Optimization of Preparation and Detection Methods for Pollen Tube Cell Wall by Atomic Force Microscopy

doi:10.11983/CBB24063

Abstract

Abstract: Atomic force microscopy (AFM) is a powerful tool for studying the ultrastructure and mechanical properties of plant cell walls, while good sample preparation is essential for AFM data acquisition. As micron-scale plant samples, pollen tubes are typical experimental materials for studying the structure and function of cell walls. Due to the difficulty in sample preparation, it is hard to obtain in situ AFM data in physiological state of pollen tubes as pollen tubes are hard to attach to the substrate firmly in fluid. In this study, the AFM preparation and detection methods are optimized using Nicotiana tabacum pollen tubes as the experimental materials. Thin solid medium is used as the adhesive, thereby pollen tubes are attached to the glass slide during germination and elongation in fluidic environment. Compared with the conventional drying-rehydration method, the optimized liquid-attaching method allows observation directly in fluid without drying treatment, which avoids the shrinkage and denaturation of pollen tubes caused by drying-rehydration process. Accordingly, liquid-attaching method can be applied for pollen tubes of different species and sizes, providing support to obtain high-resolution in situ AFM data of pollen tube cell walls in native physiological state under aqueous conditions.

Key words: atomic force microscope, pollen tube, cell wall, in situ, mechanical properties

CLC Number:

Siying Qin, Yan Luo, He Zhang, Jun Hu, Jugou Liao. Optimization of Preparation and Detection Methods for Pollen Tube Cell Wall by Atomic Force Microscopy[J]. Chinese Bulletin of Botany, 2024, 59(5): 783-791.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24063

https://www.chinbullbotany.com/EN/Y2024/V59/I5/783

Figures/Tables 4

Figure 1 Schematic diagram of pollen tube atomic force microscope (AFM) preparation and detection (A) Liquid-attaching preparation method; (B) Drying-rehydration preparation method; (C) Test areas of glass slides (bar=1 cm); (D) Optical bright field image of AFM probe at work (bar=100 μm); (E) AFM detection under aqueous conditions

Figure 2 Pollen tube atomic force microscope (AFM) imaging data of drying-rehydration and liquid-attaching preparation methods (apical dome and distal region) (A) AFM-mapping of three-dimensional topography of pollen tubes (colors represent the height); (B) Three-dimensional topography of pollen tubes overlaid with Young’s modulus (colors represent the elasticity). Bars=3 μm

Figure 3 Effects of different preparation methods on the Young’s modulus of pollen tube apical dome and distal region (A)-(D) Typical force-distance curves obtained from apical dome (A) and distal region (C) of drying-rehydration method, apical dome (B) and distal region (D) of liquid-attaching method; (E) Young’s modulus of pollen tube apical dome; (F) Young’s modulus of pollen tube distal region (colors represent the elasticity, dashed boxes (side length 3.5 μm) are the areas for calculating the average Young’s modulus); (G) The statistical results of Young’s modulus of pollen tube apical dome and distal region using drying-rehydration and liquid-attaching preparation methods (*** represent extremely significant differences among different treatments at P<0.001). Bars=3 μm

Figure 4 Effects of different preparation methods on the cross-section height of pollen tube apical dome and distal region (A), (D) The height images of pollen tube apical dome (A) and distal region (D) (colors represent the height, and dashed lines represent the position of the cross-section); (B), (E) The cross-section height of pollen tube apical dome (B) and distal region (E) using different preparation methods (the arrows indicate the position of cross-section height); (C), (F) The statistical results of cross-section height (Z) of pollen tube apical dome (C) and distal region (F) using different preparation methods (*** represent extremely significant differences among different treatments at P<0.001). Bars=3 μm

References 42

[1]	Adhikari PB, Liu XY, Kasahara RD (2020). Mechanics of pollen tube elongation: a perspective. Front Plant Sci 11, 589712.
[2]	Binnig G, Quate CF, Gerber C (1986). Atomic force microscope. Phys Rev Lett 56, 930-933.
[3]	Boudaoud A (2010). An introduction to the mechanics of morphogenesis for plant biologists. Trends Plant Sci 15, 353-360.
[4]	Burri JT, Vogler H, Munglani G, Läubli NF, Grossniklaus U, Nelson BJ (2019). A microrobotic system for simultaneous measurement of turgor pressure and cell-wall elasticity of individual growing plant cells. IEEE Robot Autom Lett 4, 641-646.
[5]	Cameron C, Geitmann A (2018). Cell mechanics of pollen tube growth. Curr Opin Genet Dev 51, 11-17.
[6]	Dufrêne YF, Ando T, Garcia R, Alsteens D, Martinez- Martin D, Engel A, Gerber C, Müller DJ (2017). Imaging modes of atomic force microscopy for application in molecular and cell biology. Nat Nanotechnol 12, 295-307.
[7]	Dumais J (2021). Mechanics and hydraulics of pollen tube growth. New Phytol 232, 1549-1565.
[8]	Gao BH, Xu L, Sun HJ, Xuan Y, Tang Y (2018). Research progress on atomic force microscopy in wood science. J Jiangsu For Sci Technol 45, 54-57. (in Chinese)
	高步红, 徐莉, 孙海军, 宣艳, 唐颖 (2018). 原子力显微镜在木材科学研究中的进展. 江苏林业科技 45, 54-57.
[9]	Geitmann A, Parre E (2004). The local cytomechanical properties of growing pollen tubes correspond to the axial distribution of structural cellular elements. Sex Plant Reprod 17, 9-16.
[10]	Guan DS, Li HY, Tong PE (2020). Experimental methods and recent progress in biomechanics using atomic force microscopy. J Exp Fluid Mech 34(2), 57-66. (in Chinese)
	关东石, 李航宇, 童彭尔 (2020). 原子力显微镜的生物力学实验方法和研究进展. 实验流体力学 34(2), 57-66.
[11]	Hu JR, Chen SB, Huang DD, Zhang Y, Lü SQ, Long M (2020). Global mapping of live cell mechanical features using PeakForce QNM AFM. Biophys Rep 6, 9-18.
[12]	Kozlova L, Petrova A, Ananchenko B, Gorshkova T (2019). Assessment of primary cell wall nanomechanical properties in internal cells of non-fixed maize roots. Plants 8, 172.
[13]	Krieg M, Fläschner G, Alsteens D, Gaub BM, Roos WH, Wuite GJL, Gaub HE, Gerber C, Dufrêne YF, Müller DJ (2019). Atomic force microscopy-based mechanobiology. Nat Rev Phys 1, 41-57.
[14]	Landrein B, Ingram G (2019). Connected through the force: mechanical signals in plant development. J Exp Bot 70, 3507-3519.
[15]	Leszczuk A, Kozioł A, Szczuka E, Zdunek A (2019). Analysis of AGP contribution to the dynamic assembly and mechanical properties of cell wall during pollen tube growth. Plant Sci 281, 9-18.
[16]	Li HY, Nie PC, Guan DS (2021). A hand of exploring the micro- and nano-scale world—atomic force microscope. Mech Eng 43, 806-811. (in Chinese)
	李航宇, 聂鹏程, 关东石 (2021). 探索微纳米世界的手——原子力显微镜. 力学与实践 43, 806-811.
[17]	Li M, Xi N, Wang YC, Liu LQ (2018). Applications of multiparametric imaging atomic force microscopy in probing cellular and molecular mechanics. Prog Biochem Biophys 45, 1106-1114. (in Chinese)
	李密, 席宁, 王越超, 刘连庆 (2018). 基于多参数成像AFM的细胞及分子力学特性探测研究进展. 生物化学与生物物理进展 45, 1106-1114.
[18]	Li TH, Wang T, Ren HY (2023). Advances in the study of cytoskeleton system regulating pollen tube development. Sci Sin Vitae 53, 763-774. (in Chinese)
	李彤辉, 王婷, 任海云 (2023). 细胞骨架系统调控花粉管发育的研究进展. 中国科学: 生命科学 53, 763-774.
[19]	Majda M, Grones P, Sintorn IM, Vain T, Milani P, Krupinski P, Zagórska-Marek B, Viotti C, Jönsson H, Mellerowicz EJ, Hamant O, Robert S (2017). Mechanochemical polarization of contiguous cell walls shapes plant pavement cells. Dev Cell 43, 290-304.
[20]	Mirabet V, Das P, Boudaoud A, Hamant O (2011). The role of mechanical forces in plant morphogenesis. Annu Rev Plant Biol 62, 365-385.
[21]	Nezhad AS, Geitmann A (2013). The cellular mechanics of an invasive lifestyle. J Exp Bot 64, 4709-4728.
[22]	Qi JY, Wu BB, Feng SL, Lü SQ, Guan CM, Zhang X, Qiu DL, Hu YC, Zhou YH, Li CY, Long M, Jiao YL (2017). Mechanical regulation of organ asymmetry in leaves. Nat Plants 3, 724-733.
[23]	Qian L, Zhao HW (2018). Nanoindentation of soft biological materials. Micromachines (Basel) 9, 654.
[24]	Riglet L, Rozier F, Kodera C, Bovio S, Sechet J, Fobis-Loisy I, Gaude T (2020). KATANIN-dependent mechanical properties of the stigmatic cell wall mediate the pollen tube path in Arabidopsis. eLife 9, e57282.
[25]	Routier-Kierzkowska AL, Weber A, Kochova P, Felekis D, Nelson BJ, Kuhlemeier C, Smith RS (2012). Cellular force microscopy for in vivo measurements of plant tissue mechanics. Plant Physiol 158, 1514-1522.
[26]	Shamsudhin N, Laeubli N, Atakan HB, Vogler H, Hu CZ, Haeberle W, Sebastian A, Grossniklaus U, Nelson BJ (2016). Massively parallelized pollen tube guidance and mechanical measurements on a Lab-on-a-Chip platform. PLoS One 11, e0168138.
[27]	Uyttewaal M, Traas J, Hamant O (2010). Integrating physical stress, growth, and development. Curr Opin Plant Biol 13, 46-52.
[28]	Vaz Dias F, Serrazina S, Vitorino M, Marchese D, Heilmann I, Godinho M, Rodrigues M, Malhó R (2019). A role for diacylglycerol kinase 4 in signaling crosstalk during Arabidopsis pollen tube growth. New Phytol 222, 1434- 1446.
[29]	Vogler H, Draeger C, Weber A, Felekis D, Eichenberger C, Routier-Kierzkowska AL, Boisson-Dernier A, Ringli C, Nelson BJ, Smith RS, Grossniklaus U (2013). The pollen tube: a soft shell with a hard core. Plant J 73, 617- 627.
[30]	Wang MM, Zhu XP, Peng GQ, Liu ML, Zhang SQ, Chen MH, Liao ST, Wei XY, Xu P, Tan XY, Li FP, Li ZC, Deng L, Luo ZL, Zhu LY, Zhao S, Jiang DG, Li J, Liu ZL, Xie XR, Wang SK, Wu AM, Zhuang CX, Zhou H (2022). Methylesterification of cell-wall pectin controls the diurnal flower-opening times in rice. Mol Plant 15, 956-972.
[31]	Wu JZ (2011). Ultrastructure of pollen tube cell wall in Torenia fournieri L. observed by FESEM and AFM. J Anhui Agric Sci 39, 21802-21804. (in Chinese)
	吴娟子 (2011). 蓝猪耳花粉管细胞壁超微结构的FESEM和AFM比较研究. 安徽农业科学 39, 21802-21804.
[32]	Wu QQ, Li Y, Lyu MH, Luo YW, Shi H, Zhong SW (2020). Touch-induced seedling morphological changes are determined by ethylene-regulated pectin degradation. Sci Adv 6, eabc9294.
[33]	Xiao L, Fang YY, Zhang H, Quan MY, Zhou JX, Li P, Wang D, Ji L, Ingvarsson PK, Wu HX, El-Kassaby YA, Du QZ, Zhang DQ (2023). Natural variation in the prolyl 4-hydroxylase gene PtoP4H9 contributes to perennial stem growth in Populus. Plant Cell 35, 4046-4065.
[34]	Zerzour R, Kroeger J, Geitmann A (2009). Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties. Dev Biol 334, 437- 446.
[35]	Zhang BC, Gao YH, Zhang LJ, Zhou YH (2021a). The plant cell wall: biosynthesis, construction, and functions. J Integr Plant Biol 63, 251-272.
[36]	Zhang H, Guo ZL, Zhuang Y, Suo YZ, Du JM, Gao ZX, Pan JW, Li L, Wang TX, Xiao L, Qin GJ, Jiao YL, Cai HQ, Li L (2021b). MicroRNA775 regulates intrinsic leaf size and reduces cell wall pectin levels by targeting a galactosyltransferase gene in Arabidopsis. Plant Cell 33, 581- 602.
[37]	Zhang LJ, Gao CX, Mentink-Vigier F, Tang L, Zhang DM, Wang SG, Cao SX, Xu ZP, Liu XL, Wang T, Zhou YH, Zhang BC (2019). Arabinosyl deacetylase modulates the arabinoxylan acetylation profile and secondary wall formation. Plant Cell 31, 1113-1126.
[38]	Zhang M, Zhang YZ, He QZH, E YL, Li Y (2023). Advances in plant cell wall structure and imaging technology. Biotechnol Bull 39(7), 113-122. (in Chinese)
	张曼, 张叶卓, 何其邹洪, 鄂一岚, 李晔 (2023). 植物细胞壁结构及成像技术研究进展. 生物技术通报 39(7), 113- 122.
[39]	Zhang Y, Yu JY, Wang X, Durachko DM, Zhang SL, Cosgrove DJ (2021c). Molecular insights into the complex mechanics of plant epidermal cell walls. Science 372, 706-711.
[40]	Zhang YG, Yuan XY, Zhang GF, Li YJ, Yin JH, Lin JX, Li XJ (2023). The application of click chemistry reactions in plant cell labeling. Chin Bull Bot 58, 956-965. (in Chinese)
	张御格, 袁笑妍, 张贵芳, 李雨健, 殷金环, 林金星, 李晓娟 (2023). 点击化学反应在植物细胞标记中的应用. 植物学报 58, 956-965.
[41]	Zheng L, Chen YJ, Ding LP, Zhou Y, Xue SS, Li BY, Wei JH, Wang HZ (2023). The transcription factor MYB156 controls the polar stiffening of guard cell walls in poplar. Plant Cell 35, 3757-3781.
[42]	Zu YG, Zhang YL, Liu ZG, Wang YB, Liang HL, Liu HM (2006). Application of atomic force microscope in plant bio- logy research. Chin Bull Bot 23, 708-717. (in Chinese)
	祖元刚, 张宇亮, 刘志国, 王延兵, 梁慧丽, 刘红梅 (2006). 原子力显微镜在植物学研究中的应用. 植物学通报 23, 708-717.