Cell Wall‐Denaturing Molecules for Plant Gene Modification

Zwitterionic molecules, such as zwitterionic liquids (ZILs) and polypeptides (ZIPs), are attracting attention for application in new methods that can be used to loosen tight cell wall networks in a biocompatible manner. These novel methods can enhance the cell wall permeability of nanocarriers and increase their transfection efficiency into targeted subcellular organelles in plants. Herein, we provide an overview of the recent progress and future perspectives of such molecules that function as boosters for cell wall‐penetrating nanocarriers.


Introduction
Plant genetic engineering is an emerging approach for elucidating plant gene function and overcoming agricultural difficulties, such as abiotic/biotic stress and crop productivity. However, the plant cell wall stands as a barrier for gene delivery tools in efficient plant genetic engineering. [1] In terms of DNA delivery into plant organelles, nanomaterial-mediated gene delivery is considered a beneficial method compared to conventional delivery methods, such as genotype-dependent Agrobacterium infection and particle bombardment, which requires specialized equipment. [2] Since nanocarriers possess unique physical and chemical properties due to their small sizes, they enable exogenous plasmid DNA (pDNA) or doublestranded RNA (dsRNA) to be directly delivered into intact plant cells, species-independently, without specialized equipment. [3] For example, nanocarriers including carbon nanotubes (CNTs) and peptide-based nanocomplexes successfully delivered pDNA to specific organelles (i. e., mitochondria and chloroplasts) in intact plants, while conventional delivery methods cannot target such specific organelles. [4] The cell wall permeability of nanocarriers depends on the plant species, the nano/microstructure of plant tissues, and the properties of nanocarriers, such as shape, size, surface charge (zeta potential), and amphiphilicity. [5] The recalcitrant plant cell wall network restricts such nanocarriers from penetrating and prevents nanocarriers from reaching their targeted subcellular organelles. [6] To eliminate the effect of the cell wall barrier, treating isolated protoplasts with polyethylene glycol (PEG) solution enabled exogenous DNA/RNA transfer for genetic modification. [7] However, plant regeneration from protoplasts requires tremendous time, tedious procedures and large quantities of DNA/RNA. [8] Thus, to promote efficient nanocarrier internalization for the modification of organellar genome(s) in intact plant cells, alternative approaches that allow them to overcome the cell wall barrier are needed. One possible method is to loosen the cell wall network. Certainly, there are mechanical treatments, chemicals (i. e., calcium ions, EDTA, detergents) or enzymes that hardly disrupt the cell wall network. However, these intensive methods might weaken cell wall mechanical properties and induce cytotoxicity that prevents plant growth. [9] Thus, new wall loosening methods should overcome the cell wall barrier by partially relaxing the cell wall network by removing the embedded matrix or loosening the tightly packed cellulose network in the cell wall to maintain plant cell mechanical stability and viability.
In this study, we address an emerging approach that relaxes plant cell wall networks to boost the cell wall permeability and transfection efficiency of nanocarriers ( Figure 1). Herein, zwitterionic molecules, especially zwitterionic liquids (ZILs) and zwitterionic polypeptides (ZIPs), that have a high hydrogenbond cleaving ability are introduced, and their perspectives are discussed. By utilizing their properties as excellent cellulose dissociating solvents, they are able to partially disrupt the cellulose network and function as a booster of nanocarriers for cell wall penetration in plant cells. [10] In addition, the latest Figure 1. A new cell wall loosening strategy to overcome barriers and enhance the internalization of nanocarriers into specific organelles of plant cells. The cartoon illustration depicts the cell wall loosening method that is enabled by cell wall-loosening molecules described in this study, including a zwitterionic liquid (OE 2 imC 3 C), zwitterionic peptide (poly(GlyHis zw Gly)) and the natural protein expansin (EXLX1, bacterial expansin of Bacillus subtilis, PDB: 2BH0). [19] applications of the cell wall-loosening ability of zwitterionic molecules are shared to further improve the transfection efficiency via nanomaterial-DNA complexes. [10] In conclusion, further perspectives for improving the cell wall-loosening ability of zwitterionic molecules are discussed in terms of mimicking or utilizing the natural cell wall-loosening protein expansin and other structural proteins that regulate cellulose morphology in the plant cell wall.

Plant Cell Wall Architecture
Before introducing cell wall-loosening molecules, the structure of the cell wall should be described to explain how the cell wall prevents nanocarriers from penetrating the cell wall. In contrast to animal cells, plant cells are enveloped by cell walls and provide mechanical strength to protect plants from physical/ biochemical stress to survive even under high-stress conditions. The cell walls of all plants contain cellulose microfibrils. Focusing on such cellulose fibrils, they are made with (1!4)-β-D-glucan chains and laterally stacked with van der Waals forces and hydrogen bonds to form nanofibrils. [11] By tightly packing cellulose glucan chains, cellulose forms a cellulose crystal phase called cellulose I in its fibrils. The unique feature of cellulose I crystals is that they have both hydrophilic and hydrophobic planes in the same crystal structure. The former is composed of the polar equatorial hydroxyl groups of glucose, whereas the latter is composed of the compiled nonpolar axial CÀ H bonds of glucose. [12] Indeed, these two opposite features of cellulose crystals enable them to interact with hydrophilic or hydrophobic molecules to maintain biological cell wall functions.
This complicated crystal rearrangement of cellulose fibrils is the first physical/chemical barrier for nanocarriers. In the primary cell wall (PCW) of most plants, these recalcitrant cellulose fibers are intricately oriented and accumulate in layers embedded with amorphous polysaccharides (i. e., pectin and hemicellulose) and structural proteins (i. e., hydroxyproline-rich glycoproteins and glycine-rich proteins). [13] This cross-lamellar structure in PCWs is the basic structure of the nanocarrier barrier ( Figure 2). [14] Since the composition of the PCW (i. e., type of hemicellulose, presence of aromatic molecules) differs between monocots and dicots, the cell wall permeability of nanomaterials varies among plant species. [15] Furthermore, the orientation of cellulose bundles in living plant cell walls also differs between the outermost layer and successive inner lamellae. [16] In certain cell types, depending on plant development, lignified robust secondary cell walls are elaborately combined by acidic phenylpropanoids or lignin networks. [17] This cell wall heterogeneity is also a challenge for nanocarriers to penetrate the cell wall barrier of living plant cells to reach their destination to achieve genetical modification.

Natural Cell Wall-Loosening Protein
Cell walls dynamically change their cell shape, cell size and cell function by providing sufficient mechanical strength during cell elongation in plant growth. This process is enabled by expansins, endogenous proteins in the cell wall that manipulate Risa Naka received her BSc (2022) from Kyoto University. Since 2022, she has been studying as a master's course student under the guidance of Prof. Keiji Numata in Kyoto University. Her research interest is the development of biocompatible cell wall loosening peptides for application to plant gene modification.

ChemBioChem
Review doi.org/10.1002/cbic.202200803 recalcitrant cell wall networks. Especially in the dicot cell wall, α-expansin, a nonenzymatic protein, works as a zipper for the separation of fibril bundles from each other without deteriorating the mechanical properties of the cell wall to induce the acidic creep that is essential to cell wall enlargement (plastic loosening) and plant growth ( Figure 3A). [18] The protein structures of expansins mainly consist of two domains, the Nterminus domain (D1) for the cell wall loosening catalytic domain and the C-terminus domain (D2) for the carbohydrate binding module (CBM) ( Figure 3B). [19] Considering how these two modules function, it is hypothesized that the aromatic planner of CBM binds with cellulose microfibrils, followed by the aspartic acid (Asp82) of the His-Phe-Asp (HFD) motif involved in the entanglement of embedded polysaccharides ( Figure 3C). [20] Considering the excellent cell wall-loosening properties of expansins, this would be the ideal molecular design to develop plant cell wall-loosening molecules.

Cellulose-Loosening Low Molecular-Weight Molecules
To breakdown the recalcitrant cellulose network barrier, ionic liquids (ILs) were developed as a nonderivatizing solvent of cellulose that can loosen cellulose fibrils under mild conditions. [22] Recently, various ILs enabling cellulose to be highly dispersed at room temperature have been utilized as solvents for cellulose in various fields. [23] ILs commonly include imidazolium, pyrrolidinium, pyridinium and sulfonium as cations (hydrogen bond acceptors) with phosphate, carboxylate, chloride and halogen as anions (hydrogen bond donors, β-value�0.8) ( Figure 4A). By including such moieties that can interact with the hydroxyl groups in cellulose, especially excellent hydrogen-bond donors, those moieties efficiently interact with the hydroxyl group of cellulose fibrils to dissolve them in solution ( Figure 4B). [24] Although ILs are believed to be low-toxicity solvating chemicals for cells, unlike surfactants or physical treatments, Stepnowski et al. first reported that ILs harm living cells by rupturing the cell membrane, which is fatal to microbial/animal cells. [25] For terrestrial plants, several groups further reported that ILs can harm photosynthesis and plant growth, suggesting that ILs should not be applied to plant cells. [26] To overcome this obstacle of cytotoxicity to be  [19] EXLX1 is thought to be a homolog of αexpansin derived from dicots and used as a model to elucidate the cell wall loosening mechanism. Amino acids that are essential for cell wall loosening are colored: Asp82 for cell wall loosening catalytic activity (red). Aromatic planner (W125, W126, Y157, orange) for cell wall binding. C) Aromatic moieties of EXLX1 interacting with cellotetraose by hydrophobic interactions (PDB; 4FG2). [20] [29] applicable to the cell wall in living cells, ILs completely comprised of bioderived ions (i. e., choline cation, amino acid anion) were developed and presented less toxicity than imidazolium-based ILs. [27] Kuroda et al. designed ZILs as cell wall dissociating solvents and dramatically improved the cytotoxicity of ILs. [28] Introducing anion moieties (i. e., carboxylate and sulfonate) at the terminus of alkyl chains that were covalently linked with the imidazolium cation successfully suppressed the alkyl chains from piercing the cell membrane by hydrophobic interactions and selectively dissociated cellulose fibrils in the cell wall of microbes (Figure 4C). [29] In particular, OE 2 imC 3 C, a ZIL, which is liquid under a mild temperature, showed great biomembrane compatibility (EC 50 = 158 g L À 1 ), improving cell viability by 6 times compared to that of a common cellulose-dissolving solvent (LiCl/N,Ndimethylacetamide) to achieve the growth of recombinant Escherichia coli. This novel cell wall-loosening ZIL has potential applications in various fields, especially plant gene modification.
There are two challenges to solve for practical ZIL use in nanocarrier-mediated gene delivery in plants. One is the high cost of synthesizing ZILs. The reagent 1-bromo-2-(2-methoxyethoxy) ethane, which comprises the oligoether moiety of OE 2 imC 3 C, is expensive and not stably supplied. Recently, Sharma et al. developed a new synthetic strategy that uses benzenesulfonate as a good leaving group instead of bromide and enabled the development of OE 2 imC 3 C using a general low-cost reagent. [30] This might allow further application of ZILs in plant gene engineering. Another challenge is the strong electrostatic interactions between ILs (ILs and ZILs) and water. The ionic moieties of ILs tend to ionically interact even with a small amount of water. Thus, by adding excess water to ILs, they mostly interact with water and lose the ability to dissolve cellulose. [31] Since water is essential for plant growth and cannot be substituted by aprotic solvents (i. e., dimethyl sulfoxide, dimethylformamide, 1,3-dimethyl-2-imidazolidinone), the development of ZILs that can effectively dissociate cellulose fibrils in protic water is also needed. [32] Considering that the cellulosedissolving ability of ZILs can be tuned by molecular structure design (i. e., alkyl chain length, spacer length, electron donation effect toward the anionic moiety, electron withdrawal effect of the cationic moiety), the optimization of the ZIL structure should improve its cellulose dissociation ability. [33]

Increasing Cell Wall Permeability with ZILs
By utilizing the cellulose network-loosening ability of the ZIL OE 2 imC 3 C, Miyamoto et al. reported that the ZIL successfully unraveled cellulose bundles to decrease fibril density and augmented the cell wall permeability of peptide-based nanocarriers encapsulating DNA to increase their transfection efficiency in specific organelles (chloroplast and nuclei). [10] First, seedlings of Arabidopsis thaliana were preincubated in OE 2 imC 3 C solution, which allowed the disruption of the amorphous region of cellulose with decreasing cellulose density in the cell wall and enhanced cell wall porosity without affecting cell viability. Next, to evaluate the effect of ZIL pretreatment on the nuclear transfection of intact plant cells, a cell-penetrating, peptide-decorated micelle complex (CPP-MC) was applied as a nanocarrier for pDNA cargo to specifically deliver into subcellular plant nuclei ( Figure 5A). As a result, ZIL pretreatment augmented the delivery efficiency of the peptide/ DNA micelle complex (CPP-MC-S) with a size of approximately 100 nm to the specific plant organelles and significantly improved the nuclear transfection efficiency in intact plants (up to 2-fold compared to untreated seedlings) ( Figure 5B, C). However, pretreatment with a relatively high concentration of ZIL solution made the seedlings brittle, as seen from the reduced crystallinity of the cellulose fibrils, and those seedlings no longer had the potential for further growth. Thus, it is essential to develop cell wall-loosening molecules that can selectively interact with regions that do not significantly affect cell wall mechanical properties to give plants sufficient mechanical strength to grow.

Cell Wall Network-Dissociating Zwitterionic Polymers (ZIPs)
We reported that a ZIP also showed cellulose network loosening ability. [34] A histidine-containing polypeptide was converted into a ZIP, poly(GlyHis zw Gly), by modifying the imidazole moiety of histidine into a zwitterionic structure (55 % modification) (Figure 6A). In contrast to 1-butyl-3-methylimidazolium acetate (BMImAc, a typical IL for cellulose dissolution), poly(GlyHis zw Gly), presenting a "polymer effect", was effective for cellulose dissociation in a dilute aqueous solution ( Figure 6B). Covalently linked zwitterionic moieties were localized near the cellulose surface to increase the ionic strength and enable cellulose fibril dispersion even at a low concentration (0.5 mg mL À 1 ) in water. In addition, by treating cellulose nanocrystals with poly(GlyHis zw Gly), the larger increment of elasticity was confirmed by indentation measurement than treating with the same concentration of IL, BMImAc. Finally, ZIP treatment of BY-2 suspension cells of tobacco (Nicotiana benthamiana) was observed by high-speed atomic force microscopy (HS-AFM). It was confirmed that the ZIP loosened the cell wall without affecting the viability of plant cells ( Figure 6C, D). Only one ZIP sequence was reported in this study, and the suitable structure of the ZIP for cell wall loosening was not studied in detail. Recently, polypeptides that periodically contain histidine residues were further synthesized to obtain various zwitterionic polypeptide precursors on a large scale. Dipeptide-based poly(HisGly) was likely to form a β-sheet structure, while tripeptide-based poly[GlyHis(BuCO 2 Et)Gly] or poly[GlyHis(BuCO 2 Bzl)Gly] formed random structures in the solid-state ( Figure 6E, F). [35] Since the secondary structures of peptides are well known to determine all peptide traits, different peptide structures might result in distinctive interaction abilities with cellulose. Thus, the postzwitterionic modification of these peptides might allow the design of new ZIPs that have fine secondary structures. The structural variety of ZIPs would allow us to find improved ZIPs to effectively interact with cellulose fibrils with a greater specificity and higher affinity (low concentration), enabling the efficient dissociation of cellulose networks to loosen cell walls. Further investigation on the quantitative analysis of cell wall loosening analysis of cell wall loosening ability among dipeptide-based and tripeptidebased ZIPs is ongoing.

Summary and Outlook
In this study, we demonstrated a new concept of relaxing the cell wall network to enhance the internalization of nanocarriers in plant cells. In particular, pretreating plant cells with zwitterionic molecules, ZILs and ZIPs, which have high cell viabilities, would be a powerful way to effectively disrupt the cell wall barrier. To prove this concept, we first demonstrated that treating A. thaliana with a ZIL successfully dissolved the cell wall and augmented the cell wall penetration of nanocarriers (~200 nm), resulting in an increase in nuclear transfection efficiency and chloroplast transfection efficiency.
A few future perspectives should be taken into consideration to design and develop novel cell wall-loosening molecules that allow cellulose network dynamic movement similar to expansins. One is optimizing the molecular design for cellulose network loosening inspired by expansins so that zwitterionic molecules can attain specificity while interacting with the cell wall network. Recently, computational analysis revealed that the CBM structure of α-expansin purportedly possesses an aromatic flat surface comprised of tryptophan and other aromatic residues that allow interaction with cellulose. [36] Furthermore, bacterial expansin, which also has cell wall-loosening activity, was also reported to have an aromatic planner that is involved in cellulose binding, as described above (see Section 3). [37] Considering the numerous reports of aromatic planners facilitating cellulose binding affinity, introducing aromatic planners (i. e., tryptophan and tyrosine) in zwitterionic molecules that can interact with the axial CÀ H bonds of cellulose linear chains might allow them to effectively bind to cellulose to better dissociate cellulose without significantly denaturing the cell wall morphology to avoid cell wall weakening. [38] Another possibility is to utilize the biological activity of natural products, such as α-expansin and auxins. Recently, a plant hormone (auxin) based on tryptophan (Trp) was intensively studied and proven to be involved in cell wall loosening to increase cell wall extensibility. [39] Synthetic auxin with a Trpbased zwitterionic structure was found to have a higher auxin activity. Considering that treating plants with the Trp-based molecule fostered plant growth, introducing the Trp moiety to wall-loosening ZILs/ZIPs might not only chemically loosen the cell wall but also biologically loosen the cell wall, which might boost nanocarrier cell wall penetration. [40]