In the last 20 years, nitric oxide (NO) has been identified as an important chemical messenger in plants. Using a combination of biochemical and bioinformatics approach EU-funded research has identified thousands of potential target proteins and specific binding sites, opening the door to tests of function.
Living organisms have a very complex network of biochemical signalling
to carry out virtually all functions from the cellular level up to the
level of the organism. NO plays a role in regulation of plant functions,
including disease resistance, gas exchange, seed germination and root
development.
Many of the biological functions result from interactions of NO (or
more generally, the nitric oxide family (NOx)) with proteins. EU-funded
scientists launched the project 'NO-dependent protein translocation and
S-nitrosylation of nuclear proteins in Arabidopsis thaliana' (
PRONITROARAB) to determine targets that mediate effects with a focus on nuclear proteins.
One of the most important ways NO regulates functions in plants is
to attach itself covalently to cysteine (S) residues of other molecules
(S-nitrosylation). First, the team employed computational methods (the
GPS-SNO 1.0 software, a recently developed S-nitrosylation site
prediction programme) to survey the entire proteome of A. thaliana (27
416 proteins). The results were impressive.
Candidate S-nitrosylation proteins were found in all cellular
compartments (e.g. membrane, chloroplast, etc.) in large quantities.
When focusing on the most likely candidates (more stringent statistical
probability), the team identified 3 190 total S-nitrosylation sites in a
total of 3 005 target proteins, primarily in chloroplast, the
intracellular compartment and plasmodesmata. They represented 5–17 % of
total protein content per compartment.
Then the team had a closer look on S-nitrosylation of nuclear
proteins in-vivo. To find S-nitrosylated nuclear proteins that are
related to plant defence response the team exposed A. thaliana to a
pathogen. This small flowering plant is a model system in plant biology.
Proteins were then extracted from the nuclei and subjected to the
biotin switch assay to identified S-nitrosylated nuclear proteins.
Nuclear extracts exposed to an NO donor were used as control. Of the 195
candidates identified, 57 % (111) were nuclear proteins. These proteins
serve a myriad of functions demonstrating the extensive reach of the NO
pathway in regulation.
Understanding regulatory mechanisms in plants is important on a
number of levels, from acquiring fundamental knowledge to applications
related to crop growth or disease resistance, and in pointing to similar
pathways in other systems. PRONITROARAB contributed new knowledge to
the field of plant regulation by S-nitrosylation of proteins and opened
the door to numerous experiments and a bright career investigating these
phenomena.