidermal differentiation during embryo development. In collaboration
with four European and partners and supported by the AFGN program (AL 387/5 SCHA 591/3), we seek to understand
the function of additional members of this large gene family. Our functional studies involve the characterization of lossof-function
T-DNA insertional mutants, and expression analyses for the entire gene family.
To date, for plants grown under standard conditions, no phenotypic changes were observed within the 90 T-DNA
insertion lines corresponding to 49 individual subtilase genes.
Spatial and temporal subtilases gene expression was specified for 33 members of the family by analyzing β-
glucuronidase staining patterns in transformed Arabidopsis plants. The majority of the transgenics displayed complex
yet distinct patterns of reporter gene expression, SBT3.14 being the only one so far that is specifically expressed in the
inner integument of the developing Arabidopsis seed coat. For this line, GUS and GFP signals were first detected at the
micropylar end of ovules at stage 12 i.e. before floral bud opening. The expression persisted until stage 17, at which it
also extended to the chalazal area.
While the expression pattern of SBT3.14 suggests a role in seed development, no morphological differences could
be identified between wild-type and mutant ovules or seeds. In order to test whether the loss of SBT3.14 function results
in a “molecular” phenotype, Microarray experiments were performed using RNA extracted from stage 12-17 wild-type
and mutant gynoecium. Progress on different aspects of the project will be presented.