4 Root cell type GRN adaptations to drought: Impermeabilization of endodermis and exodermis
The evolution of hydrophobic extracellular biopolymers was a critical innovation for plant terrestrialization, contributing to permeability and water transport control. Some of these compounds include complex phenolic-derived polymers such as lignin, cutin and suberin (Niklas, Cobb, & Matas, 2017). Lignin generally works in the reinforcement of secondary cell walls, cutin is commonly found as part of the impermeabilizing cuticle of aerial parts and was recently reported as being also part of the root cap cuticle, and suberin is found in many tissues including specialized root cells, tubers, fruit skin, and seed coat (Berhin et al., 2019; Niklas et al., 2017; Philippe et al., 2020; Renault et al., 2017). In general these compounds function in mechanical support, defence against pathogens and herbivores, and in the control of the movement of water, nutrient and gases (Niklas et al., 2017; Philippe et al., 2020; Renault et al., 2017; Pei Wang et al., 2020).
Convergent evolution has also played an important role in the appearance and shaping of the biosynthetic pathways of some of these biopolymers across diverging plant taxa. For example, the ancestral green algae and red-algae have the ability to produce “lignin-like” compounds, leading to the hypothesis that ancient biosynthesis pathways have been rewired in the vascular plant lineage (Delwiche et al., 1989; Labeeuw et al., 2015; Martone et al., 2009). Furthermore, lycophytes and spermatophytes have independently developed the ability to produce monomers necessary for lignin biosynthesis and assembly (Renault et al., 2017; Weng et al., 2010; Weng et al., 2008). It is possible that convergent evolution also played a role on the appearance of cutin and suberin across distantly related plant species. Both cutin and suberin share similar initial biosynthetic steps and lipid precursors, however different enzymes belonging to the same superfamily and with different mechanisms of action work on their modification and assembly (Philippe et al., 2020; Pollard et al., 2008). A recent large-scale comparative genomic study has revealed that the ability to synthesize precursor molecules of cutin and suberin emerged prior the evolution of land plants, however the subsequent steps of the biosynthetic pathway may have evolved independently across land plants (Cannell et al., 2020). Still, little is known about how the other parts of the pathway, such as polymerization and distribution in plant cells, evolved in land plants (Niklas et al., 2017; Philippe et al., 2020; Pollard et al., 2008).
The diversification of biosynthetic pathways leading to the production of impermeabilizing hydrophobic compounds has also contributed to the evolution of distinct cell types important for adaptation to terrestrial life and for resilience to drought. For example, the roots of all vascular plants contain an endodermis surrounding the vascular tissues (Doblas, Geldner, & Barberon, 2017; Enstone et al., 2002). The endodermis cell layer forms a diffusion barrier for water, gases and nutrients due to the presence of two cell wall modifications: the Casparian strip and suberin lamella (Barberon et al., 2016; Doblas et al., 2017; Seago & Fernando, 2013; Vishwanath, Delude, Domergue, & Rowland, 2015). Casparian strip is composed of lignin, deposited in the walls of endodermal cells at their junctions, dividing the layer into outward and inward polarities and forming an effective barrier to the apoplastic movement of molecules into the stele and preventing their backflow (Barberon, 2017; Enstone et al., 2002; D. Roppolo et al., 2014; Daniele Roppolo et al., 2011). The suberin lamella is a secondary cell wall modification deposited in the inner surface of the primary cell walls, usually after the Casparian strip is formed in the mature endodermis (Barberon, 2017; Enstone et al., 2002). Different from Casparian strip, the suberin lamella may not form in every root nor in every endodermal cell (the so-called passage cells) (Andersen et al., 2018; Barberon et al., 2016; Enstone et al., 2002; Holbein, Shen, & Andersen, 2021). Despite debate in the past years, the role of suberin lamella as an apoplastic barrier for water and nutrient uptake from the apoplast to the endodermis cytoplasm has been demonstrated (Barberon et al., 2016; Ranathunge & Schreiber, 2011; Peng Wang et al., 2019).
The roots of several species also develop an exodermis below the epidermis, which is a specialized type of hypodermis with Casparian bands and suberin lamellae depositions (Enstone et al., 2002; Perumalla et al., 1990). The exodermis function as a dynamic barrier not only against water loss under drought and salinity, but also against loss of oxygen under anoxic conditions, against penetration of ions and heavy metals, and against pathogen infections (Aloni, Enstone, & Peterson, 1998; Damus, Peterson, Enstone, & Peterson, 1997; Ejiri & Shiono, 2019; Enstone et al., 2002; Líška et al., 2016; Namyslov, Bauriedlová, Janoušková, Soukup, & Tylová, 2020; Ranathunge, Lin, Steudle, & Schreiber, 2011; Tylová, Pecková, Blascheová, & Soukup, 2017). At the same time, the development of exodermis barriers has its downside as it may impair the uptake of nutrients and interaction with beneficial microbes (Kamula, Peterson, & Mayfield, 1994). To cope with this problem, many plant species developed the ability to induce an exodermis dynamically in response to abiotic stresses, such as drought (Enstone et al., 2002; Kreszies et al., 2020; Líška et al., 2016; Reinhardt & Rost, 1995; Taleisnik et al., 1999). Interestingly, the development of the exodermis may vary among closely related species displaying distinct stress response phenotypes (Ejiri & Shiono, 2019), indicating that this cell type contributes to plant plasticity and acclimation and may also help plants to adapt and colonize dry environments.
Regardless of its adaptive role, the evolution of exodermis in plants still remains untangled. Perumalla et al. (1990) surveyed 181 species from 53 families of plants from different ecological groups (hydrophytic, mesophytic, and xerophytic) to determine the presence of hypodermis with Casparian bands (exodermis). As the majority (156) of the species assessed presented an exodermis with suberin only (hypodermis) or with both suberin and lignin, the authors hypothesized that the presence of a modified hypodermis is ancestral to flowering plants, and has been retained in many species (Perumalla et al., 1990). Furthermore, the authors found that festucoid grasses lack Casparian bands despite presenting cells with similar shape and packing as species with hypodermal Casparian bands, leading to the hypothesis that their recent ancestor may have lost the trait. Interestingly in seminal roots of modern cultivars of barley (a festucoid species) the exodermis fails to develop even upon severe osmotic stress (Kreszies et al., 2019), while in wild barley the exodermis is induced in response to osmotic stress (Kreszies et al., 2020). On the other hand, in other crop grasses (non-festucoid), such as rice and maize, an exodermis is present and develops faster in response to stress (Ranathunge, Schreiber, Bi, & Rothstein, 2016; Schreiber, Franke, Hartmann, Ranathunge, & Steudle, 2005).
Understanding how the exodermis evolved in plants can help in the identification of the underlying regulatory networks responsible for its induction in response to drought. To obtain more knowledge about the evolution of exodermis, we compiled the current information about their presence in plant species based on literature search (Figure 2) (Bani, Pérez-De-Luque, Rubiales, & Rispail, 2018; Barrios-Masias, Knipfer, & McElrone, 2015; Barykina & Kramina, 2006; M. Brundrett, Murase, & Kendrick, 1990; Calvo-Polanco, Sánchez-Romera, & Aroca, 2014; Damus et al., 1997; Demchenko, Winzer, Stougaard, Parniske, & Pawlowski, 2004; Eissenstat & Achor, 1999; Ejiri & Shiono, 2019; Enstone et al., 2002; Ghanati, Morita, & Yokota, 2005; Kosma, Rice, & Pollard, 2015; Liu et al., 2019; Perumalla et al., 1990; Ranathunge et al., 2017; Reinhardt & Rost, 1995; Ron et al., 2013; Schreiber, Franke, & Hartmann, 2005; Schreiber, Hartmann, Skrabs, & Zeier, 1999; Shiono & Yamada, 2014; Thomas et al., 2007; Zhang, Yang, & Seago Jr, 2018). Based on this analysis, the exodermis with suberin first appeared in early land plants (lycophytes) but it is missing from other seedless vascular plants and all but one gymnosperm (Damus et al., 1997). Interestingly, four species in the lycophyte genus Selaginella contain exodermis with lignified Casparian strips (Damus et al., 1997). Most flowering plants contain an exodermis with suberization only (hypodermis), while a lignified exodermis appears in about a third of the species. The scattered appearances of the exodermal lignification indicates that it has evolved independently multiple times, suggesting a high evolutionary pressure and pre-conditioning for the characteristic to arise. Species with no exodermis have been identified in seven clades (in purple, Figure 2), and the most parsimonious explanation for presence/absence of exodermis is the loss of the cell type in these lineages. However, the evolutionary hypotheses are restricted by the sparse sampling in families of interest. This is highlighted by the relevant literature containing contradictions (e.g. pea Bani et al. (2018); Perumalla et al. (1990); Taleisnik et al. (1999)), likely due to the dynamic nature of the exodermis.
Evolutionary studies focused on characterizing the exodermis, e.g. by staining suberin and lignin or using barrier property assays (Supplementary Table 1) will contribute with important information about how this cell type has appeared or disappeared multiple times across the plant lineages. Coupling that with comparative genomics and transcriptomics of phylogenetically close species (e.g. from the same family) but with different phenotypes (e.g. non-exodermal, constitutive and stress-inducible exodermis) will be key to identify the origin of the regulatory networks and how master regulators underlying exodermis development and suberization in response to drought evolved. It is possible that the sporadic appearance of exodermis during plant evolution was possible through rewiring regulatory networks of Casparian strips and suberin lamellae formation in endodermis or similar lignin and suberin biosynthetic pathways from other cell types. The coexistence of endodermis and exodermis in roots has complicated transcriptomic approaches, and to investigate the exodermis gene expression and GRNs in individual species, methods such as laser capture microdissection (LCM) and translating ribosome affinity purification (TRAP) have been used (Kajala et al., 2021; Shiono et al., 2014). The exodermis-specific gene expression patterns and GRNs are a foundation for understanding the genetic underpinnings of the cell type and enable investigating its evolutionary paths.
Recent studies are showing the importance of distinct clades of the MYB transcription factor family as conserved regulators of suberin deposition in response to osmotic stress in different cell types and in phylogenetically distant plants, linking their evolution with colonization of dry terrestrial environments by early land plants (Capote et al., 2018; Cohen, Fedyuk, Wang, Wu, & Aharoni, 2020; Gou et al., 2017; Kajala et al., 2021; Kosma et al., 2014; Lashbrooke et al., 2016; Legay et al., 2016; Shukla et al., 2021; To et al., 2020; Wei et al., 2020). Two of the possible scenarios are: (1) osmotic stress-inducible regulation of suberization diversified from pre-existing developmental pathways, (2) the regulation of suberization in response to drought was re-activated as plants colonized drier environments. Taking that into account, further evolutionary/phylogenetic study of exodermis is needed and selection of a good clade(s) to dissect the gain/loss events is key for understanding exodermis development and how it evolves so readily.