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Cornell s Jocelyn Rose on Studying Cell Walls Via A Proteomics Approach

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At A Glance

Name: Jocelyn Rose

Position: Assistant professor, department of plant biology, Cornell University, since 2000.

Background: Postdoc peter Albersheim and Alan Darvill’s research group, Complex Carbohydrate research Center, University of Georgia, 1997-2000.

PhD, Alan Bennett’s laboratory, University of California Davis, completed 1997.

 

What did you study during your early years as a plant biologist, and how did you get into proteomics?

I’m from England but I came out to the US to do my PhD in Davis, California. There’s a big center of agricultural biotechnology there. So I got very interested in studying plants’ cell walls. In plants, to date there are multiple projects where researchers are studying the subcellular proteome of chloroplasts and nuclei and peroxisomes and so on, but no one was really tackling the cell wall as a subcellular compartment. And in plants, there are some very distinct challenges and features of the cell wall that are unique to plants. So plants are sessile — they can’t run away from stress, they can’t run away from attackers — so, there’s a very substantial amount of the proteome that is dedicated to the cell wall space. And the cell wall structure itself is extremely dynamic — it’s extremely plastic, and there are many enzymes involved in its assembly and reorganization. So, the cell wall responds to many stimuli. There are huge numbers of enzymes that are also signaling molecules transducing information across the cell wall. And therefore, consequently, it’s very important to try to understand what the proteome of that compartment is.

I was studying a couple of research problems. One of them was how are the cell walls taken apart and modified in processes such as fruit softening? So I was studying the molecular basis of how fruit softened, and why, and how can we alter this? When you buy fruits, they have a limited shelf life. This is important for many reasons. One of them is from a nutritional standpoint. For example, most tomatoes are picked when they are green, and then ripened with a gas called ethylene. They do this so they can ship them long distances and get them into stores before they soften. You can improve their shipping quality. The problem with that is if you pick a tomato when it’s green, it hasn’t had time to accumulate all the nutritional compounds. So the idea is if we can understand how a fruit softens and perhaps limit or regulate how it softens, we can not only improve the texture, but massively improve their nutritional quality.

One thing that became clear through my research was that looking at individual proteins and how those influence the cell wall structure gives you a very limited amount of information, and that a proteomics strategy is far more likely to give you a sophisticated understanding.

Also, I was interested in another project which relates to how plants defend themselves against bacteria and fungi. There are many, many defense-related proteins in the cell wall that not only are involved in surveillance to detect the presence of pathogens, but also they attack the pathogen. So, I was very interested in understanding again, populations of proteins and using proteomics as a tool to find the new proteins.

Was this while you were doing your PhD?

Well, my PhD really revealed to me how complex the cell wall was, and that current approaches to try to understand cell wall biology were very limited by not having a good catalogue of the cell wall proteome. At that time, about 60 percent of the cell wall proteome had not been identified. We were trying to understand a very complex biological problem with very limited information.

So for my postdoc, I went down to a place called the Complex Carbohydrate Research Center at the University of Georgia, and they have a very good mass spectrometry facility down there. At that point, proteomics was really coming into fashion — this was starting in 1997. Proteomics was really starting to come up in the vocabulary of plant biologists. The plant world really lagged behind animal and bacterial, obviously. But at that point in the mid-to-late ‘90’s, it was becoming accepted more as a potential approach. And I thought hey, we’re trying to understand this complex biology — why not use these proteomics tools. And so I started to get involved in some mass spectrometry and thinking about how to use the technology to study the [cell] wall.

Where did you go from there?

Well, I saw that Cornell was trying to recruit faculty in different aspects of genome-scale biology, and so I interviewed and decided that my research program would be involved in using proteomics as a platform to study the cell wall and trying to catalogue the proteome to understand how it changes during different development processes in response to different stimuli.

Were you one of the pioneers in that field?

In terms of looking at the cell wall proteome, there were very few groups that were doing it, yes. There’s probably one or two other groups that have really started to take this in a serious way. So in terms of pioneering an approach, I think looking at the cell wall in a more global approach certainly, yes, there are very few groups.

Are you still working on the fruit softening problem?

We’re still working on the fruit softening, absolutely. We’ve now been cataloguing during fruit development hundreds of extracellular proteins and trying to find new ones and to look at the functions of those.

Do you use a particular plant?

We’re working on tomato. That’s going to be the first fruit plant that’s going to be sequenced. The NSF has just put in some money to fund an international effort to sequence the tomato genome. There’s an international consortium, but the central part of this is based at Cornell. The initiative has just kicked off, and it’s going to be a two to four year timeline. A number of countries have been assigned a chromosome.

What have you found by studying the cell wall proteome?

First, we’ve found that there’s a substantial amount of post-translational modification in cell wall proteins. So there are very large numbers of extracellular proteases and protease inhibitors, and what that says to us is that regulation of gene function in the cell wall is very substantially influenced by proteolytic modification. And that could be activation or inactivation.

We’ve also seen that there is a very large number of cell wall binding proteins. We’re finding some very interesting proteins with modules that suggest that they bind to the wall that have yet to be identified. There are a lot of proteins involved in signaling and protein-protein interactions — and those could be protein complexes within the wall, or they could be binding and interacting with proteins secreted by plant pathogens and symbiomes.

One of the areas that we’re trying to get into now is — there’s been very little known about protein complexes in cell walls of plants. That’s one of the areas we’re actively targeting now.

Do you see any of these findings being applicable to the problems you were talking about with fruit softening?

I think very much so. For example, if we try to uncover new cell wall modifying proteins, it ought to give us some new insights. One of the problems with working with cell wall biology is you have a very complex polysaccharide matrix, and the interactions are very intricate and very hard to resolve. It’s very hard to find those proteins, and in many cases their activity is very hard to assay. So we’re limited I think in terms of looking at assays. If we can start to do the reverse approach by cataloguing those proteins and looking for modules that appear to bind to polysaccharides, and to screen in that way, that can give us very useful insights.

What is also very interesting is we’re finding a very significant number of proteins in the wall that have not been predicted to be wall localized. So in many cases there are plant proteins present as families, and in many cases we’re finding isoforms of proteins whose orthologues, or members of the same family, are localized in intracellular compartments — the chloroplast form of cytoplasmic form. And we’re finding that there are very closely related proteins that are also secreted to the wall. We have no idea what their function is, and in many cases it’s very hard to predict why they would be there. What that suggests to us is in many cases, many of these proteins are multi-functional. They may have completely other roles. I think that’s going to be a very fascinating field to start to tease apart.

What kind of proteomics techniques are you using?

One of the challenges we have is when you’re looking at an organelle, for example, you have a nice little organelle that’s encapsulated by a membrane, and there are nice ways in which you can fractionate tissues and purify organelles to get nice, clean fractions. When you’re looking at a wall, it’s more of a continuum. So we’ve put a lot of effort into trying to isolate cell wall protein fractions while minimizing contamination with intracellular proteins. That has been a big challenge. But we do have some very nice approaches now we’ve developed.

We’ve started to use now the DIGE, or Difference in Gel Electrophoresis, approach that Amersham sells. We’re going after the protein in the wall in that way. In addition to that we have a functional screen that pulls out genes that encode proteins that are secreted in a secretory pathway, and that’s been extremely helpful for us. So for example, if find a protein in a wall-associated fraction, what we can do is then clone that gene into the secretion system and test that it really is secreted. We’ve been coupling with some GFP analysis as well. This functional screen is relatively high throughput.

In addition to that, we try to use computational tools to predict the presence of signal sequences. We’ve found that some of the software that currently exists is relatively effective, but by no means perfect. So one of the things we want to do is use our functional screen to develop a better predictor for secretion of plant proteins. I think the coupling of multiple techniques is really going to be very important to us.

Have you found any proteins in particular that might be very applicable to solving problems, such as the fruit-softening problem?

Yes, we found some proteins that have some interesting new domains. Some of the proteins have some domains that suggest that they bind to some signaling molecules that have not really been characterized in plants before. These signaling molecules have been very well characterized in animals, but they have absolutely no known function in plants.

What we’re seeing really is that just by shot gunning and approaching this proteome in a very untargeted way, we’re getting a large number of proteins, and it’s throwing out all sorts of questions that we wouldn’t even have considered addressing before. I think using this non-hypothesis driven approach is really exciting.

What are you looking to work on in the future?

We’re looking at transcript profiling. What I want to do is couple proteomic analysis with microarray analysis. There’s a guy here who has really started to look at gene expression in depth during ripening, and we think it’s important to look at proteomic analysis in parallel with that so we can look at post-transcriptional regulation as well.

 

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