SEB Bulletin January 2006 - NO Mysteries: Nitric Oxide effects in plants
The air pollutant nitric oxide (NO) plays a role in many cellular processes in mammalian systems1, and recent research shows plants to be no exception. Ranging from hormonal signalling to gene control, NO in plants seems to have a hand in many vital internal processes, as highlighted by the session at the SEB Annual Main Meeting 2005 in Barcelona2.
NO Defence
In 1998, two papers by Delledonne et al.3 and Durner et al.4 showed the importance of the role NO plays in plant-pathogen interactions. The invasion of a plant cell by a pathogen triggers the production of the reactive oxygen intermediates superoxide (O2-) and hydrogen peroxide (H2O2) and leads to the initiation of cell death. This is known as the hypersensitive disease resistance response, and kills infected cells to help prevent the spread of pathogens throughout the plant5,6 . Both NO and reactive oxygen species on their own are not enough to kill the cell, however together they work effectively. “Nitric oxide appears to boost the effect of reactive oxygen species during cell death” says Prof. Delledonne, currently at the University of Verona7. “[NO] also affects the anti-oxidant machinery in the cell ensuring the effectiveness of the hypersensitive response”.
NO also acts as an important signalling molecule, triggering the expression of defence genes as demonstrated by Durner et al. Expression of these genes was also induced by cGMP and a molecule called phenylalanine ammonia lyase (PAL), both of which also act as second messengers during NO signalling in mammals. “The first genes identified were all defence genes, but in the last two years the focus has switched to looking at the whole genome” says Dr. Durner, who is currently based at the GSF Research Centre in Munich8. Using DNA microarrays9, researchers have demonstrated that NO induces a number of unexpected genes, including those involved in growth, development and cell elongation.
NO Modification
A relatively new area of research is studying a process called S-nitrosylation10, which is the modification of an existing protein to change its function. NO acts with other proteins either directly or via an intermediate to modify the amino acid cysteine by swapping the sulphur group with S-NO. This results in a reversible conformational change in the protein which severely affects its function. A similar mechanism, protein phosphorylation, has a similar effect and is a key process in regulating cell signalling11. Phosphorylation occurs with the help of two enzymes, one of which adds the phosphorous on, and the other which removes it. S-nitrosylation is different, however, because it appears that it can happen without enzymes and is self regulating through the addition and subtraction of NO, depending on the redox state of the cell.
“Proteins that are involved in signal transduction chains are changed in their activity by S-nitrosylation, which means that NO is playing a role in modifying signalling pathways in the plant,” says Dr. Durner. Proteins in the TCA cycle in plants are also regulated by S-nitrosylation, showing that NO can also influence metabolism, a feature that has already been described in animal systems.
Another layer of complexity is added to the role of NO in plant-pathogen interactions, because S-nitrosylation could regulate the balance of reactive oxygen species and NO in the cell during the hypersensitive response. This reaction is probably influenced by the s-nitrosylation of critical anti-oxidant proteins. “This reduces their power, meaning that reactive oxygen species have a greater effect on the cell” says Prof. Delledonne, meaning that NO has an effect at many different levels on the response of the plant to pathogen attack. Whilst high levels of NO are very useful for defence, they can cause problems for a healthy cell. “Plant haemoglobins are molecules that act as housekeepers” says Prof. Delledonne mopping up excess NO, they prevent the plant being damaged unnecessarily by this toxic molecule12.
NO production
One of the mysteries that researchers are currently puzzling over is how NO is made in plants. In animals, NO is made by 3 different nitric oxide synthases (NOS). iNOS makes high levels of NO which are used in immune responses by macrophages to kill invading pathogens within a cell. eNOS and nNOS make low levels of NO, which are mostly used for cell signalling and are regulated post-translationally by calcium. All the mammalian NOS make NO from arginine with citrulline as a by-product13.
In plants, however, there are many different sources of NO, only some of which are beginning to be elucidate. Dr. Nigel Crawford's lab at the University of California in San Diego14 are interested in how NO is made and whether the process is similar in plants to that already described in mammals. There were no signs, however, of mammalian-type NOS activity in plants, and searches of the Arabidopsis genome for conserved markers that might indicate the presence of a NOS were also unsuccessful.
Crawford and his colleagues Mamoru Okamoto and Fan-Qing Guo then shifted their attention to a gene called AtNOS115, which was first described in the snail Helix, and is involved in the production of NO in the central nervous system. The protein product of this gene presented a puzzle as it was much smaller, at 60 kD, than mammalian NOS at 130-150 kD. There were also none of the features present that you would enable the enzyme to make such as a haem group that provides a site for the binding of arginine and cofactors to produce NO16. The only identifiable feature of the gene was a site for GTP binding proteins. “By knocking out the gene biochemically, we could see that it was critical for producing low levels of NO, which could be used in cell signalling” says Dr. Crawford - “mutants also have greatly reduced NO production”. Whilst the gene does not have a similar sequence to a mammalian NOS gene, the process by which NO is produced does rely on arginine. The sequence of the gene gives no clues as to the mechanism by which it makes NO.
The evidence indicates that AtNOS1 is a NOS, but this has not been comprehensively proved yet. “I feel that until we do that we can't say with complete confidence that this is an NOS” says Dr. Crawford. Despite this, there are some more intriguing similarities and differences between AtNOS1 and mammalian NOS. In mammalian systems, eNOS and nNOS are involved in signalling and regulated by calcium. Results indicate that AtNOS1 is similar it is also regulated by calcium and produces low levels of NO which are used in cell signalling. In contrast with mammalian systems, where iNOS fulfils the defence role, the gene is probably also involved in defence responses.
NO influence on stomatal guard cells
Another important process that makes NO in plants is via nitrite dependent pathways, using the enzyme nitrate reductase. Professor Steve Neill and colleagues at the University of the West of England17 found that mutants in the nitrate reductase enzyme play a role in the function of stomatal guard cells. These are very complicated cells on the surface of leaves, joined at the top and the bottom end and are important for regulating water-gas exchange18. When the plant takes up water, the stomatal guard cells swell, bending outwards to open the stomatal hole. “They are very useful cells as they respond to a massive range of signals by swelling and shrinking” says Prof. Neill. Looking at these responses is a useful assay for determining the effects of different molecules on the plant.
When the roots of a plant are short of water, abscisic acid (ABA), a plant stress hormone19, is produced and transported to the leaves in the transpiration stream. This causes the guard cells to shrink and flop inwards so the stomatal hole closes, hence conserving water. Adding NO to guard cells by the means of NO donors increases ABA production, showing that NO is involved in stomatal hole closure. Removing NO by the means of scavengers that mop it up in the leaves means that guard cells do not close in response to ABA. By using a fluorescent dye called DAF-2 researchers can visualise NO levels in leaves and cells. Guard cells in plants with mutations in the nitrate reductase enzyme produce a lot less NO and also have reduced closure responses to ABA.
There is, however, a final twist to the tale which hasn't yet been resolved. Dr. Crawford also wanted to know if the AtNOS1 gene was involved in hormonal signalling, and the most obvious candidate for looking at this problem were stomatal guard cells and their ABA responses. “In AtNOS1 mutants, ABA induced NO synthesis was greatly reduced, as was closure of the guard cells by ABA” he says. This showed that the gene AtNOS1 is involved in producing the NO needed for ABA induced stomatal signalling and was a similar, but contradictory result to that seen in nitrate reductase mutants. “It is not clear yet which pathway for producing NO is dominant in this system” explains Dr. Crawford, “It is possible that any number of conditions could determine which pathway is dominant”.
It is likely that the study of NO in plants will continue to throw up many surprises, but for now researchers have plenty of problems to keep them busy.
Laura Blackburn
University of Cambridge
References
1) http://www.sebiology.org/Publications/pageview.asp?S=7&id=391
2) http://www.sebiology.org/Meetings/pageview.asp?S=2&mid=&id=473
3) Delledonne, M. et al., (1998) Nature 394:525-7
4) Durner, J. et al., (1998) PNAS 95:10328-33
5) http://www.aber.ac.uk/plantpathol/celldeath.htm
6) Agrios, G. (1997) Plant Pathology, 4th Ed. Academic Press.
7) http://profs.sci.univr.it/~delledon/
8) http://www0.gsf.de/biop/en/mitarbeiternoenglisch.phtml
9) http://www.sebiology.org/Publications/pageview.asp?S=7&id=356
10) Lindermayr, C. et al. (2005) Plant Physiol 137:921-30
11) http://en.wikipedia.org/wiki/Phosphorylation
12) http://home.cc.umanitoba.ca/~rhill/
13) http://www.sacs.ucsf.edu/home/Ortiz/res-nos.htm
14) http://www-biology.ucsd.edu/faculty/crawford.html
15) Guo, F.-Q., et al. (2003). Science 302:100-103.
16) http://metallo.scripps.edu/PROMISE/NOS.htmlV
17) http://science.uwe.ac.uk/research/homePage.aspx?pageId=cripsHome
18) http://www.mna.hkr.se/~ene02p7/twig.htm; http://www.fhsu.edu/biology/thomasson/stomate.htm
19) http://cbr-rbc.nrc-cnrc.gc.ca/abscisicacid/; http://www.plant-hormones.info/abscisicacid.htm
