Scattering and tapping on soft-hard-open nuts

Project Information

SCATAPNUT

Grant agreement ID: 681885

DOI

10.3030/681885

Project closed

EC signature date 31 May 2016

Start date 1 September 2016

End date 31 August 2021

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 993 606,25

EU contribution

€ 1 993 606,25

Coordinated by

UNIVERSITAET FUER BODENKULTUR WIEN
Austria

Periodic Reporting for period 4 - SCATAPNUT (Scattering and tapping on soft-hard-open nuts)

Reporting period: 2021-03-01 to 2021-08-31

Summary of the context and overall objectives of the project

With the ERC-consolidator grant “Scattering and tapping on soft-hard-open nuts” we elucidated how plants build strong shells (seed coats) for packaging the next generation, the seed. The hard encapsulations protect the developing embryos and ensure survival in many different environments, including harsh conditions from dry to wet, from hot to cold and from short to very long periods. Nut-producing plant species have evolved a number of traits that facilitate dispersal by certain rodents and corvids and repel parasites. The big nutritious kernel inside attracts, while the hard outer shell enhances the probability of the nut to get buried in the soil instead of being cracked and eaten immediately.
To understand the different functionalities of nut shells we had to dive deep into the micro-and nanostructure of these sclerenchyma tissues and their chemistry by state-of-the-art methods. Sclerenchyma derives etymologically from the Greek word sklērós (= ‘hard’), indicating that hardness is a central feature. We wanted to understand the properties of the hard nutshells, but also elucidate the “production process” from the soft to the hard state by including samples from different developmental stages. To derive general and specific design principles of the nutshells and structure-function relationship we investigated and compared different nut species.
Understanding these packaging concepts of plants, will inspire biomimetic material approaches as well as the use of nutshells for new biomaterial developments. Nutshells will be available in increasing amounts as waste, because nuts are more and more recognized as valuable, healthy natural food resources.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Investigating different types of seed coats we elucidated two different packaging concepts. For some of the nutshells (e.g. Macadamia, hazelnut, pecan) we confirmed different cell types (sclerenchyma fibers and stone cells), arranged in layers, while some relied only on one type of sclerenchymatic cells (e.g. pine seed, walnut, pistachio). We suggested a new classification into “multi cell type” and “single cell type” plant shells (Huss et al. 2020). In the latter ones we discovered a new cell type: the 3D puzzle cell (Antreich et al. 2019). We visualized these polylobate unit cells with concave and convex parts in walnut and pistachio. In both species the irregularly lobed cells interlock with 14 neighbors into a 3D puzzle that cannot be disassembled (see videos: http://wordpress.bionami.at/2019/06/12/the-puzzle-of-the-walnut-shell/). Mechanical testing revealed a significantly higher ultimate tensile strength of these interlocked cell tissues compared to the sclerenchyma tissue of a pine seed coat (single cell type) lacking the lobed cell structure and the “multi cell type” shells. The higher strength of walnut and pistachio shells was explained by the observation that the crack cannot simply detach intact cells, but has to cut through the lobes due to the interlocking. Pistachio shells showed beside high strength also remarkable high energy absorption and we revealed ball-joint like structures as one reason behind (Xiao et al. 2021). Additionally to topological interlocking we found geometric stiffening as a complementary strategy for strong plant shells (Huss et al. 2020)(Huss et al. 2021).

To understand the formation of the nutshells, from isodiametric to polylobate and from soft to hard, we sampled walnut at different time points from catkin-formation (May) to harvest (October). By serial block face-scanning electron microscopy, Raman and fluorescence microscopy we found the secrets behind shape formation: multiple loops of cellulosic thickenings in cell walls, act as stiff restrictions during cell growth and leading to the lobed cell shape (Antreich et al. 2021). After shape morphogenesis the transformation from the soft to the hard state is fulfilled by adding cell wall material and impregnating with hydrophobic polymers (lignin) and aromatic components (Xiao et al. 2020). The cell wall is added continuously in layers and could be visualized as helicoidal deposition of cellulose fibrils (Xiao et al. 2021). The suture, the line along which the seedling will push the nut apart, shows different composition (high pectin content) and by this different swelling properties to achieve the opening (Xiao et al. in preparation).

Results have been published in multidisciplinary, material science and plant journals with high impact and have been picked up by several news outlets. Elucidating these packaging concepts of plants, will inspire biomimetic material approaches as well as the use of nutshell waste for sustainable material solutions.

5 most important publications
Antreich, SJ; Xiao, N; Huss, JC; Horbelt, N; Eder, M; Weinkamer, R; Gierlinger, N;
The Puzzle of the Walnut Shell: A Novel Cell Type with Interlocked Packing. Advanced Science. 2019; 6(16):1900644
Altmetric 130: blogged by 3, picked up by 12 news outlets, tweeted by 17, on 1 facebook page

Xiao, NN; Felhofer, M; Antreich, SJ; Huss, JC; Mayer, K; Singh, A; Bock, P; Gierlinger, N
Twist and lock: nutshell structures for high strength and energy absorption. ROY SOC OPEN SCI. 2021; 8(8), 210399
Altmetric 107: blogged by Science, picked up by 7 news outlets, tweeted by 53

Huss, JC; Antreich, SJ; Bachmayr, J; Xiao, N; Eder, M; Konnerth, J; Gierlinger, N; Topological Interlocking and Geometric Stiffening as Complementary Strategies for Strong Plant Shells..
Advanced Materials. 2020; 32(48):e2004519
Altmetric 37: picked up by 1 news outlets, tweeted by 35, on 1 facebook page

Huss, JC; Gierlinger, N; .(2021): Functional packaging of seeds. New Phytologist. 2021; 230(6):2154-2163
Altmetric 25: blogged by 1, tweeted by 31, on 1 facebook page

Antreich, SJ; Xiao, N; Huss, JC; Gierlinger, N; A belt for the cell: cellulosic wall thickenings and their role in morphogenesis of the 3D puzzle cells in walnut shells.. J Exp Bot. 2021; 72(13):4744-4756
Altmetric 9: tweeted by 14

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

A big challenge in biology and biomimetic research is to understand the design principles and physicochemical mechanism underlying the optimized biological systems as well as their developmental assembly. To tackle exactly these challenges the objectives of this project were threefold to go beyond state of the art:
1) We developed in-situ methods for in-depth characterization of seed dispersal units at the micro-and nano level: native sample preparation by cryo-microtomy, Atomic force microscopy to reveal nanostructure and mechanics, Raman imaging for microchemistry (database of vibrational spectra and multivariate data analysis approaches), X-ray tomography, fluorescence microscopy and serial block face electron microscopy for cell shape elucidation, mechanical testing and investigation of fracture surfaces by scanning electron microscopy to draw conclusions on mechanical performance.
2) We revealed the heterogeneity and common design principles by including different biological species and derived general structure-function relationships (see above).
3) We followed in-situ morphogenesis and maturation of seed coats and highlighted the selective deposition of cellulose fibrils to achieve cell shaping and impregnation with aromatic components to waterproof the final mature nutshell.

the puzzle of the walnut shell: a novel cell type with interlocked packing