**1. Introduction**

As the threat of climate change rages on, the frequency of dehydration stress and the risk posed by fungal pathogens is on the rise. These challenges are further complicated by the sessile nature of plants, which must survive a range of a combination of abiotic and biotic stresses. The cell wall serves as a barrier against stress, in addition to providing critical mechanical and structural support to the plant [1,2]. The ability of the cell wall to function in a range of mechanisms is dependent on the various components of the primary cell wall, including cellulose, hemicellulose, proteins and pectin [3]. Except for grasses, pectin is one of the most predominant components, comprising 30 to 50% of the cell wall dry matter [3]. Therefore, the composition of pectin is integral to the growth, morphology and development of the cell well, as well as in its ability to defend the plant against stress [4].

**Citation:** Forand, A.D.; Finfrock, Y.Z.; Lavier, M.; Stobbs, J.; Qin, L.; Wang, S.; Karunakaran, C.; Wei, Y.; Ghosh, S.; Tanino, K.K. With a Little Help from My Cell Wall: Structural Modifications in Pectin May Play a Role to Overcome Both Dehydration Stress and Fungal Pathogens. *Plants* **2022**, *11*, 385. https://doi.org/ 10.3390/plants11030385

Academic Editors: Penélope García-Angulo and Asier Largo-Gosens

Received: 14 December 2021 Accepted: 26 January 2022 Published: 30 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Pectin, the most structurally complex polysaccharide found in nature, is a five-member family currently considered to include: (1) Homogalacturonan (HG); (2) Rhamnogalacturonan I (RG-I); (3) Rhamnogalacturonan II (RG-II); (4) Xylogalacturonan (XGA); and (5) Apiogalacturonan (AP) [4]. HG and RG-II account for approximately 65% and 10% of all pectin, respectively, and have the unique ability to form more complex structures through cross-linkages to other elements [4–6].

Under the prevailing view, calcium ions form cross-linkages to carboxylate ions in demethylesterified galacturonic acid residues of HG [5,7]. The demethylesterifcation of HG occurs by way of pectin methylesterases (PMEs) [7] and pectin methylesterase inhibitors (PMEIs) provide negative feedback to PMEs [7]. In comparison, RG-II forms cross-linkages with boric acid, creating RG-II dimers [8]. These borate–diol ester cross link a boron atom and the apiosyl residue of side chain A in RG-II [8]. The formation of these cross-linkages maintains structural stability by reducing porosity while enhancing tensile strength and bound water in the cell wall fraction [9–16]. Increasing the quantity of bound water within the cell wall maintains tissue hydration and turgor pressure in addition to increasing cell wall rigidity [14]. Debra Mohnen's group recently proposed a new model for pectin structure consisting of a family of glycans (homoglycans, heteroglycans and proteoglycans) [17] and therefore, aspects of the current cell wall models may need to be reconsidered as more information emerges.

HG and RG-II have also been implicated as key forms of pectin in mitigating the resistance to desiccation and drought stress in several species, such as green algae and wheat cultivars [18–23]. This is likely the result of pectin forming hydrated gels, which in turn may limit damage during dehydration stress [19]. The degree of pectin methylation has also been linked to their ability to hold water, with reduced methylation increasing the water holding capacity of pectin [12]. Willats et al. (2001) also found the addition of calcium to pectin gels influences the water holding capacity, presumably as a result of "egg-box" structures [12].

The analysis of the *MUR1* gene has further implicated RG-II dimers as an important aspect of the tolerance to freezing stress, tied to dehydration stress resistance [24]. Despite these findings, there is a need for greater research regarding the role calcium and boron on the structure–function of the cell wall in relation to dehydration stress resistance.

Previous research has additionally highlighted the important role that pectin plays in allowing the cell wall to function as a barrier to *Botrytis cinerea*, a necrotrophic pathogen, and *Colletotrichum higginsianum*, a hemibiotrophic pathogen. Lionetti et al. (2007) observed increased resistance to *B. cinerea* in *Arabidopsis* as a result of the over-expression of PMEI1 and PMEI2 [25]. Other PMEIs have also been reported to increase plant resistance to *B. cinerea* [26]. Interestingly, PMEs play a beneficial role in the immunity against *B. cinerea*, despite the opposing nature of PMEIs and PME [27]. More generally, pectin has been identified as a main target during a *B. cinerea* infection [28]. Petrasch et al. (2019) reported that, during the process of a *B. cinerea* infection, the pathogen heavily degrades the cell wall and, in particular, pectin [28].

PMEIs also play a role in the immune response during a *C. higginsianum* infection [29]; however, research within this area is limited. A study conducted by Engelsdorf et al. (2017) found *Arabidopsis pmei6-2* mutants had reduced susceptibility to *C. higginsianum*. Reduced susceptibility may be indicative of a connection between the establishment of a *C. higginsianum* infection and pectin content [29]. Nonetheless, further exploration into the role of PMEIs and *C. higginsianum* is required.

Boron application has also been shown to play a positive role in *B. cinerea* infections in a variety of species [30,31]. Qin et al. (2010) found boron application reduced the germination of *B. cinerea* spores, reduced germ tube elongation and mycelial spread in table grapes [30]. When paired with boron, calcium increased resistance to *B. cinerea* in strawberry plants [31]. In addition, while there is no apparent research on the impact of boron on *C. higginsianum*, borate was found to inhibit the germination of *Colletotrichum gleosporioides* spores in mangos [32,33].

*Allium fistulosum* and *Allium cepa* were selected based on their contrasting freezinginduced dehydration stress tolerance. *A. fistulosum* is extremely tolerant to freezing stress, withstanding temperatures as low as −40 ◦C, while *A. cepa* lacks this ability [34,35]. Both species have easy to peel epidermal cell layers with a large cell size, making them an ideal species for this investigation. *Arabidopsis* was selected based on the wide range of genotypic mutant lines available and its close genetic relationship to *Brassica rapa*.

Since plants are often exposed to multiple stresses in the field, this study aims to gain a further understanding into the influence of calcium, boron and PMEI on pectin cross-linkages, and in turn, on the ability for the cell wall to act as a barrier against both water loss and fungal pathogens. We hypothesized the application of calcium and/or boron results in cell wall structural changes and increased resistance to both abiotic and biotic stress in *Allium* species and *Arabidopsis thaliana.*
