Researchers from VIB and Ghent University have used targeted proteomics to map the interactions of roughly 100 proteins involved in regulatory pathways controlling cell division in Arabidopsis thaliana.
The study, which was published this month in the online edition of Molecular Systems Biology, offers insight into the great diversity of core cell cycle proteins in plants and could help scientists understand systems essential to increasing agricultural yield, Geert De Jaeger, a researcher in VIB's department of plant systems biology and one of the paper's authors, told ProteoMonitor.
Compared to most other eukaryotes, plants have a large number of proteins involved in core cell cycle functions. For instance, while yeast and human genomes contain 15 and 23 genes for cell cycle regulatory proteins, respectively, the Arabidopsis genome contains 71.
These 71 genes code for cyclin-dependent kinases, kinases whose substrate specificity is determined by interaction with various cyclins. Despite these proteins' key roles in plant cell cycle regulation, relatively little is known about them and their interactions.
In an effort to better characterize them, the VIB and GU researchers selected 102 Arabidopsis cell cycle proteins and put them through tandem affinity purification in order to isolate and analyze their interactions with other proteins.
Using TAP, followed by MALDI mass spectrometry on Applied Biosystems 4700 and 4800 Proteomics Analyzer machines, the researchers identified a total of 857 non-redundant interactions involving 393 proteins. Of these interactions, 150 matched known or predicted protein-protein complexes. A search of public databases revealed that the remaining 707 interactions had not yet been documented. They also identified roughly 100 genes involved in cell cycle pathways previously not thought to be tied to cell proliferation.
With this interaction data, the team was able to build a rough map of the basic cell cycle pathways in Arabidopsis, De Jaeger said.
"By using available knowledge combined with data coming from gene expression analysis, we were able to analyze the network [of protein interactions] and actually build up a kind of core cell cycle map where we were able to place the [CDK] complexes and their regulators," he said. "We could predict where these kinases are active [and] at which timepoint in the [cell cycle] process these kinases are doing their job."
This information gave the researchers insight into the wide diversity of cell cycle proteins in Arabidopsis.
"There are many, many more cyclins in plants than in animals, and a lot of these cyclins are actually reflecting genome duplications," De Jaeger said. "Arabidopsis has had four genome duplications during its evolution, and these duplications have caused this huge amount of cyclins."
A major question, he noted, has been whether these cyclins – many of which have quite similar amino acid sequences – are simply redundant, or whether they actually have distinct functions. According to De Jaeger, the researchers' cell cycle map suggests the latter.
"Our map shows that the cyclins have very high functional diversification," he said. "Each cyclin, even if they belong to the same subclass and are quite close at the amino acid level, interact with different regulators or are active at different points in the cell cycle. It's not that plants have duplicated their cyclins for redundancy to make the system robust – it's that each cyclin present in the plant has a particular function reflecting the huge complexity of cell cycle regulation in plants."
This complexity, De Jaeger suggested, is likely necessary because of plants' need to adapt to their constantly changing environments.
"A plant can't run away from a difficult situation in the environment," he said. "It has to go through periods of cold stress, drought stress, attacks from predators. When seasons change they have to make new leaves or let their roots grow to pick up water. So environmental adaptation is actually built into [plants'] developmental plan, and cell proliferation and the cell cycle play a very important role there."
Cyclins act as an entry point for these external inputs, De Jaeger said, linking signaling pathways tied to conditions outside the plant to protein pathways involved in cell proliferation. The next step, he added, is to develop a better understanding of how these two types of pathways fit together, with the ultimate goal being the development of methods for improving plant yield.
"The big thing in plant research right now is yield," he said. "The [United Nations] has predicted that by 2050, the amount of food that can be grown on a hectare has to double, so there's a lot [of research] about finding genes that can actually steer growth in crops or are able to steer growth in crops under difficult environmental conditions."
Cell proliferation is one of the main factors in determining plant organ size and, consequently, yield, De Jaeger said, making an understanding of the process key to the yield question.
"We want to understand why, for instance, a banana plant makes an extremely big leaf and why another plant like Arabidopsis makes a very small leaf, and cell proliferation is playing a very important role there. So one of the main secrets of the organization of plants that we have to figure out is how cell proliferation is regulated," he said. "It will all fall back on trying to figure out these protein networks."