Repeated colonisation of alpine habitats by Arabidopsis arenosa involved parallel adjustments of leaf cuticle traits

Abstract

Introduction

When the first plants conquered the land 500 to 450 million years ago (Becker, 2013), several key innovations enabled them to cope with the challenges posed by the new environmental conditions. These challenges included, among other factors, a desiccating atmosphere, higher light intensities and greater temperature fluctuations. One of the innovations was the evolution of the cuticle, an impermeable, highly hydrophobic outermost layer of leaves, young shoots and other aerial parts (Kong et al., 2020). The cuticle covers the epidermal cells and acts as a physical barrier against uncontrolled water loss (Burghardt & Riederer, 2006; Kong et al., 2020) and also confers protection against various other environmental stress factors. It represents the first barrier to the entry of pests and pathogens (Serrano et al., 2014), influences surface properties such as wettability and water run-off, plays a central role during development by establishing organ boundaries and may be involved in the screening of UV light in some species (Yeats & Rose, 2013). It also helps create a suitable microenvironment for certain microorganisms, the phyllosphere (Kerstiens, 2006; Riederer, 2006). Accordingly, cuticle traits can vary within a plant species, depending on the habitat (Xue et al., 2017).

Plant cuticles are composed of two highly hydrophobic components, cutin and cuticular waxes, which are assembled in several layers. While cutin mainly provides mechanical strength, cuticular waxes determine water permeability, leaf wettability and light reflectance, and thus play important roles in the adaptation to environmental stress factors, including drought, temperature fluctuations and plant–pathogen interactions. Cuticular waxes are embedded within the cuticle and also deposited as crystals on the surface (Bernard & Joubes, 2013). Key functional traits of cuticles, especially cuticle permeability for water vapour, are not directly related to cuticle thickness or to the total amount of waxes, but rather to the composition of its layers, and specifically to the accumulation of very-long-chain aliphatics (VLCA) (Jetter & Riederer, 2016; Seufert et al., 2022). The main classes of VLCAs found in cuticular waxes are alkanes, aldehydes, primary and secondary alcohols, ketones and esters (Yeats & Rose, 2013), which may contribute to reducing transpiration to varying degrees (Grncarevic & Radler, 1967). For instance, the water vapour permeability determined for films of pure compounds was lower for aldehydes and very long chain alcohols than for alkanes of similar carbon chain length (Leyva-Gutierrez & Wang, 2021). Pathways for the biosynthesis of cutin and cuticular waxes have been partially characterised in Arabidopsis thaliana (Bernard & Joubes, 2013; Philippe et al., 2020), the differential regulation of which may affect cuticle function, in particular the control of water loss (Asadyar et al., 2024).

One measure of water deficiency in leaves is the water saturation deficit (WSD), which represents the proportion of water missing to reach full saturation, reflecting how well the cuticle protects the leaf tissue from water loss after stomatal closure (Larcher, 2001). To reduce water loss, higher plants close their stomata. However, they still transpire through the cuticle and incompletely closed stomata (Schuster et al., 2017; Duursma et al., 2019), together constituting the leaf minimum conductance (g_min). This trait varies greatly among species, with some phenotypic plasticity, and is adjusted in relation to the micro-climate, especially by abiotic factors that can influence the evaporative demand such as wind, drought and temperature (Fernández et al., 2017; Duursma et al., 2019; Körner, 2021). Furthermore, foliar water balance is influenced by leaf surface wettability, that is, how easily a liquid such as water spreads over the leaf surface (Barthlott & Neinhuis, 1997), which is affected by the abundance, nature and density of trichomes (Brewer et al., 1991). Leaf wettability decreases with increasing elevation in relation to both changing species composition and intraspecific adjustments (Aryal & Neuner, 2010), and confers important leaf functions, affecting both water uptake through leaf surfaces and transpiration (Goldsmith et al., 2017). However, surface water can also reduce gas exchange and carbon assimilation (Brewer & Smith, 1995; Ishibashi & Terashima, 1995), promote pathogen growth (Evans et al., 1992), lead to leaching of leaf nutrients, and can increase biomechanical stress (Cape, 1996). In addition, the probability of extrinsic ice nucleation increases with the amount of water retained on leaves (Wisniewski et al., 2002), hence an increased leaf water repellency may be beneficial in alpine environments (Yumoto et al., 2021). In summary, environmental conditions during plant growth and development apparently influence cuticle composition, g_min, and leaf wettability. However, in spite of the known importance of cuticle traits for plant adaptation to the external environment (González-Valenzuela et al., 2023), the extent to which the interplay between the environmental factors and intraspecific genetic variation, possibly resulting from evolutionary adjustments, shapes these leaf traits remains poorly understood.

Naturally replicated cases of evolution encompassing parallel evolution of specific traits provide an opportunity to distinguish adaptive traits from neutral changes (Bolnick et al., 2018; James et al., 2023). In Arabidopsis arenosa, the repeated emergence of an alpine A. arenosa ecotype from a broadly distributed foothill ecotype, in at least three European mountain ranges, was accompanied by parallel changes in multiple morphological, anatomical and functional traits such as petal size, plant height (Knotek et al., 2020), leaf thickness, trichome density (Bertel et al., 2022) and cold acclimation potential (Kaplenig et al., 2022). This congruent ecotypic differentiation has been associated with a higher fitness of populations in their local vs foreign habitats (Wos et al., 2022). Aiming to uncover the genomic and transcriptomic basis of parallel adaptation to the alpine habitat in A. arenosa, previous studies identified 151 candidate genes that were repeatedly differentiated between foothill and alpine populations (Bohutínská et al., 2021) and these changes likely led to broad, partially parallel, transcriptomic responses (Wos et al., 2021). However, the extent to which genetic changes affected cuticle synthesis pathways, potentially affecting cuticle composition, g_min and leaf wettability, remains unknown.

Leveraging the established case of parallel evolution in A. arenosa, we explored the relevance of cuticle traits in the adaptation to the alpine habitat, accounting for ecotypic differentiation based on detailed knowledge of population genetic differentiation (Knotek et al., 2020). We used six foothill and six alpine populations, representing three independent evolutionary origins of an alpine ecotype, for which other phenotypic traits have already been linked to a fitness advantage in their local habitat (Wos et al., 2022). In line with previous studies, we used a reciprocal transplantation experiment at elevations where the alpine and foothill A. arenosa ecotypes occur, to test whether alpine populations show heritable and consistent changes in cuticle eco-physiological traits compared to foothill ones. We also compared the composition of cuticular waxes to determine whether biochemical changes could explain the ecotypic differentiation. Furthermore, to elucidate the genetic basis of cuticle trait differentiation in A. arenosa, we re-analysed available genomic data (Bohutínská et al., 2021, 2023; Wos et al., 2021), combining selection scans and differential gene expression analysis between foothill and alpine populations, focusing on a set of known cuticle-related genes.