Organogenesis in plants is a dynamic process that occurs in meristems, where stem cells are maintained. A new paper in Development characterises the real-time dynamics of floral organ primordia initiation and floral meristem termination. We caught up with the authors, Ya Min, Stephanie Conway and Elena Kramer, to find out more about the paper and the story behind the research.

L-R: Ya Min, Stephanie Conway and Elena Kramer

Elena, can you give us your scientific biography and the questions your lab is trying to answer?

EK: I did my undergraduate at Brandeis University working with Jeff Hall on Drosophila behavioural neurogenetics, but when I went to graduate school at Yale, I discovered my love of plant development in Vivian Irish's lab. After doing a short postdoc with Gunter Wagner at Yale, I started my position at Harvard in 2000. Today, our laboratory is very broadly interested in the evolution of plant reproductive morphology. We use molecular, developmental and phylogenetic approaches to study how plants (mostly flowers) have changed over the course of evolutionary time. Research projects in the lab cover a range of topics, including comparative developmental and transcriptomic studies of novel morphological features; the consequences of gene duplication for the evolution of gene function as well as floral morphology; and the developmental and genetic mechanisms that underlie morphological diversification, especially in petals.

Ya Min and Stephanie, how did you come to work in Elena's lab and what drives your research today?

YM: I first met Elena as a master's student when I was doing a rotation in the lab for a semester. At that time, I didn't know what evo-devo was, and the research projects going on in the Kramer lab opened my eyes and deeply intrigued me. Plus, an obvious bonus of working in the Kramer lab is being surrounded by beautiful Aquilegia flowers. So, it was a very easy decision to pursue a PhD with Elena to study floral development in Aquilegia. In the first two years of my PhD, with help and guidance from Elena, I discovered my passion for understanding the earliest phases of making a flower (e.g. floral meristem regulation, floral organ determination and initiation) and formulated it into a PhD dissertation focusing on floral meristem termination in Aquilegia. I'm still deeply curious about the molecular networks controlling these early developmental processes and excited to learn more.

SC: I met Elena when I was a visiting graduate student at the Arnold Arboretum of Harvard University. I was really drawn to her lab's work because it investigated plant form and function through the powerful lens of molecular biology, and I loved the ability to integrate plant biology across all of these frameworks. A few years later, I was lucky to become a postdoc with Elena and was able to work with beautiful Aquilegia flowers, which has been a joyful learning experience. My research has always been driven by a love of plant morphology, and I am now trying to use as many tools as possible – histology, microscopy, molecular, genetic – to think about how plants have evolved their shapes in order to adapt to and succeed in every corner of our planet.

Before your work, what was known about the dynamics of floral meristem development?

YM, SC & EK: How meristems function and develop has been a subject of interest for a very long time, tracing back to the early 1800s, when the first microscopes were invented and people saw the meristems of various plants for the first time (e.g. Sprengel, 1793; Payer, 1857). Since then, we have gained a lot of knowledge about the properties of meristems and their fundamental importance to plant growth and reproduction. We know that floral meristems (FMs) normally have a similar overall structure to the vegetative meristems that produce leaves. They harbour a population of stem cells in the centre, which is surrounded by organogenic cells that will give rise to organ primordia. On the one hand, just like other meristems, FMs need to maintain homeostasis of their stem cell pools to ensure the successive production of organs. On the other hand, this proliferation must be terminated at a specific time point during floral organ initiation, a process called floral meristem termination (FMT). FMT results in the loss of pluripotency of all the cells that remain in the FM, which will then be incorporated into production of the innermost organs of the flower.

However, despite all of this progress, our understanding of how meristems develop is still fragmentary because most of the information was obtained from single time points, using methods such as scanning electron microscopy and histology, but meristem development is a continuous and dynamic process. This is especially true when we seek a comprehensive understanding of meristem regulation by connecting knowledge of gene expression with growth patterns. Very often, we do not know how the expression of a gene directly impacts the local growth parameters and the physical property of a cell, because we do not have the information of how either gene networks or physical growth are behaving at cellular resolution both spatially and temporally. Our current study sought to examine how cellular dynamics change during the last phases of FM regulation at high resolution, which will be fundamental to our eventual in-depth understanding of FM regulation.

Can you give us the key results of the paper in a paragraph?

YM, SC & EK: Using live confocal imaging, we carefully examined the developmental dynamics of floral organ primordia initiation and FMT in Aquilegia coerulea. We found that the initial outgrowth of stamen, staminode and carpel primordia is driven by a layer of cells that are located at the abaxial-most position of the incipient primordia, and these high growth rates are primarily driven by highly anisotropic cell expansion. However, the organs also exhibited identity-specific patterns. For instance, we observed differences in growth dynamics between the two whorls of staminodes, which share the same floral organ identity. The growth rate of the inner whorl of staminodes appears to be lower than that of other organs in the same developmental interval. We also examined the early developmental processes of the carpel primordia in detail, capturing a transition from growth that is mainly driven by strong anisotropic cell expansion to one that is promoted by concentrated cell divisions. This suggests that there are two sets of molecular programmes acting during early carpel primordia growth: one for the earliest phase of primordia initiation that is common to all floral organs and a subsequent programme that is specific to sculpting carpel primordia. Finally, we analysed the cellular behaviours during FMT. We detected a higher number of cell divisions in the centre of the floral apex during the initiation of the carpel primordia, which suggests that FMT can be discernible from carpel initiation.

Do you think that the burst of cell division is necessary for FMT or just for carpel morphogenesis?

YM, SC & EK: Well, that's a really good question. It's just a guess, but our current hypothesis is that the shift in cell division patterns in the central zone reflects a transition of identity. When these cells cease to become central zone cells and start to shift to carpel primordium identity, their rates of cell division shift. So, in some ways it's both!

A TP5-TP6 flower bud

A TP5-TP6 flower bud

What impact do mechanical forces play in organ morphogenesis, and do you think this will apply at all floral transitions?

YM, SC & EK: We know that mechanical forces are very important for plant development because of the way that their cells are bound together by their cell walls. Mechanical forces appear to influence cytoskeletal orientation and auxin trafficking, which in turn can affect a wide array of factors, such as cell wall extensibility, phyllotaxy and merosity. There's every reason to believe that these processes are acting in all plant meristems, including flowers.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

YM: My first, but also one of the most memorable eureka moments, was when we found out our little meristems were growing happily on the culture medium. Being able to grow the meristems through the developmental window of interest and to recapitulate all the morphological landmarks of each stage in vitro is the foundation to this study, and we had no idea whether we could make it work or not. We experimented with different base media and hormones and combinations of different concentrations of ingredients and had little success in the beginning. It was only when we tried the current formula that we were pleasantly surprised and very relieved to discover that the tiny meristems had grown into much bigger buds with all the floral organs looking very normal. Another thing I want to add here is that the TP5-TP6 carpels (which look like a flat star when the edges of the primordia have just started to elevate) of Aquilegia are among the cutest things I've ever seen under a microscope. Whenever I see one, it makes me gasp and I will never get tired of it.

SC: The moment I knew that we could make this project work was when we realised the meristems were producing new primordia in tissue culture. I was shocked, but in a good way! Seeing those carpels develop was so satisfying. And then that joy was doubled when we realized that even after multiple rounds of staining and imaging the meristems still produced new organs. Pure joy!

The moment I knew that we could make this project work was when we realised the meristems were producing new primordia in tissue culture.

And what about the flipside: any moments of frustration or despair?

YM: This project was conceived in 2017 and was funded by the Society of Developmental Biology in early 2018. It was a winding journey filled with frustration and despair for at least the first two years, which is why the final publication of this project is incredibly rewarding. Since we needed to develop the tissue culture and live-imaging protocol first before conducting any analysis, there have been countless moments of failures and troubleshooting. Initially, we tried many ways of making the medium, staining, avoiding contamination, and keeping our tissues alive. It also took me a while to get good at dissecting the tiny meristems. So many times, the meristems looked totally fine under a dissection scope, but when we stained them and looked at them using a confocal, often parts of the meristem apex had been destroyed. Once we finally got more familiar with tissue culture and imaging, we started the long journey of becoming familiar with MorphoGraphX. We are particularly grateful to Dr Mingyuan Zhu (Duke University), Dr Daniel Kierzkowski (University of Montreal) and Dr Anne-Lise Routier-Kierzkowska (University of Montreal) for patiently giving us almost step-by-step tutorials; also to Dr Richard Smith (John Innes Centre) and Dr Soeren Strauss (Max Planck Institute for Plant Breeding Research) for being incredibly approachable and actively answering all our questions posted on the MorphoGraphX online forum ( Two years passed by, but when we finally felt that we got a handle on things and could start collecting some data to analyse, COVID-19 arrived and the university was shut down. When labs and the imaging centre were allowed to partially reopen again in the summer, Steph and I had to divide our roles in the project because of the low occupancy requirement and lack of access to workstation computers. I would go to the lab first to do dissection, then Steph would do the imaging every other day for at least a week, and I would process all the images at home. At that time, the only accessible computer that was powerful enough to run MorphoGraphX, was my husband's, Weilin Meng, which he also needed for his full-time work. We are very grateful that he shared his computer with me so that we could proceed with this project.

SC: This project was definitely a long process of trial and error from the very first day. For me, the hardest part was getting the staining protocol to work. It was a balancing act of staining enough so that we could properly analyse the cell behaviours, but not so much to damage the tissue. In the beginning, it was frustrating to make it halfway through the time series and then find out that the tissue was damaged. But the most frustrating part was when we finally had all the different parts of the protocol working perfectly and then COVID-19 shut down the lab for months and we lost all our plants. We had to wait months to start again!

What next for you after this paper?

YM: I defended my PhD in August and became a postdoc researcher at Yaowu Yuan's lab at the University of Connecticut in October 2021, studying the molecular basis of floral organ initiation and patterning in Mimulus flowers. I'm very excited to apply the skills and techniques that we developed for this project to Mimulus and to look at how gene expressions control cell behaviour in real time during organ initiation.

SC: I am currently working on a floral transcriptomic project in the Kramer Lab and am soon moving back to Australia to continue my career there. I am excited to apply our live-imaging protocol to other plant lineages, hopefully to discover and add to our understanding of the diversity of meristem growth across land plants.

Where will this story take your lab next?

EK: Well, this is really Ya Min's project – she had the insight to recognize that Aquilegia is uniquely well suited to study the FMT process. I hope that she and I will be able to continue collaborating as she pursues her postdoctoral research and establishes her own lab in the future. We are also hoping to use the live-imaging approach to visualise development in other organs, particularly the Aquilegia petal spur.

Finally, let's move outside the lab – what do you like to do in your spare time?

YM: I love travelling. Normally, every year I will take time off to explore new places and countries that I haven't been to before and visit friends all over the world, but unfortunately it has not been possible since the COVID-19 pandemic. Music is also very important to me. I used to go to a lot of rock concerts, but of course this is also a hobby that had to be put on hold during the pandemic. I have an electric piano at home that really helps me to relax and clear my mind.

EK: It may sound cliche for a plant biologist, but I love gardening. I don't have a lot of space in our yard because I've given most of it over to our dogs, but it's fun to see how many different things I can manage to grow in our cold climate. I also spend a lot of time hiking in local nature reserves with my two hound dogs, one of whom is good enough to come into the office with me, while the other is not office material.

SC: I enjoy making things with my hands; lately, that has been pottery. I was always on the search for the perfect coffee mug and then I realised I could make my own! And so now I have many misshapen mugs and a couple of really good ones. I also love knitting, a craft that my grandma introduced me to. She got me hooked on making my own socks. Now I am trying to knit a sweater, which is as hard as it sounds and turning out misshapen, just like my mugs.

Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.


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Quantitative live imaging of floral organ initiation and floral meristem termination in Aquilegia