QnAs with Caroline Dean

As winter gives way to spring, many plants erupt in a riot of colors in a bid to attract pollinators and set seed. As if by clockwork, these species time their bloom, delaying flowering until the onset of spring, when conditions favor fertilization and seed development. Researchers have long known that plants delay flowering until they have experienced a period of prolonged cold, a process termed vernalization. For decades, plant breeders have exploited this cold-dependence to develop varieties of cereals and vegetables sown in the winter and spring, extending crop range to ensure year-round supply. The molecular basis of vernalization, however, is just beginning to come into view. The question of how plants sense the passing of winter and prepare to bloom preoccupies plant molecular biologist Caroline Dean at the John Innes Centre in Norwich, United Kingdom. Dean’s work and its implications for agriculture have gained increasing currency in the face of a changing climate. A foreign associate of the National Academy of Sciences, Dean received the 2018 L’Oréal-UNESCO For Women in Science award for her contributions to the molecular understanding of vernalization. Dean spoke to PNAS about the work that led to the award.

Caroline Dean. Image courtesy of Thierry Bouët (photographer).

PNAS: How did you become interested in vernalization?

Dean: I grew up in the north of England and enjoyed the distinct seasons, so when I went as a postdoc to California, I missed the clear seasonal transitions. In California, I bought some tulip bulbs, and the person in the shop said to me “Put them in the fridge for 6 weeks.” I was so struck by the comment that I went and read about why I would put them in the fridge, and realized that [tulips] actually need the cold to bloom. That got me thinking generally about how plants use seasonal cues to time their development. Around this time, the Arabidopsis phenomenon was happening [plant geneticists had discovered the utility of focusing on one species as a model organism], and we could now clone genes important for complex traits. So when I started my own laboratory, I chose to tackle the molecular basis of vernalization in Arabidopsis.

PNAS: We are in the midst of cherry blossom season in Washington, DC, and the cherry trees burst into flower within days to weeks of each other seemingly on cue. Is that vernalization in action?

Dean: A related process; trees arrest their buds in autumn, with both temperature and photoperiod being important to break that arrest and promote the spring bloom that everyone enjoys. We know that many of the same molecular components are required to control flowering in trees and Arabidopsis, but still have to fully elucidate the regulatory network.

PNAS: What are some examples of commercially important plants that undergo vernalization?

Dean: Vernalization is important for many of our major crops. Winter and spring-sown varieties have been bred for many cereals, [for example], wheat and barley. On the other hand, for vegetable Brassica varieties, [such as] broccoli and cauliflower, breeding has focused on differing cold requirements that lead to flowering at different times throughout the winter, ensuring a continuous supply to the supermarkets.

PNAS: Before we delve into the molecular biology of vernalization, could you briefly explain how plants adapt to different lengths of cold exposure before flowering?

Dean: We are still dissecting exactly how plants register winter exposure. Clearly, plants have to distinguish a short period of cold in autumn from full-blown winter. Different varieties of Arabidopsis thaliana show different cold requirements, presumably as an adaptation to the climate they are growing in. Typically, varieties from southern Europe only need 4 weeks of cold to maximize acceleration of flowering, whereas those from northern Sweden need 10 to 12 weeks to do the exact same thing.

PNAS: And the variation is genetically encoded, correct?

Dean: Yes, a small number of polymorphisms in the gene that underpins vernalization dictate the length of cold exposure a particular variety needs to flower.

PNAS: You have uncovered the central role of the gene FLOWERING LOCUS C (FLC) in vernalization. What does the gene do?

Dean: The FLC gene was cloned almost simultaneously by two other groups. FLC represses flowering by blocking genes required to switch the plant’s apical meristem to a floral fate. We came to FLC through our interest in the mechanism of vernalization. Many natural variants of Arabidopsis have a winter annual habit and need prolonged cold exposure to flower. We mutagenized the winter annual variant to obtain vernalization mutants that could no longer respond to cold. These mutants affected the silencing of expression of FLC, which occurs gradually with cold exposure.

We found that silencing of FLC expression happens through chromatin regulation: modifications of histone tails and chromatin remodeling [which are common routes of chemical modification that induce conformational change in the genetic material to switch off the expression of certain genes]. This particular type of gene-silencing mechanism is faithfully inherited through cell division (an epigenetic mechanism) and is conserved in plants, animals, and humans.

But if cold exposure silences FLC by essentially switching off the gene’s expression, we wondered why it was gradual with increasing cold exposure [the length of cold exposure tracks time to flowering]. Through collaboration with computational biologist Martin Howard, we showed that there is an ON–OFF switch at each individual FLC gene. It takes all winter for all of the genes to be switched OFF, with the gradual silencing being a reflection of the proportion of cells in which the FLC gene is switched OFF. So, naturally, if you ground up the whole plant and looked at RNA expression, as we had been doing, it would look like a nice, slow decrease. This cold-induced epigenetic silencing is then maintained through many hundreds of cell divisions, even when plants return to warm conditions in the spring; in other words, it is epigenetically silenced. The FLC gene does not switch back on again until seed development.

PNAS: But what is upstream of FLC? How does cold trigger the silencing of FLC?

Dean: We are finding that temperature influences a large number of steps in FLC silencing. The major temperature steps characterized so far include early cold-induced up-regulation of antisense RNA transcripts at FLC, and up-regulation of the VIN3 protein required for the epigenetic switch. In the cold, VIN3 associates with a protein complex at the FLC locus to promote the epigenetic switch.

PNAS: Your recent article in Nature Communications (1) explores how plants register fluctuating temperatures normally encountered in the field during vernalization.

Dean: What we showed in this paper is that plants are monitoring distinct aspects of the fluctuating temperature to regulate different parts of the vernalization process. For example, FLC down-regulation occurs relatively quickly when temperatures drop below 10 °C. In contrast, VIN3 accumulates gradually with time over a relatively wide temperature range (0–15 °C), but is degraded by transient high temperatures (above 15 °C). So, it is the absence of warmth that is an important trigger enabling accumulation of VIN3 and epigenetic switching. This temperature registration strategy enables plants to “read and interpret” daily temperature fluctuations, which often are as extreme as those over a whole season.

PNAS: Was that a surprising finding?

Dean: It wasn’t expected. Until recently, it was thought that plants slowly integrated cold temperature exposure to judge winter progression. In fact, horticulturalists have used day-degree calculations to judge the progression of vernalization in the field. We now know these models will only work well when the peak high and low temperatures are not too extreme. Instead of just averaging temperature over time, the plants are constantly monitoring maximum and minimum daily temperatures to judge seasonal progression.

PNAS: Against the backdrop of climate change, does that finding have any commercial implications for food crops?

Dean: The crop varieties that we grow are unlikely to cope well with the predicted future extreme temperature fluctuations. Commercially, we have seen the consequences of such extremes. Last winter, for example, the unusually cold temperatures in the south of Spain triggered poor broccoli production. This led to a severe shortage in the supermarkets around Europe.

PNAS: The late molecular biologist Susan Lindquist at the Whitehead Institute in Cambridge, Massachusetts, reported the role of a prion-like protein called LUMINIDEPENDENS (LD) in mediating plants’ memory of winter. Does the protein play a role in vernalization?

Dean: LD does affect FLC, but its role appears to be chiefly in the warm. Since mutants defective in LD vernalize efficiently, it seems unlikely to play a major role in cold sensing.

PNAS: Would you care to share your thoughts on winning the L’Oreal-UNESCO For Women in Science award?

Dean: I was thrilled. I just came back from the awards events in Paris, and it was wonderful getting to know the other laureates and the rising talents: young, talented, and enthusiastic women scientists from all over the world, who [received fellowships and] were also invited to the event. My hope is that this prize encourages other women to stay in research long-term; you can combine a fulfilling, high-level scientific career with a relatively normal personal life.