Markus Klein on Characterizing a Plant MATE Transporter
Fast Moving Front Commentary, September 2011
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Article: The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+-antiporter active in proanthocyanidin-accumulating cells of the seed coat
Authors: Marinova, K;Pourcel, L;Weder, B;Schwarz, M;Barron,
D;Routaboul, JM;Debeaujon, I;Klein,
M |
Markus Klein talks with ScienceWatch.com and answers a few questions about this month's Fast Moving Fronts paper in the field of Plant & Animal Science.
Why do you think your paper is highly cited? Does
it describe a new discovery, methodology, or synthesis of knowledge?
Would you summarize the significance of your paper in layman's terms?
How did you become involved in this research?
Why is this paper highly cited? In my view this is because it was the first biochemical characterization of a plant MATE transporter demonstrating function with flavonoids, and because it addressed a missing element in flavonoid biosynthesis. This was already very well characterized based on genetic evidence in several model plants; however, the "transport" question had not been solved before.
When we started to work on the Arabidopsis Transparent Testa12 (TT12) transporter, we brought several pieces of evidence together and came up with the first biochemical characterization of a transporter for flavonoids. Flavonoids are one class of so-called secondary metabolites. A prominent and very "visible" example of flavonoids are the anthocyanins, which are pink to blue plant pigments e.g. in flowers or fruits that are attracting insects or birds for pollination and seed dispersal.
In most cases, flavonoids are synthesized in a species-, tissue-, or even cell-specific manner in the plant cell cytosol. In order to act as plant "sunscreen pigments" for UV protection, defense reactions, insect attraction or as deterrents, usually high amounts of these substances are needed because these functions are often dose-dependent. At the same time, flavonoids have protein denaturing and other properties that are harmful or toxic to the cell when high concentrations are synthesized. Consequently, as an important part of a "self-defense" mechanism, flavonoids and many other secondary metabolites with toxic side-effects are very efficiently excluded from the cytosol.
"Considering the scientific field, it is very obvious that secondary metabolite transport has expanded and made a leap forward in recent years."
Apart from export into the extracellular space, the major site of accumulation of flavonoids is in the large plant cell vacuole where they can reach concentrations in the upper milli-molar range. Thus, there have to be very efficient and specific transporters at work that generate these concentration gradients. However, while many important transporters for primary metabolites and ions where known when we started to work on these processes, the molecular basis of secondary metabolite transport was—and still is—in its infancy.
During my Ph.D. study performed in the team of Gottfried Weissenboeck at the University of Cologne, we found that a minimum of two different transport mechanisms for transport of barley and rye flavonoids into vacuoles have to exist. Depending on the chemical structure and the species tested we characterized directly energized, ATP-driven flavonoid "pumps" or secondary energized transporters using the steep proton gradient between the acidic vacuolar lumen and the more basic cytosol.
This discovery was somewhat similar to the situation for alkaloids—another important class of secondary metabolites—where either ABC (ATP-binding cassette) transporters or proton-dependent transport systems were described for the plasma membrane and the vacuolar membrane, respectively.
Through the characterization of Arabidopsis mutants in ABC transporters performed in the lab of Enrico Martinoia at the University of Zurich, we hoped to put our hands on the "secondary metabolite pumps." However, although we obtained a couple of other interesting phenotypes leading to an understanding of important processes controlled by these membrane proteins, we did not find our flavonoid transporters.
At that time, in 2001, Isabelle Debeaujon characterized the Arabidopsis tt12 mutant which lacks the proanthocyanidin flavonoids of the seed coat and could map the TT12 gene which turned out to be a MATE (multidrug and toxic extrusion) transporter. MATE transporters were—and still are—only poorly characterized in plants. However, in bacteria and mammals they turned out to be secondary active transporters for a large variety of compounds. So, we suspected that TT12 could be our first flavonoid transporter and teamed up with Isabelle to put together the molecular and biochemical evidence to demonstrate this.
How would you describe the particular challenges,
setbacks, and successes that you've encountered along the way?
One very positive experience during this discovery was our productive and open collaboration with Isabelle Debeaujon from the Institut National de la Recherche Agronomique in Versailles, France. We came from different corners and with different experience: Isabelle represented the genetics side and also contributed her knowledge on the Arabidopsis seed experimental system. The Zurich team had the experience on transporter characterization and membrane biochemistry. I was extremely proud at that time that this discovery was based on our teams working efficiently together, complementing each other fruitfully and that we could finish this project with a publication in a very respected journal.
One major challenge was establishing a biochemical transport assay using baker's yeast membranes (microsomes) as a heterologous system. All our prior experience with the so-called "rapid filtration yeast membrane assay" was based on using radioactive substrates for the transport processes to characterize. These radioactive substances could be measured with high sensitivity and accuracy. For the TT12 characterization, we only had the unlabeled flavonoids. The radio-labeled equivalents were not in the catalogs of the standard radiolabeled chemicals suppliers. On the other hand, we could not afford custom-labeling or synthesis of our test compounds.
We thus had to apply higher amounts of substrates, more of the yeast membranes, and combine it with the chromatography and spectroscopic detection methods we had around. It needed many liters of yeast cultures and lots of drive to convince ourselves that we were really following a transport process and not just observing non-specific binding. Taken together, this took a long time—and it is the perfect example of the one important and finally successful dataset where nobody can see afterwards how much time, personal effort, sweat, and intermediate frustration it took to get there.
Where do you see your research leading in the
future? Do you foresee any social or political
implications for your research?
"As we start to expand our understanding from the mechanistic side (how the transporter works) towards approaches and tools helping to "design" transporters for specific substances, new applications will be in the realms of possibilitie."
Personally, I have now moved out of this field and also academia. I now work for Philip Morris International, and my team is involved in developing and breeding tobacco varieties with improved properties. In this environment and with the objectives that we use to guide our research, a very sound understanding of plant biochemistry and metabolism is needed. Transporter processes, however, are only one of the many elements we are evaluating at this time.
Considering the scientific field, it is very obvious that secondary metabolite transport has expanded and made a leap forward in recent years. Several transporters were characterized—as well ABC transporters as further MATE transporters. Today, we have a much better understanding of, e.g., sequestration and transmembrane transport of alkaloids, including nicotine.
We also understand more about the way that epidermal cells on plant surfaces excrete the building blocks of the extracellular wax layer reducing water loss and protecting the plant. Here the expectation could be that this leads to tangible strategies to increase the water use efficiency of plants and therefore reduces the need to artificially water crops, especially in arid regions.
I have enjoyed seeing that based on our TT12 characterization, a couple of similar processes have been characterized: the anthocyanin transporters in red grapes or the proanthocyanidin precursor and flavonoid transporters in Barrel Medic, an important forage crop, are all MATE transporters related to the Arabidopsis TT12 protein. Are there direct implications or applications from all these results? We have to see whether this research can—among other applications—be used to improve red wine quality, increase crop resistance to UV irradiation or leads to ornamentals with a new spectrum of flower colors that consumers may value.
An exciting breakthrough in my eyes was the finding that an agronomically important plant mechanism to deal with aluminum toxicity in soils and to acquire iron via roots needs the activity of MATE transporters that are excreting carboxylic acids which bind these metal ions. Thus, this pretty "newly discovered" family of transporters mediates essential processes and contributes to crop productivity on acid soils. Consequently, this may be the most direct "social implication"—it would be great to see that the MATE knowledge is applied to develop or breed crops that can better grow under such unfavorable soil conditions ultimately resulting in higher crop productivity.
On the contrary, many results from several research groups also confirm the "dilemma" when studying so-called "multidrug" transporters such as ABC or MATE transporters: it is hard to predict functions and roles because the substrates are chemically extremely diverse—so we need to look at the processes in some detail because general assumptions may not be valid. Here, the recent elucidation of the X-ray crystal structure of the bacterial NorM MATE transporter by Geoffrey Chang and team will for sure strongly influence the understanding of structure-function relationships.
As we start to expand our understanding from the mechanistic side (how the
transporter works) towards approaches and tools helping to "design"
transporters for specific substances, new applications will be in the
realms of possibilities. The similarity and homology between plant-specific
transport processes for secondary metabolites such as flavonoids and
alkaloids and the way that similar transporters are used to detoxify
"man-made" chemicals (xenobiotics) such as herbicides has the potential to
lead to new strategies targeting the design of more specific, i.e.
species-specific, crop protection agents. These new agents could reflect
the understanding of the mechanisms involved in the removal of these toxic
products from the plant cell cytosol.
Markus Klein, Ph.D.
Philip Morris International
Neuchatel, Switzerland
KEYWORDS: ARABIDOPSIS MATE TRASPORTER TT12, VACUOLAR FLAVONOID, H+ ANTIPORTER, PROANTHOCYANIDIN-ACCUMULATING CELLS, SEED COAT, MULTIDRUG EFFLUX PROTEIN, VIBRIO PARAHAEMOLYTICUS, GENE ENCODES, PLANT CELLS, DOMAIN PROTEIN, BIOSYNTHESIS, FAMILY, MEMBER, RESISTANCE, EXPRESSION.