Controlled buckling as a mechanism to regulate cuticle patterning in plants

The outer surfaces of plants are covered in a thin layer of a water-repellent material called the cuticle. The cuticle protects the plant from dehydration when the air is dry, and it also protects the plant's delicate tissues from damage by insects and other animals and from invasion by pathogens such as bacteria and fungi. Having a cuticle is absolutely essential for the survival of all plants on land, and the evolution of the cuticle was a key step when plant life first moved onto the land, having begun in the sea.

We are interested in the patterns that we find on the surface of plant cuticles. These patterns are formed from ridges, wrinkles or waves in the cuticular material. They can appear random, or can follow very organised arrangements such as sets of long lines or star shapes. They may also be absent, giving an entirely smooth cuticle. All of these different cuticle patterns affect the way that the plant interacts with the world around it, depending on the type of pattern and the organ on which it forms. For example, long ridges on the petal cuticle, if organised in a regular way, can interfere with light arriving at the plant surface and produce iridescent effects. These can be important on flowers to attract pollinators. A different example is the larger scale ridges found on some leaves, which make the leaf surface slippery for insects that grip with adhesive pads on their feet. As a result, these plants are protected from some of the beetles that would otherwise eat them. Cuticle patterns can also affect the wettability of the plant surface, and one of the least wettable plants, the Sacred Lotus, has provided the inspiration for the design of paints and other surface coatings that shed water (widely known as the "Lotus effect").

Nobody knows how these different patterns form on the plant surface, but our preliminary data suggest that they are produced by buckling of the cuticular material as the plant cells grow. We have developed a model which suggests that buckling forces arise because the plant cells grow more in one direction than another, and this stretches and compresses the cuticle on top. Our model suggests that buckling can only occur if the cuticle is of the right stiffness, which will be a result of the detailed chemical make-up of the cuticle. The model suggests that we can think of the patterns on the plant surface as a result of the emergent properties of the plant cells and their cuticle - if cell growth, cuticle chemistry and cuticle production come together in a specific way, then buckling will occur and patterns will be formed.

In this proposal we would like to test our model, to gain a detailed understanding of how cuticle patterns form. We believe that this understanding will be important in a range of ways, providing inspiration for the production of artificial surfaces with different roles, suggesting ways of improving crop yield by manipulating the plant's interaction with the environment, and even providing input into biodiversity and conservation work by explaining how plants interact with their environments in more detail.

To test our model we will use a range of different approaches to change or perturb the growth of plant cells, the amount of cuticle produced, and the chemistry of cuticle. We will then analyse how these changes influence cuticle buckling and pattern formation. Many of our approaches will rely on altering the activity of the genes controlling cell growth and cuticle production or chemistry, but we will also use physical stretching of plant tissues and pharmacological (chemical) disruption of cell growth to give a wide range of different cuticle patterns. Our data will feed back into our model and provide a strong understanding of this important biological process.

Enormous progress has been made in recent years in understanding the genetic basis of plant cuticle synthesis, secretion and assembly. However, we know almost nothing about how the surface of the cuticle is patterned. Many plants display distinct cuticle patterns, such as stellate wrinkles or rows of elongated ridges. These different patterns play important roles in the plant's interaction with the biotic and abiotic environments. Cuticular patterns can influence the behaviour of light, water and animals. As a result, the light capture and optical appearance of the tissue can be affected, its wettability is influenced, and herbivorous and pollinating insects can experience grip or slip.
We hypothesise that the patterns on the cuticle form as a result of buckling forces generated when a stiff substrate (the cell wall) grows anisotropically beneath a soft material (the cuticle). The patterns can therefore be thought of as an emergent property of the cell+cuticle system, and will form as a consequence of particular combinations of cell growth, cuticle production and cuticle chemistry (which determines stiffness).
Our aim in this proposal is to test this model. We will use genetic, mechanical and pharmacological approaches to perturb the cuticle and to establish how fine-tuning of those parameters can lead to different organisation of the wrinkles and to the production of a range of patterns. This will involve the use of transgenes to manipulate cell growth, total cuticle production, and specific aspects of cuticle chemistry. We will also use tissue stretching and pharmacological approaches to perturb cell growth. All of the manipulated plants will be analysed using a range of techniques to describe cell stretch (confocal microscopy), cuticle thickness (TEM), cuticle chemistry (LESA-MS), cuticle stiffness (AFM) and cuticle patterning (SEM). These data will allow us to quantify the effects of each of these parameters on buckling, and revise our model iteratively.

IMPACT STATEMENT Understanding plant cuticle buckling may be important in maintaining crop productivity, particularly as climate changes and interactions with herbivorous and pollinating animals change. Many manufacturing and design processes are inspired by biology, and cuticle buckling has the potential to be used in biomimetic work. Understanding how plants control their surface patterning and therefore their interactions with the biotic and abiotic environment may have important implications for conservation of biodiversity. Our project therefore has potential impact in several sectors, as well as providing great opportunities for public engagement.

AGRIFOOD IMPACT We will disseminate data to farmers and breeders, and increase our engagement with stakeholders, through activities at the University and at NIAB. We will target outreach to these stakeholders through BJG's current contacts with Syngenta and a number of plant breeders. Results will be demonstrated at NIAB Innovation Farm Open Days and Symposia. Additional opportunities to interact with industry will be through the University's "Enterprise Tuesdays", where research can be presented to a range of interested companies.

BIOMIMETIC IMPACT We will disseminate data to academics and companies working in bio-inspired design and manufacturing through BJG's current contacts through the Cambridge NanoScience Centre and the Heriot-Watt University Nature Inspired Manufacturing Centre. Both centres provide the opportunity to showcase work through symposia, workshops, poster sessions and shared students. Al Crosby, Professor of Polymer Science and Engineering at University of Massachusetts Amherst, is expert in artificial generation of buckling patterns and has offered support in reaching out to relevant industry (see letter).

CONSERVATION IMPACT We will disseminate data to conservation and biodiversity stakeholders through the Cambridge Conservation Initiative (CCI). The CCI links a number of conservation charities with the University of Cambridge. We will link our project to the CCI webpage, provide updates on the News section of the webpage, and advertise seminars through their diary section.

IMPACT THROUGH PUBLIC ENGAGEMENT is nationally and internationally increasingly important. Public concerns, fuelled by media coverage, are an important factor in dialogue at all policy levels concerning our management of environmental and agricultural systems. Current activities include presentations at public engagement events including National Science Week, the Cambridge Festival of Plants and the Cambridge Festival of Ideas. Enhanced public impact activities will include linking our project to the Cambridge Conservation Initiative webpage, providing News updates and advertising seminars through their diary section, and setting up a project-specific webpage giving project details and accessible introductions to the concepts involved. In addition, we will work with the Horticulture, Education and Interpretation staff at Cambridge University Botanic Garden to develop a living display with appropriate interpretation material (and boards and linked through QR codes to online material) to explain the project to a general audience. Many plants with nanoscale surface patterning are highly charismatic (eg. iridescent Hibiscus trionum, superhydrophobic Nelumbo nucifera), and the surface patterns produced are intriguing when viewed on a scanning electron microscope, so we anticipate that an engaging display should be easy to design with appropriate horticultural support. The Cambridge University Botanic Garden has 250,000 casual visitors per year, plus 10,000 schoolchildren on arranged visits, so this display will reach a large and varied audience.