Grant Cramer, University of Nevada at Reno
Keynote Talk, Afternoon Session, 1 September (11th MGED Meeting, 1-4 September, 2008)
Why interested in this type of stress? Cold is a major problem for grapes, salt tolerance would be useful (over time salts remain in the soil when the water evaporates after very long-term irrigation), and want to know more about drought stress. We want to stunt growth so that most of the effort goes to the fruit. Therefore, grapes can be quite drought tolerant.
General intro to systems biology
Not just grapes make money: wine sales, tourism, etc brings it to $50 billion in California annually. Also, there are 200+ phenolics, (anti-Alzheimer's – interferes with plaque formation, anti heart disease) and other human health benefits. Also, wine tastes good 😉
They are using transcriptomics (affy chips), proteomics (2d-gels) and metabolomics (primarily gcms, but also lcms) data and are integrating that information into MetNet. Goals include annotation of genes, map molecular networks, build models to describe physiology and development, and manipulate and improve fruit quality and stress resistance.
Proteomics: they are using 2d-page with maldi-tof-tof. Transcript data is available publicly. Currently 8461 out of 39423 genes have been mapped, with 120 pathways.
Abiotic stress effects on shoots
They've done a long-term stress experiment. Start with potted 2-year-old, own-rooted, Cabernet Sauvignon clone 8 (don't normally grow from seed as can be very different from the original). Pruned to one shoot. Grown in a greenhouse. Most salt experiments are osmotic shock experiments, where you stick them in salt water all of a sudden. But in the environment, salinization happens very gradually. Salinity affects plants via osmotic removal of water and also have an aspect of ion stress. What he did was to get the osmotic water effect was just to stop watering. To do the salt stress, you have to measure the water deficit of the leaves and then add salt to the roots of the plants to get it to have the same water deficit response as with water deficit only. They harvest the growing shoot tip. Over time, the control was steady, but the pressure of the water potential in the leaves for both the salinity plants and water-deficit plants were virtually identical, so was able to mimic the water deficit well.
The salt and drought-stress plants slow down their growth prior to the drop in water potential: that is, the growth is almost more sensitive then their ability to measure the water potential. Shoot elongation was very sensitive to stress, and in the early stages was actually more affected by water deficit than salinity.
Their microarray data came out very nicely (partly due to the use of clones). They did a gene expression time course, and for both types of stress there was an increase in the number of transcripts being upregulated and downregulated over time. First the water deficit by day 6, and then not until day 13 for salt deficit. There are large differences between the two types of stress.
They did a comparison on day 16 to see which were differentially expressed between salinity and drought stress. There are significant differences between MIPS functional categories. These include transcription, cell defence, transport mechanisms, metabolism. Also, some key hormone biosynthesis genes are affected by stress. ABA-NCED is affected by drought before salinity. Ethylene comes in much later, around day 18. The metbolism of a growth hormone goes up, reducing its amounts in the plant.
The drought plants were wilting faster. They think this is because the salinity plants were able to use the salt in controlling osmosis. There's also large changes in amino acid composition, specifically proline, isoleucine and leucine. The differences in the expression levels between the two types of stress were mainly to do with photosynthesis and ROS.
Summary for the data set: the exp indicates that water damage had larger impact (and caused larger changes in gene expression) than equivalent salinity.
Proteomics comparison to transcriptomics
So, how does this compare to the data they got from proteomics data? Grapes are problematic, e.g. due to the large amount of phenolics. When run, 84 proteins were significant out of 645 proteins quantified (took a year due to all the manual reviewing of the photos). The abundance of 40 proteins increased, 20 decreased, and 22 increased and then decreased in various ways over time.
Comparing the results wrt functional classifications is interesting. Uncharacterised in transcripts is 30%, but not in the proteome, where almost all can be identified. About 30% are involved in metabolism. Energy and protein synthesis also important.
66 of 84 proteins had a Mowse score of 7 (95% confidence that the protein is what we think it is). 57 /66 have a transcript match with 90% identity or higher (90% is an arbitrary number). 17/57 have significant Pearson correlation with the transcript profile, which is relatively low.
Proteins that have bad correlation are, for example, antioxidants. Proteins with good correlatoin are heat shock proteins, and major latex-like proteins, and a methyltransferase. Could be due to limitations of the technology.
Summary: only 30% of protein profiles correlted with transcript profiles. Early responses in energy and growth-related protein profiles are not reflected in transcripts, but late responsive protein profiles do correlate. Plants respond first with changes in proteins related to photosynthesis and growth followed by changes in transcripts in photosynthesis, photorespiration and ROS detoxification.
1. rapid growth 2. lag phase 3. ripening
Harvested every week. They did a PCA of the data at each stage. Everything was grouped nicely except those in the lag phase, which means that it's incorrect to assume, as have in the past, that nothing's happening in that phase. Metabolism is higher in phases 1 and 2, and lower in 3. Transcription is going up, in contrast.
A lot of fruits ripen due to higher levels of CO2, which causes production of ethylene. Grapes, like strawberries, are thought not to respond to ethylene. However, it seems there are some small bursts of ethylene around veraison (step 3). There is a burst at 32, which is at the beginning of the lag phase. This is consistent with ethylene usage, which is a growth inhibitor. It goes up again in grapes just before veraison.
Water-deficit effects on berries of two different cultivars
Berries were smaller when they had a water deficit for white grapes and
red grapes, though less pronounced in older plants with deeper roots.
There are definite changes in the metabolism that can't be accounted
for just by reductions in size. The terpinoid pathway is stimulated by
the chardonnay but not the red grapes. In both, fatty acid metabolism
is stimulated, which creates more volatiles (via yeast or grapes isn't
known yet). The phenylpropanoid pathway, stimulated in the Cab Sauv
(what makes red wine "healthier") in the drought.
Microarrays provide valuable insights, and SB tools are in development. Molecular network maps will soon be released. There are multiple stress responses, and future work will focus on stress survival and berry quality metrics.
These are just my notes and are not guaranteed to be correct.
Please feel free to let me know about any errors, which are all my
fault and not the fault of the speaker. 🙂