Assistant Professor
Gene expression and metabolic changes during plant defense responses
to pests
Functional genomics of plant nucleases
Secreted ribonucleases (RNases) have been well studied at
the enzymatic and structural levels, although little is known regarding
their biological functions. One family of secreted RNases, the RNase
T2 family, is particularly widespread, with members throughout various
kingdoms. In recent years, many plant members of this superfamily have
been identified. Gametophytic self-incompatibility in several plant
groups involves the activity of S-RNases, one subfamily of plant T2
RNases. Another subfamily, the S-like RNases, is found in self-compatible
as well as self-incompatible plant species. The Arabidopsis genome contains
five genes from this family, RNS1 to RNS5. Expression patterns of S-like
RNases in many species led to the suggestion that these enzymes are
involved in remobilization and recycling of phosphate from nucleic acid
sources. However, the high level of conservation of enzymes of this
family in all species examined and recent data showing regulation of
these enzymes by stress conditions where nutrient limitation is unlikely
suggest that S-like RNases could have other roles in plants and other
organisms.
To understand the biological roles of members of the T2 family, we analyzed
mutants that lack RNase activity both in yeast and in plants. Those
experiments showed that secreted RNases are important to maintain normal
growth under stress conditions, and suggested that the T2 family of
RNases may be involved in developmental processes. It is clear from
our experiments that these enzymes have multiple functions in all organisms
that are far more complex than the original role proposed (nutrient
recycling). Secreted RNases are found in all organisms, and the RNase
T2 family in particular has been highly conserved throughout evolution,
suggesting they carry out critical functions in a wide range of life
forms. Due to their plasticity and ability to adapt to adverse growth
conditions, plants are an especially suitable system to study this type
of enzyme, which is likely to have cytotoxic or morphological effects.
In particular, the wide availability of resources makes Arabidopsis
an optimal organism for these studies. However, as exemplified by the
fact that plant RNases and secreted RNases from other systems can complement
RNase T2 mutant phenotypes in yeast, we expect that our findings on
RNS functions will extend to other organisms and can guide studies on
other, unrelated secreted RNases. My long-term research goal is to understand
the physiological functions of secreted RNases in plants. The main areas
of research are highlighted below:
Functional analysis of the RNS family in Arabidopsis
This project involves subcellular localization studies, analysis of
gene expression using traditional methods and microarray technology,
and characterization of T-DNA insertion mutants and overexpressor plants,
and detailed biochemical characterization for each protein in the family.
In particular, we are studying RNSs as part of defense and other stress
responses. RNS1 is induced systemically after wounding, and preliminary
results point to a role in defense against several different pathogens.
In addition, RNS1 induction is independent of all known signals controlling
wounding responses in Arabidopsis. We have developed several tools to
dissect this novel wounding signaling pathway.
RNS roles in other plants.
The use of simpler model organisms has been useful to understand the
roles of secreted RNases, as exemplified by our work on yeast. However,
yeast is not useful to understand developmental processes or stress
responses involving cell to cell signaling and tissue coordination.
We plan to investigate the role of members of the RNS family in a lower
plant system, Physcomitrella patens. This moss is an excellent model
to study cellular organization and developmental programs, and good
genetic and molecular techniques are available. We have already identified
different RNS genes in P. patens. Since it is possible to create mutants
by homologous recombination, RNS null mutants should be easy to obtain.
Proteomics analysis
This project will identify and characterize ribonucleases involved in
developmental processes and defense responses in Arabidopsis. Secreted
RNases can have similar roles in different organisms, even in the case
of structurally or phylogenetically unrelated enzymes. We have identified
many peptides with RNase activity in Arabidopsis extracts, using an
in gel activity assay. We are implementing a functional proteomic approach
to identify and characterize Arabidopsis RNases. To obtain regulatory
information, proteomic profiles will be developed from tissues collected
during different stages of development, and from plants subjected to
different stress treatments and pathogens.
