The Best Alternative

Published in Lab Times 04-2013.

ZIFL1 in Root

Expression of ZIFL1 protein in Arabidopsis root

It’s not uncommon for a gene to code for more than one protein. Alternative splicing is one of many transcriptional events behind such biodiversity. Paula Duque is carving a niche in this field that is very new to botanists.

When the Human Genome Project spewed out the “numbers” in 2003, one thing was certain: the “one gene one enzyme” hypothesis was an oversimplification. More surprising was the discovery that the lab mouse has about the same number of genes as man, about 25,000, and an even simpler organism Trichomonas has over 60,000 genes!

It’s a true thing that if one followed Beadle and Tatum’s math, a mere 25,000 genes can barely be accountable for an organism that has a debated million odd proteins. And a parasite that has more genes is definitely not belonging to a higher or even the same level of complexity as man. Evolution always finds ways to relinquish the dogmas of mankind, so it is unsurprising that it came up with alternative phenomena to expand the repertoire of proteins or in other words, complexity, without having to deal with too many genes. And yet again, geneticists are faced with the challenge of decrypting evolution’s mastery.

When a gene is transcribed into RNA, the primary transcript is prone to further processing that controls the nature of the translated protein. Splicing or the removal of introns allows protein-coding frames or exons to be stitched together to form an mRNA. If a primary transcript displays ‘alternative’ splice sites or if it allows introns to be ‘spliced in’, a number of product mRNAs can be generated for a given gene. These mRNAs may eventually translate to different protein isoforms that differ in their amino acid sequences and most often, have varied biological roles.

Alternative splicing, a process that increases biodiversity has been extensively studied in animals. A massive 95% of multi-exon genes in man are alternatively spliced. Drosophila is also famous for its prototypes. This ubiquitous process is however, as yet unexplored in plants. Though splicing is estimated to occur in at least half the genes in plants, the functional significance of splice variants of hardly a dozen genes has been identified. Paula Duque, head of the Plant Molecular Biology group at Instituto Gulbenkian de Ciência, Oeiras, Portugal is among the pioneers in plant gene splicing. Her research on the transport protein ZIFL1 isoforms in plant physiology has been recently reported in Plant Cell (Plant Cell vol.25(3):901-26). As an ardent plant-lover and a geneticist, Paula hopes to continue exploring the functional relevance of alternative splicing in Arabidopsis.

For the love of plants

Ever since her teens, Paula has had a crush on plants. It was this passion that persuaded her to set up her own plant lab in 2007 after two successive postdocs at Rockefeller University, New York. “Upon graduating in plant biology from the University of Lisbon, my interest was drawn to abscisic acid signalling and during my first postdoc I studied stress response in plants. It involved a gene with a bromodomain, so I did a lot of nucleic acid-protein interaction experiments which in turn, introduced me to the exciting area of splicing”, Paula remarks about her flashback. She temporarily “left plants and missed them” during her second postdoc in the lab of Magda Konarska, where she performed extensive in vitro biochemistry simply to master techniques for her future splicing studies.

As a principal investigator, Paula wishes to combine her interest in plants with her knowledge of genetics. Starting a lab wasn’t easy as she had to establish genetics in Arabidopsis right from scratch. Though the atmosphere in her hometown in Portugal was conducive, the 35 groups at her institute worked on a myriad different systems and she had to set up her lab from a “bunch of wildtype seeds”. “As a starter, nobody knows you. You face the challenge of publishing your first papers and then, there are the financial constraints. The success of grant application is down to half than it was 4 or 5 years ago”, Paula recollects the rough tides as a new group leader, “but eventually as results came in, this has been a great place to work and I’ve had help in every respect”.

The Duque group started out fishing for candidate splicing factors in Arabidopsis and generated mutants in which they particularly looked out for stress phenotypes. “A neighbouring yeast lab was then working on transporter genes that mediate yeast tolerance to stress. I wondered if the Arabidopsis homologs would be of potential interest. Though this was only a secondary project, my postdoc Estelle Remy gave it a huge launch and soon, there was no turning back”, Paula narrates how their story took off.

One phenotype and one bonus

Zinc-induced facilitator-like 1 (ZIFL1) influences the export of the synthetic form of the plant hormone auxin when expressed in yeast and its paralog ZIF1 protects cells from zinc toxicity. Given these hints, Paula and her postdoc ventured to follow their instincts and went on to characterize the function of ZIFL1 in Arabidopsis. Their initial findings uncovered three isoforms of the protein that differ in structure and tissue distribution. While isoforms 1 and 2 (ZIFL1.1 and 1.2) are present both in leaves and roots, splice variant 3 (ZIFL1.3) is exclusively expressed in the leaves. To resolve the function of ZIFL1, the duo generated loss-of-function mutants. To their excitement, they found that root elongation in the zifl1 mutant is sensitive to the inhibitory effects of auxin and that the mutant root displays gravity bending defects. Surprisingly, overexpression of ZIFL1.1 but not ZIFL1.2 or 1.3 rescued the distorted phenotypes. “We were already delighted that we found an isoform-specific role of ZIFL1, when we had a second breaking point in the story”, the botanist reflects on a serendipitous discovery that ensued soon after their first Eureka. “Estelle had forgotten to water the plants one day and she came out screaming to me that the mutants were wilting while the wildtypes weren’t!” Estelle’s negligence to tend her plants actually rewarded them with another mutant phenotype – hypersensitivity to drought. It did not take them long to interrogate into this new observation and they found that it is in fact the third isoform that functions in this context. ZIFL1.3 they determined controls stomatal aperture size, a factor that prevents excessive water loss from leaves.

ZIFL1’s double role

From the work of the Portuguese group, it appears that ZIFL1.1 controls auxin-related processes in the root while ZIFL1.3 regulates transpiration in the leaves. Chatting further on the intricacies of her research, Paula elaborates “when we looked carefully, we found that the shootward transport of auxin was disrupted in our mutants. We hypothesized that ZIFL1.1 may actually have a role in auxin efflux in the root and its shootward transport and that altered auxin distribution in zifl1 mutants contributes to the root defects. But how could ZIFL1 export auxin across the plasma membrane when its expression is confined to intracellular vacuolar membranes called tonoplasts?” Their preliminary studies had shown that ZIFL1.1 localizes to tonoplasts and this seemed inconsistent with its involvement in transporting auxin across the cell membrane.

“PINs and ABCB transporters are both involved in auxin export. While the latter use ATP for their activity, the PIN proteins rely on energy from proton gradients to drive transport”, Paula remembers the little clue that sparked when they were faced with the localization paradox. ZIFL1 in their study turned out to extrude protons, so they postulated that ZIFL1-mediated changes in pH in the cytosol may indirectly influence auxin transport proteins at the plasma membrane and hence, facilitate auxin efflux. “We could show that ZIFL1’s activity affects PIN2 abundance at the membrane. The zifl1 mutants mimicked to some extent pin2 null mutants in their defective shootward auxin transport suggesting that ZIFL1 positively but indirectly regulates auxin transport”, she summarizes their findings.

Exciting prospects

Paula and colleagues have elucidated isoform-specific roles of plant ZIFL1. The functional differences may in part, arise from their diverse tissue distributions. Such specific patterns of expression kindle one’s curiosity. What may be the upstream regulators of ZIFL1.1 and 1.3 that give them their respective spatial expression profiles? “ZIFL1 may be spliced off differently in the roots and leaves depending on tissue-specific splicing factors. We also know that ZIFL1.3 is unstable in the roots. This could mean a possible tissue-specific micro RNA regulation of the transcript. This certainly becomes the most important question in the paper”, the Portuguese botanist chews on some of the prospects of their research. Another obvious open question is on the role of ZIFL1.2 which did not have any function at least in the processes they studied.

Despite all the tough luck that she had had as a start-up investigator, Paula remains undaunted. “The financial situation in Portugal is not looking good. Though it’s a great scientific atmosphere in here and my projects are funded by the FCT which is the National Funding Agency, it’s not easy when the government starts cutting down on research”, she underscores the financial crisis in Portugal.

Nevertheless, Paula is very excited about the projects that have spun off following their first papers. As a concluding note, she gives a little pointer to aspiring group leaders. “The trouble in Science is – particularly, when you are a new PI – you have to learn to strike a balance between not giving up too soon and not trying incessantly. When you are determined to find an answer but are not immediately successful, you need to hang in there. But you should also know when it is time to give up. And the truth is, as a scientist, you are never going to know how to deal with this perfectly”, she shrugs.

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