The shoot apical meristem is a key stem cell niche in plants, and proper stem cell maintenance is partly regulated by CLAVATA3 (CLV3). Without CLV3 meristems overgrow, but the mechanistic basis of this phenotype was unclear. A new paper in Development suggests that CLV3 modulates the physical properties of meristematic stem cells, and that these properties help shape meristem morphology. To learn more about the story behind the paper, we caught up with first author Léa Rambaud-Lavigne and corresponding authors Namrata Gundiah, Arezki Boudaoud and Pradeep Das.

Clockwise from top left: Arezki Boudaoud, Namrata Gundiah, Pradeep Das and Léa Rambaud-Lavigne.

Namrata, Arezki and Pradeep, what questions are your labs trying to answer?

PD: We aim to investigate different aspects of patterning and morphogenesis in plants. One key question is how organisms generate their stereotypic shapes. Unlike animals, most plants do their shape-building after embryogenesis. So, whereas young animals are littler versions of their older selves, a young plant can look substantially different from its mature version. There are obviously lots of different regulatory processes that go into making this happen, but the continued existence of a small group of pluripotent stem cells (up to thousands of years in certain species) is crucial. In parallel, we are also investigating the precise spatial and temporal control of the gene expression patterns that underlie some of these regulatory processes.

Unlike animals, most plants do their shape-building after embryogenesis

AB: We have two separate axes of research. On the one hand, we aim to understand how plants grow and establish well-defined shapes and structures, focusing on cell mechanics (properties of the cell wall – the plant equivalent of the extracellular matrix – and cell pressure, also known as turgor pressure) and responses to mechanical stress. On the other hand, we aim at unravelling how rain enables several plant species to disperse their progeny, by considering morphogenesis of specific organs, fluid mechanics of rain drop rebound from these organs and adaptation of these species to their environment.

NG: This is our first project in plant biomechanics, and I am delighted by the synergy between the model and experiments that is evident in this work. My lab studies how cells and the extracellular matrix grow and remodel in response to various mechanical cues. Specifically, we address questions on the mechanobiology of cell adhesions to substrates, and the biophysical cues involved in their migration. Often, we are required to build custom tools to create the appropriate mechanical milieu. We also use computational methods to model the phenomena observed in our data.

Léa, how did you come to work in the lab and what drives your research today?

LR-L: Pradeep was my teacher when I was a Master's student and I enjoyed very much his classes on plant development. After doing an internship in his lab it became obvious to me that I had to pursue a PhD. Flower development has always been my favourite topic and my current postdoc work is an evo-devo project that uses ovule development as a tool to study the evolutionary origins of the flower.

Tell us about the background of the field that inspired your work

PD, AB, NG & LR-L: The genetic and molecular features of aboveground stem cells have been known for over a quarter of a century. In our lab, we started using novel biophysical techniques to re-examine these cells and have shown that they have characteristic mechanical traits, in that they are more rigid than their neighbours. From this, we tried to understand what role such increased stiffness might play in stem cell maintenance and, more generally, in plant architecture. This then led us to study the mechanics of one class of stem cell mutants. The results were unexpected and led us to broaden our analysis of this mutant, including via a fantastic collaboration between the biologists in France and mechanical engineers in India that began when Pradeep was on a sabbatical in India.

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

PD, AB, NG & LR-L: Two key genes are known to regulate the stem cell population at the shoot apex. Deleting one of them leads to a thousand-fold increase in the size of the apex, such that it has always been thought that this is because stem cells overproliferate. We show that it is not as simple as that, because the cells in these massive apices do not adhere to most of the benchmarks we would normally use to define a given cell as a stem cell.

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

PD & LR-L: Yes, two! The first was when we looked at gene expression patterns in our mutant lines for stem cell regulators and found that there was no longer the clean separation of regional identities predicted based on the dogma in the field. The second was when our mechanical models showed that we could closely mimic the defects visible in mutant apices simply by varying stiffness and growth rates in the system.

Mechanical models showed that we could closely mimic the defects visible in mutant apices simply by varying stiffness and growth rates in the system

LR-L: Another ‘cool’ moment was when we identified the precise nature of the mutation in the clv3-2 allele used in our work. This canonical allele is widely used in the field, but the mutation was unknown until we found out where the DNA was altered. It was like knowing a secret that nobody else in the world knew! This was made even cooler by the fact that it isn't a simple nucleotide change but rather a chromosomal rearrangement, so we had to use some neat molecular biology tricks to reveal that secret.

Two images showing the highly complex phenotypes of clv mutants. The turtle-shaped meristem on the left appears to be mocking the slow progress made in the beginning, whereas the propellor-shaped one on the right could be hailing the quicker pace at the end! Image credit: V. Battu and P. Das.

Two images showing the highly complex phenotypes of clv mutants. The turtle-shaped meristem on the left appears to be mocking the slow progress made in the beginning, whereas the propellor-shaped one on the right could be hailing the quicker pace at the end! Image credit: V. Battu and P. Das.

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

LR-L: This work has taken a few years to get ready for publication after I left the lab and it was at times difficult to keep things going while I was working full-time on my postdoc project. Finding the right way to analyse our atomic force microscopy data was a big challenge, but we had spent too much time and energy to give up!

Why did you choose to submit this paper to Development?

PD: I love this journal! I have published several papers here, including my first first-author paper, as well as my first corresponding-author paper! I also happen to think that Development's publishing model is much healthier than many other journals. It will be my first-choice journal for the rest of my career.

What is next for you after this paper?

LR-L: Even though I was able to find postdoc positions without having published on my principal PhD project, I feel that this paper is a milestone in my career. It will legitimise my applications for permanent positions in France, and it is also a good way for me to gain more self-confidence, as I was tempted to think that my work wasn't good enough to be published.

Where will this story take your lab next?

PD: It is traditional in France to celebrate such events with a ‘pot’ (a party). This is an important business and needs proper planning and attention. Science will just have to wait. But when we do get around to it, we will be looking at the interactions between plant hormone signalling and stem cell mutants.

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

PD: Spare time? What is that? But seriously, squash has been my preferred sport for a long time and camping my preferred holiday mode. The COVID lockdowns got me to take the plunge into woodworking, which I do whenever time permits. I'd love to say I make art but, that part of my brain being non-existent, I stick to making functional things – shelves, planters, guitar stands and such.

LR-L: In my spare time, I love to look for wild orchids in spring time, to hike in quiet spaces away from big cities, and I have a passion for knitting that I practiced a lot with my lab colleagues. After several years abroad, I also value very much the time I spend with my family, especially my young nieces, to whom I like to show plants and nature's wonders.

AB: I enjoy hiking and sailing very much. These are two activities that allow me to forget about work and come back with a fresh view on ongoing projects.

NG: I like pottery, yoga, reading, travelling and spending time with friends and family in my spare time.

L.R.-L., A.B. & P.D.: Laboratoire Reproduction et Développement des Plantes, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, INRIA, Lyon, France.

N.G.: Department of Mechanical Engineering, Indian Institute of Science, Bengaluru, India.

A.B.: LadHyX, CNRS, Ecole Polytechnique, Institut Polytechnique de Paris, 91128 Palaiseau Cedex, France.

E-mail: [email protected]; [email protected]; [email protected]; [email protected]

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants