How genes simplify the architecture of a leaf

About authors

Dr. Krishna Reddy Challa completed his PhD in plant developmental biology under the supervision of Utpal Nath, an Associate Professor in the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore. The title of his PhD thesis is “Genetic and molecular analysis for the maintenance of leaf shape, size and flatness in Arabidopsis thaliana”. Here, he talks about his recent work “Active suppression of leaflet emergence as a mechanism of simple leaf development” published in Nature Plants.

Dr. Monalisha Rath has recently completed her PhD in plant morphogenesis under the supervision of Utpal Nath. Prior to this, she obtained her MSc degree in Biotechnology with a gold medal from Pondicherry University in 2015. Her doctoral research work was focused on identifying novel functions of the TCP group of proteins in seedling morphogenesis, leaf architecture and leaf senescence. Besides research, she enjoys drawing, sketching, traveling, and listening to music in her free time. Here, she talks about her recent work titled “Active suppression of leaflet emergence as a mechanism of simple leaf development” published in the Nature Plants journal.

How would you explain your research outcomes to the non-scientific community?

Leaves primarily grow in the planar dimension and form the largest biological surface on earth. They can be divided into two types based on their shape; simple and compound. Simple leaves – as found in plants such as spinach, mango, oak, and Arabidopsis thaliana – have a single leaf blade with a smooth, serrated, or wavy margin. On the other hand, leaves of tomato, neem or Cardamine hirsuta form compound structure where leaf blade is dissected into multiple leaflets. Though scientists have converted compound leaves to simpler or more complex forms by genetic manipulation, no simple leaves have thus far been converted to compound form, suggesting that our understanding on the genetic regulation of leaf shape is still fragmented.

Our recent work published in Nature Plants reports the conversion of the simple Arabidopsis leaf into a super-compound form with never-ending leaflet formation. This demonstrates that simple leaves not only possesses the morphogenetic potential to form leaflets but also can completely overcome the growth determinacy by bringing on indefinite rounds of leaf dissection. In wildtype leaf, several members of the two gene families, CIN-TCP and KNOX-II, work together to actively restrict lamina dissection, thus ensuring simple architecture. Simultaneous downregulation of both these gene families converted a simple Arabidopsis leaf into a dramatically compound form that never ceased to form new leaflets. This shows that the underlying molecular mechanism of leaf-simplifying action of the CIN-TCP-KNOX-II gene module is to irreversibly repress the stemness genes in the leaf, thus restricting the latter’s expression only within the meristem, the stem-cell zone in plants from where leaves arise.

In wild type Arabidopsis leaves, the CIN-TCP genes activate the KNOX-II genes, and they together actively suppress the KNOX-I-CUC stemness module. The basal level of CUC gene is sufficient to produce only serrations at the margin. When CIN-TCP and KNOX-II genes are simultaneously down-regulated (Mutant), the KNOX-I-CUC stemness genes are de-repressed and help produce reiterative leaflets, converting the simple leaf into a super-compound form. Scale bars, 5 mm.

How do these findings contribute to your research area?

Though compound leaves have been converted to simple leaves, no extent of gene manipulation had thus far been able to convert a simple leaf to a truly compound form, encouraging some scientists hypothesize that simple leaf architecture is regulated by a genetic mechanism that is different from that of compound-leaf development. However, this possibility has not been well accepted in the plant biology field as both simple and compound leaves are believed to have the same evolutionary origin and are expected to be governed by similar molecular mechanisms. In our study, we attempted to understand the underlying mechanism behind the regulation of simple leaf structure. We found that similar genes indeed regulate the shape of both simple and compound leaves. Robust early expression of these genes ensure leaf simplicity, and one needs to simultaneously downregulate all of them to achieve compound leaf architecture. This result provides support to the telome theory of leaf evolution, according to which leaves evolved from the stem system that branches indefinitely to fill the three-dimensional space. During leaf evolution, the terminal stem branches were first rearranged from three-dimension to two-dimensional space and then the gaps between the branches were filled by thin photosynthetic tissue to convert it to a leaf. A prediction of this theory would be that inactivation of the genes involved in leaf evolution should convert it to an ever-branching structure. We have converted a simple leaf into a form that never stops branching, thus providing support to the telome theory.

We have converted a simple leaf into a form that never stops branching, thus providing support to the telome theory.

What was the exciting moment during your research?

Krishna Reddy Challa: Phenotypic alterations by gene manipulation have never stopped surprising me and the CIN-TCP genes have never disappointed me in this regard. The most exciting moment in this work was when I first observed a 60-day old Arabidopsis leaf with downregulated CIN-TCP and KNOX-II genes: its simple architecture was converted to such a messy compound form that one has to swear that it is an Arabidopsis leaf. I took a picture of it and showed it to my supervisor, and his first expression was, “I have never seen such a dramatic phenotype in my life!”. Later, I framed that picture and presented it to him as my lab-parting gift.

Monalisha Rath: Noticing the reiterative leaflet initiation in our mutant, I left a few plants to grow for a long time. A wild-type Arabidopsis plant usually senesces and dies within a couple of months. To my surprise, I noticed that the mutant plants were growing even after 6 months, and their leaves continued to produce new leaflets at their margin, making the total number of leaflets uncountable! This is when I realized that we have converted a simple determinate leaf to a truly ever-growing organ with endless branching potential. Activating just one of the CIN-TCP genes (TCP4) in this ever-branching leaf at various stages of growth produced varied number of leaflets: earlier the trigger of TCP4 activation, simpler was the leaf structure. In other words, we now can produce a designer leaf with the extent of complexity that you choose! This was the real exciting moment for me.

What do you hope to do next?

There are indications that, in simple leaves, CIN-TCPs convert the on state of the stemness genes to the off state by epigenetic means; we would like to test this experimentally. We are also interested in investigating whether the function of the CIN-TCP-KNOX-II gene module is conserved in other simple-leaved species.

Where do you seek scientific inspiration from?

Krishna Reddy Challa: Nature is the primary inspiration for my research. Since I came from a village with an agricultural background, I keenly observed plants and animal that surrounded me in my childhood. I am glad that I was mentored by wonderful teachers throughout my career, who ignited the scientific flare in me and encouraged me pursue my PhD in plant biology.

Monalisha Rath: How genes and their network give rise to biological patterns and their natural diversity has always fascinated me. My supportive parents and inspiring teachers have always motivated me to follow my scientific interest and career goal in academia. After joining IISc, I realized how molecular genetics can elegantly address the pattern diversity in biological systems such as Arabidopsis. Throughout my Ph.D., I received constant inspiration from my supervisor to pursue my scientific interest with determination, no matter how many hurdles come by our way.

How do you intend to help Indian science improve?

Krishna Reddy Challa: I believe that every child has an inherent curiosity in science and technology as they face the world around them. The grown-ups can identify the direction of that curiosity and ignite the scientific flare in them. I would like to teach school children how to question every incident they come across and help them find their own answer.

Monalisha Rath: What we see around us is a marvel of nature, and for me, understanding the fundamentals behind the functioning of any object is science. Science need not necessarily have social implications; it satisfies the curiosity of the human mind in understanding the very existence of nature. And as a researcher, I would like to spread this to the non-scientific community through our thought-provoking scientific discoveries and social communications.


Challa, K.R.#, Rath, M.#, Sharma, A.N. et al. Active suppression of leaflet emergence as a mechanism of simple leaf development. Nat. Plants (2021). (# equal contribution)

Edited by: Sukanya Madhwal

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