The biomineralization of silica induced stress tolerance in plants: a case study for aluminum toxicity

ABSTRACT

Biomineralization in plant roots refers to the process of cell-induced self-assembly to form nanostructures on the root surface. Silicon (Si) is the second most abundant element in soils, and beneficial to plant growth. Meanwhile, silicon is shown to participate in the process of biomineralization, which is useful for improving mechanical strength and alleviating biotic and abiotic stress, for example silicic acid polymerizes to form amorphous silica (SiO₂–nH₂O) in the process of growing to resist fungi and environmental stress. This process alters physical and chemical properties of cell wall. However, the mechanistic basis of this process remains unclear. Aluminum toxicity is a major constraint affecting plant performance in acid soil. This paper summarizes recent research advances in the field of plant biomineralization and describes the effects of silicon biomineralization on plant aluminum tolerance and its adaptive significance, using aluminum toxicity as a case study.

KEYWORDS:

• Biomineralization
• aluminum toxicity
• acid soil
• biosilicification

1. Introduction

The process of biomineralization refers to cell-induced self-assembly to form nanostructures, both in living and human-made structure, and has been a subject of great interest to biologists and material chemists

For decades. It uses small amounts of organic macromolecules (polysaccharides, proteins, glycoproteins, etc.) as templates for molecular manipulation in a process that controls the nucleation growth of mineral phases and their ordered assembly into inorganic materials.^1–3 Biomineralized particles have a unique function superior to the bulk phase when they are nano-sized, and nanoparticles can enter cells and interact with cells, which makes the most striking difference between nanomaterials and ordinary materials.

Currently, more than 40% of the world’s agricultural cultivated land is affected by aluminum toxicity, which is the main constraint for crop growth in acid soils. Many crops display toxic reactions to micromolar concentrations of aluminum ions.⁴ Several studies have shown that silicon, as a beneficial element in plants, can mitigate aluminum toxicity to plants.^5,6 Silica oxide, when deposited on the cell wall and intercellular spaces, can form nanostructures induced by SiO₂ self-assembly due to the presence of various hydrophilic substances at these sites; this improves the resistance of cell wall to environmental constraints.^7,8 However, it is still unknown if and how the nanoparticles can enhance the resistance of roots to Al toxicity in acid soil. Filling this gap in the knowledge is the main aim of this work.

2. The concept, function, and bionics of biomineralization

2.1. The concept of biomineralization

Biomineralization is a common phenomenon in nature and a strategy used by organisms to support their physical structure.⁹ During the past tens of billions of years of natural evolution, plants and animals have mutated their structures to cope with adversity, and biomineralization is widespread in unicellular plant diatoms, multicellular organisms sponges, bacteria, shells, and higher mammals, including humans.^1,10 In living organisms, biomineralization refers to the process of mineral deposition directed by cellular activities.¹¹ First, cells secret a small amount of organic macromolecules (polysaccharides, proteins, and glycoproteins etc.) as templates to provide nucleation sites for biomineralization. Then, by adjusting the concentration of anions and cations in the environment, cells can transport ions required for mineralization to the deposition site, promoting nucleation and growing complex specific products. Although the ingredients that makeup diatom cell walls, shells, teeth, and other biological minerals are very common, these materials have a complex and highly ordered structure and special physical properties, as well as special species- dependent and genetically controlled characteristics. Plants undergo biomineralization, based on silicon and calcium, in which silicon is deposited as silicon dioxide in cell walls and cells of various types, while calcium is mainly deposited as calcium oxalate in the vacuole of specialized cells.^9,12 Biosilicification is an important biomineralization process, and biomineralization events are common in marine, lacustrine, and terrestrial photosynthetic organisms; the most representative of which is diatom with highly silicified cell walls. In diatom vesicle, silicon is first deposited to form nanoparticle spheres (less than 100 nm in diameter), and then interacts with organic catalytic substances and further polymerizes, forming different morphological structures and performing various functions.¹³ Analyzing the structure and process of this biomineralization can facilitate the research of silicified biomimetic materials and provide clues for the biomineralization of silicon in other organisms.^14,15

2.2. Functions of biomineralization

Biomineralized SiO₂ particles have a unique function superior to the bulk phase when they are nano-sized, and nanoparticles can enter cells and interact with cells, making the most striking difference between nano-materials and ordinary materials. Applications of nanotechnology in agriculture are currently experiences a boom. One of these fast growing trends is applying simulated biosynthesis strategies and developing new chemicals to understand and improve plant resilience.^2,16,17 Wang et al found that colloidal SiO₂ could deposited on the cell wall of rice after the application of nano-silica, and which significantly inhibited the entry of Cd²⁺ across the plasma membrane into plant cells due to its high specific surface area and strong adsorption ability.¹⁸ This nano silicon preparation can promote the plant to have deeper roots and larger leaves and can form nanostructures in cell walls and intercellular spaces to cope with various environmental stresses. In fact, in the long process of evolution, organisms will respond to transformation by changing their structure, such as diatoms.¹⁹, mollusks.²⁰, and arthropods.²¹ During their growth, they form a SiO₂ shell to resist fungi, environmental stress, toxicity, and natural enemies, and even regulate life activity information.^2,8 For example, bacteria also can reduce the toxicity of Cd²⁺ by regulating the expression of related genes and inducing mineralization, which is a basic bioremediation strategy to deal with the heavy metal pollution from the perspective of gene regulation.²² Yet some scholars have discussed the possible contribution of bacterial biomineralization to the global silicon cycle.²³ In addition, by studying silicon dioxide deposits in sorghum leaves, Kumar et al found that not only these deposited minerals can improve the stress tolerance of plants, but also silica deposition is an active and physiologically regulated process rather than being a simple precipitation.¹² At present, Silicanin-1 (Sin1) has been identified in diatoms and Siliplant1 (Slp1) and other transmembrane proteins have been identified in Sorghum bicolor.^24,25, although little is known about the role of biofilms in mineralization.

2.3. Biomineralization and bionics

Inspired by nature, many researchers have used physical, chemical, or biochemical methods to imitate the sheath structure outside biological cells, especially chemical modification methods. One of them is a technology that makes polyelectrolyte multilayer coating by alternately depositing cationic and anionic polyelectrolytes on the solid surface through electrostatic interaction.^26–28 Chemical modification has been widely used in biological surface modification and other related fields because of its simplicity and its film-forming capability. Importantly, this process is not affected by the structure of the substrate material and can greatly protect the activity of proteins, enzymes, and other biomolecules in the film-forming components.^7,29–31 For example, nanomedicine nanoparticles, a new drug carrier, can carry a variety of drugs and bioactive macromolecules, such as peptides, proteins, and genes. Therefore, nanoparticles have been widely used in drug targeting and controlled release, immunodetection, and diagnostics.^27,32,33 Now, chemical modification methods have been extensively used in the fields of bioengineering, gene transfer, and sensors, but their application in agriculture has just started.

Chemical modification methods are widely used in biomimetic mineralization, such as layer-by-layer self-assembly (LBL) and polyethyleneimine (PEI) nano induction technology. LBL is a technology that makes polyelectrolyte multilayer coating by alternately depositing cationic and anionic polyelectrolytes on the solid surface through electrostatic interaction.^3,26,29; The principle of polyethyleneimine nano induction technology is in employing a typical polyamine polymer which PEI has high adsorption capacity and selectivity for metal ions, and amine groups have good reaction activity and are easy to be modified. In recent years, researchers have applied this technology to root border cells (RBCs), showing that nano silica deposition confers higher aluminum resistance to the cells.³ PEI induced nano silicon deposition was also applied to yeast cells and rice suspension cells. The results showed that the silica nanoshell could inhibit the entry of cadmium ions into cells and effectively alleviate the toxicity of cadmium on cells.^34,35 RBCs are a group of special living single cells that are produced from the root cap meristem and gathered around the root tip. Their production is regulated by genetics and plays a range of biological functions.^3,36,37 Herein, the method of assembling nano-SiO₂ particles onto the cell wall through LBL and PEI technology by using RBCs as model cells is illustrated in Figure 1.

Figure 1. Two principles of biomineralization of RBCs.

3 Biomineralization and mechanism of plant aluminum tolerance

3.1. The role of silicon in plant aluminum tolerance

Aluminum toxicity is the main reason for limiting crop growth in acid soil. Many crops have toxic reactions to the micromolar concentrations of aluminum.³⁶ At present, more than 40% of cultivated land in the world is endangered by aluminum.⁴ The main site of the toxic effect of aluminum on root growth is the root tip.³⁸, and aluminum in cells is mainly located in the cell wall.^39–41 Aluminum in the cell wall is mainly bound to negatively charged pectin which is critical to aluminum toxicity.⁴² The early effects of aluminum toxicity is the disruption of the ordered organization of the cytoskeleton resulting in reduced stability.^43,44 Therefore, the cytoskeleton-cell-membrane extracellular matrix continuum is considered to be the target of aluminum toxicity. According to the different action sites of aluminum toxicity to plants and the response of plants to aluminum toxicity, the mechanisms of plant aluminum tolerance can be divided into two categories: internal tolerance mechanism and external exclusion mechanism. Internal tolerance means that plants can tolerate accumulation of high concentrations of aluminum in plants by reducing the binding of aluminum to sensitive metabolic sites as much as possible through a series of strategies. An external exclusion means the extracellular chelation and exclusion of aluminum by plants.⁴⁵ For most plants, external exclusion is the main way for plants to deal with aluminum toxicity. As the first barrier for plant cells to respond to the external environment, the fixation of aluminum by the cell wall is one of the most important mechanisms of plant aluminum tolerance.⁴⁶

Many studies have reported that silicon, as a beneficial element of plants, can alleviate plant aluminum toxicity.^5,18,41,47 However, the mechanism of silicon operation in alleviating aluminum toxicity is still unclear. At the same time, “silicon-loving” cereals such as rice are not sensitive to aluminum toxicity. Whether it is caused by the massive deposition of silicon in the cell wall or the space of the plasmodesmata, remains to be explored in depth. Although many facts have shown that silicon confers many special functions to different plants, the understanding of the corresponding mechanism is very limited. It is mainly because most of the plant biological research on silicon is at the plant organ or tissue level.

Ion fluxes across cellular membranes are the earliest event in the cellular response to the environment. This allows to use ion flux kinetics for rapid diagnosis and prediction of cellular responses to diverse environmental stresses, which will exclude bias in plant macroscopic assay results due to tissue- and cell-speificity of plant adaptive responses. The detection of ion flux by noninvasive ion selective microelectrode technology(NMT) enables the study of the dynamic process of nutrient uptake at the microscopic scale, with high spatial and temporal resolution.⁴⁸ Thus noninvasive MIFE (microelectrode ion flux estimation) and NMT has become an emerging and effective tool in the research of plant nutrition and physiology.^29,49,50 Now, some well-developed cytological methods can also be used for real-time in situ observation. For example, transmission electron microscopy can observe the thickness of cell wall.⁵¹, and atomic force microscopy (AFM) can observe the fine changes of microfibrils at the nano level.^52–55 In particular, AFM can quantitatively measure various physical properties of fine structures on the cell surface in real-time, such as roughness, surface potential, elasticity, hardness, friction, etc.⁵⁶ (Figure 2). AFM has been well used in the studies of protein crystal nucleation.⁵⁷ and the effects of different functional groups on the nucleation process of silica.⁵⁸ The combination of the front edge technologies such as MIFE, AFM, transmission electron microscope(TEM), and scanning electron microscope(SEM) may be very helpful in disclosing the mechanisms of silicon in the protection of plant cells to stresses like Al toxicity taking advantages of RBCs, a population of single living plant cells.

Figure 2. Mechanism of biomineralization induced by nano silicon.

3.2. Biosilicification and aluminum tolerance mechanisms in plants

As an important biomineralization process, silicification is common in the leaves of gramineous plants such as wheat and rice. After silicon is absorbed and transported to the aerial part by silicon-loving plants, most of them are deposited in specialized silicon cells or apoplast space in the form of biological minerals, and a few are deposited on the cell wall of the outer leaf epidermis.^59,60 In roots, silicon is mainly deposited on the cell wall, and its main forms are hydrated amorphous silica (SiO₂ • nH₂O) and silica (SiO₂), followed by silicic acid and colloidal silicic acid.⁶¹ Therefore, the study of a cell wall structure and its chemical components will be the basis for understanding the biological role of silicon. There is a hydrophilic matrix in the cell walls, intercellular space, and vessel elements, such as the hydrophilic surface of the cell membrane, cellulose polysaccharide hydroxyl system of the cell wall, and various hydrophilic groups of glycoprotein. This hydrophilic matrix can induce the self-assembly of SiO₂ to deposit in these cellular spaces to form nanostructures.^2,62 In addition to SiO₂ deposited in the cell wall, various morphologies of SiO₂ can also be formed in the cell. The forming process usually starts with the degradation of cytoplasm, the disappearance of the nucleus, the death of differentiated mature cells, enlargement of vesicles, and finally SiO₂ filling the intracellular lumen to form phytoliths of various shapes.^60,63 Simpson et al found that nanostructured SiO₂ exists in various unicellular algae, bacteria, spongy and higher plants.⁶⁴ This deposition enhances the rigidity of the cell wall, improves the resistance of the cell wall to external forces, and also facilitates the growth of roots in a solid medium.

Feng et al successfully induced the deposition of cell nano silicon structure based on layer-by-layer self-assembly, and this deposition is conducive to the accumulation of activated aluminum on the surface of the cell wall, to alleviate the cell toxicity of aluminum.³ Further, X-ray photoelectron spectroscopy(XPS) analysis showed that the main form of a combination of aluminum and silicon after nano-silicon deposition is aluminosilicate nanostructure. Ma et al proposed that the mitigating effect of silicon on aluminum toxicity may be to reduce the toxic Al³⁺ concentration in the solution by forming an aluminum-silicon complex.⁴⁰ Wang et al confirmed that silicon treatment can form aluminum silicon coprecipitation in the apoplast and proposed that aluminum-silicon coprecipitation is the mechanism to alleviate aluminum toxicity.¹⁸ The structure and molecular composition of the Al-Si co-deposit, however, remains unknown. The special structure and reactivity of aluminosilicate nano minerals in nature may strongly affect the processes of the Earth’s material cycle in which they participate. Especially the interfacial reactivity of nano minerals will be combined with other factors to show a high diversity of geochemical reactivity. Therefore, the specific structure and chemical composition of this mineralized structure is worth studying, and the factors that affect the mineralization rate also need to be further clarified. In addition, when plants are subjected to various organic and inorganic environmental pressures including heavy metal stress, they will generate reactive oxygen species.⁶⁵ For example, aluminum toxicity can lead to the increased levels of reactive oxygen species (ROS) in cells resulting in oxidative damage, and the presence of large amounts of ROS can induce a series of cascade reactions in mitochondria, ultimately triggering programmed cell death. Silicon reduces the production of reactive oxygen species and enhances the expression of antioxidant enzymes thereby reducing oxidative stress in plants.³⁶ Therefore, whether the distribution of aluminum and silicon in cells and the precipitation mechanism can strengthen the plant stress defense system by reducing oxidative damage and changing the activity of antioxidant enzymes may represent an important physiological mechanism waiting to be investigated (Figure 2).

4 Problems and prospects

In recent years, researchers have explored the mechanisms of plant aluminum tolerance under biomineralization mechanisms through a broad range of approaches, and have made breakthroughs in the study of the mechanisms of silica in mitigation of plant aluminum toxicity. However, there are still many issues to be solved regarding how to regulate the internal and external tolerance mechanisms of plants under biomineralization that need to be addressed. The first question is how to build an external “barrier” based on biomineralization to alleviate cell aluminum toxicity, and how to properly build a silica layer outside the cell through biomimetic mineralization to form an “armor” and protect the cell without inhibiting cell viability. The second task is to decipher the mechanism of aluminum-silicon coprecipitation formed by biomineralization in alleviating aluminum toxicity, focusing on the structure or molecular composition of aluminum-silicon coprecipitation. Considering that the special structure and reactivity of aluminosilicate minerals in nature may greatly impact the Earth’s material cycle process, especially the interface reactivity of nano minerals in combined with other factors, so the specific structure and chemical composition of this mineralized structure is worth studying. The third issue to be studied is the role of biomembrane in biomineralization. Biomineralization is usually carried out in a special compartment surrounded by a lipid bilayer membrane. However, overall we still barely understand the role of membranes in biomineralization. Furthermore, an in-depth study of the relationship between biomineralization and aluminum tolerance at the molecular level, including the discovery and identification of key functional genes in the aluminum tolerance, will help to clarify the fundamental mechanism of biomineralization so as to improve plant aluminum tolerance.

Acknowledgments

This research was funded by the National Natural Science Foundation of China, Ministry of Science and Technology of China, Science and Technology Department of Guangdong Province, Higher Education Department of Guangdong Province, and Natural and Fundamental Research Funds for the Central Universities of China. The above materials were provided by Department of Horticulture, Foshan University,and Figure 2 was supported (in part) under the state contract of the Ministry of Science and Higher Education of the Russian Federation (Project No. 122050400128).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 42077150, 32172672, 31672228, 31172038), Ministry of Science and Technology of China (CB02-07, G2022030079L, G2021030008L, G2021030014L, DL20200230002,), the Science and Technology Department of Guangdong Province (Grant No. 2018A050506085, 2015A040404048, 2019A1515110059, 163-2018-XMZC-0001-05-0049, 2022B1212010015, 2017-1649), the Higher Education Department of Guangdong Province (Grant No. 2020KCXTD025), the Natural and Fundamental Research Funds for the Central Universities of China (Grant No. 2662019PY013).