3 posts tagged “grapes”

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.

Berry development

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. :)

...or, all you ever wanted to know about wine yeast, but were afraid to ask
Duccio Cavalieri
Plenary Talk, Afternoon Session, 1 September (11th MGED Meeting, 1-4 September, 2008)

Volatile organics in: Grapes = 466 , Wine = 644, Difference= 178. The most ancient evidence from 3150 BC, in Egypt. However, the ecology of yeast has been mainly unknown. The probability of S.cerevisiae on a pristine grape is .0005, while per damaged grap is 25%, which means 10^4 - 10^5 (probably from clonal expansion). Proportion of damaged grapes is 1/1000. When there are many organisms (above 4%), cerevisiae is the only survivor. 88% of the S288c gene pool derives from EM93 isolated from rotten figs in Mercedes, California in 1938. Most regulatory genetic variation is due to a high rate of cis acting alleles and a small number of trans acting alleles.

Exploring the genome-environment interaction, and looking at mendelian segregation of expression profiles. One strain's colony, after 4 days' growth, produces an interesting and cohesive shape ('filigree'). Compared the parent to the first segregants. They found that 2 alleles were controlling the expression of 378 genes, or 6% of the genome. They tried to clone these genes from the recessive mutation, but failed after screening 300 false positives. Then, a few years later, tried to get the genes responsible for the phenotype via pathway analysis via a Fisher exact text followed by pathway correction. If you compare with the wt, there isn't a single significant pathway that segregates except the cell cycle stuff. Then he applied Bagel Analysis to extract absolute values and probabilities using bayesian statistics - discovered significance for amino acid transporters. They then did the same experiment, but with rich amino-acid medium, and with a minimal aa medium. The filigree strain has a mutation in SSY1, which causes a truncation. This means you lack the sensing part of the protein on the outside of the membrane, which means that the strain is blind to the amino acids in the media. This makes the yeast produce its own amino acids.

Only a small number of genes were differentially expressed based on the filigree phenotype. Ammonia mediates signalling between these cells. MEP1, MEP2, and MEP3, and PHD1 three were overexpressed in the strain. M28 is homozygous for a mutation causing non-disjunction of cells after cell division affecting AMN1 and GPA1. The daughter cell doesn't divide immediately after division, which creates 3d structures.

Means that the potential for differences in the various wine strains is quite high. The estimate of the relatedness between By4743 and Em93 (parent strain) is 90% similar. Most of the variation is not evenly distributed. Mutations in the aa, nitrogen sensing, and hap genes are highly pleiotropic. HDAC and SIR-SAS gene expression control provides the cell with a mechanism of epigenetic buffering of variation.

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. :)

Claudio Moser, Department of Genetics and Molecular Biology, E. Mach Foundation - IASMA
Plenary Talk, Afternoon Session, 1 September (11th MGED Meeting, 1-4 September, 2008)

The grapevine berry ripening process

It takes 4 months to go from a flowering cluster to a fully-ripened cluster. The development of the grape is in three phases: 2 growing phases divided by a phase where the berry doesn't grow anymore. Berry formation followed by berry ripening. There are three major tissues: skin, pulp, seeds.

Why is it important?

Economics: It is the most important fleshy fruit from an economic point of view. There are 8 million hectares of vineyards worldwide with an annual turnover of more than 20 billion US $. There is increased interest in grape-derived anti-oxidant compounds. In Trentino, grapes and apples represent the two most relevant crops.

Biological relevance: The ancestor of the cultivated version climbed trees, reached the top of the canopy, and then flowered. Domestication has passed from separated-sex flowers (male flowers and female flower) to a hermaphrodite flower.

The grape transcriptome

Transcript profiles are studied to understand the molecular dynamics of berry ripening and regulation. Research can be done with the Affymetrix Vitis GeneChip. This chip contains 14,000 unigenes. Applications of the research improve management practices (via lower inputs, lower costs, and higher quality), use gene transfer to create new varieties or improve traditional ones. There are two ways of getting new genes in: gene transfer (direct) or marker-assisted selection.

They used pinot noir grapes, and sampled them at three different stages of the life cycle, with 3 biological replicates for each time point, then repeated the analysis in 3 different seasons. The 2003 data was quite different - this can probably be explained by 2003 having a very hot summer. Further, the analysis separated out the ripe berry time point from the earlier two time points. They ended up with ~1800 genes that will form the berry-ripening core set.

What have we learned about the biology?

9 GO classes were statistically significantly different from the affy chip, with 6 of those being overrepresented. Ripening is finely programmed before veraison and its transcriptional modulation reflects berry biochemical changes. In the first phase, the berry goes through a re-programming phase before moving on to the next growing phase. A consistent fraction of the isolated genes are devoted to the control of the developmental program. (This has also been found in tomatoes.) Major TF families include zinc finger, oxin-related, and others.

He looked more closely at genes related to ROS metabolism. Noticed there was a burst of hydrogen peroxide at the veraison phase. Seasonal influences produces changes that involve light signalling, ripening-related genes, and non-ripening-specific isoforms

Future directions

Wants to look into ethylene, as it is probably quite important in berry development. Exogenous ethylene application can modify the ripening curve. Endogenous ethylene can be measured just before veraison. Tried to measure these things on their pino noir grapes. They got some noisy data, but think they saw two peaks. In 2007 there were two different genome sequences of pinot noir grapes published. They found about 30,000 genes. They want to do more analysis on it.

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. :)