Aluminum localization in tissues of Eriophorum vaginatum and its effect on root growth and recovery

Highlights

• We mapped Al distribution in Al-tolerant E. vaginatum using confocal microscopy.

• We identified a potential mechanism to sequester Al in E. vaginatum tissues.

• We recorded stimulatory and adverse effects of Al on E. vaginatum root growth.

• E. vaginatum should be investigated to phytoremediate metal polluted wetlands.

Abstract

Previous observations of Eriophorum vaginatum L. growing in its natural habitats suggest that this perennial graminoid is highly tolerant of aluminum (Al). Eriophorum vaginatum is unusually productive in the bogs surrounding Sudbury (Ontario, Canada) where many years of intensive mining and industrial activities resulted in strong acidification of the environment and wide-scale deposition of metals including highly phytotoxic Al, which in acidic soils is readily available for biological uptake. The ability to accumulate Al within the plant body might be the way of detoxification and can play an important role in Al tolerance by E. vaginatum. To detect Al distribution in leaves, corms and roots of E. vaginatum, plants were collected from a highly polluted site and the fluorescence intensity from the Al-lumogallion complex was observed using a confocal laser scanning microscopy (CLSM). A strong fluorescence signal was detected, demonstrating dense Al accumulation mainly within the cell walls of the analysed plant tissues. It suggests that E. vaginatum can sequester the phytotoxic Al away from the metabolically active tissues so it cannot interfere with the normal cellular activity. Additionally, a hydroponics study was conducted to examine Al effects on root growth and recovery using in vitro regenerated E. vaginatum plants not previously exposed to Al. Root growth was stimulated by 5 and 10 µM Al, but was inhibited by 50 and 100 µM Al. However, all roots returned to control growth rates within 48 h after being transferred to Al-free media, regardless of initial exposure concentration. Based on the ability of this species to tolerate and sequester Al, our results support investigation into E. vaginatum to phytoremediate metal polluted wetlands.

Introduction

Highly toxic to plants, aluminum (Al) is the most abundant metal and third most abundant element in the Earth's crust. High level of soil pollution with Al, caused by either natural processes or by human activities, is one of the most serious environmental problems with regards to plants that are exposed to it (Dos Reis et al., 2018). At higher pH, Al is immobilized in clay minerals, bound by ligands or occurs in other non-phytotoxic forms such as aluminosilicates and precipitates. However, under acidic conditions Al becomes soluble, and readily available for biological uptake, being extremely toxic to plants even at submicromolar levels (Achary et al., 2013; Yu et al., 2019).

Aluminum is one of the most important factors to restrict plant growth, mainly by inhibiting root growth and interfering with the uptake and transport of water and nutrients (Rodrigues et al., 2017; Dos Reis et al., 2018; Riaz et al., 2018b). However, despite the negative growing conditions, some native species have developed compensatory mechanisms that allow them to successfully colonize and vigorously grow in acidic, metal polluted soils (Aihemaiti et al., 2017; Guarino et al., 2019; Guterres et al., 2019). Recently, there is considerable interest in such plants to understand their Al tolerance mechanisms and their potential use in the process of revegetation and reclamation of toxic metal contaminated environments (Ha et al., 2019; Rossini-Oliva et al., 2019; Afonso et al., 2020).

Native species that are well adapted to high levels of Al in soil are capable of removing excess metal ions from the cytosol to prevent vulnerable tissues from experiencing the effects of metal toxicity (Arroyave et al., 2018; de Souza et al., 2018a; Tahara et al., 2018; Muhammad et al., 2019). Although Al has been shown to be a toxic metal, the molecular mechanism of its toxicity to plants is still not well understood. In general, tolerance presented by Al-resistant species may be the result of two mechanisms, the exclusion of the metal from the tissues and / or accumulation within the plant body in a chemically inert form (Singh et al., 2017; Arroyave et al., 2018). Exclusion is often accomplished by the secretion of organic acids, e.g. oxalate, citrate or malate by roots to externally or internally neutralize Al and other phytotoxic metals. It has been suggested that Al exclusion from roots and leaves is an important mechanism of plant resistance to Al (Valentinuzzi et al., 2016; Yang et al., 2020). In contrast, Al-accumulator plants have developed mechanisms such as the detoxification of Al ions by producing Al-ligands and other non-phytotoxic complexes, or by compartmentalization to alleviate Al toxicity in their tissues (de Souza et al., 2018a; Fan et al., 2020).

Cotton-sedge (Eriophorum vaginatum L.) can be described as an Al-tolerant species. Eriophorum vaginatum is a perennial sedge, forming tussocks, native to circumpolar habitats that range from nutrient limited peatlands of the boreal forest to wet sedge tundra of the Arctic (Chapin III et al., 1979; Souther et al., 2014; Schedlbauer et al., 2018). Numerous traits have been identified that enhance the competitive ability and allow E. vaginatum to thrive in cold, relatively infertile environments. Several leaves remain green over the winter months and are capable of photosynthesis at first opportunity in the early spring. New tiller formation involves the sequential development and senescence patterns of leaves during the growing season, and expansion occurs at the expense of nutrients that are translocated from the older leaves. A very efficient vascular system allows for rapid transport and deposition of the solutes within the developing leaves (Cholewa and Griffith, 2004). Additionally, E. vaginatum is one of the very few native plant species that can survive in highly Al-contaminated wetlands, such as those near Sudbury (Ontario, Canada) (Szkokan-Emilson et al., 2014), where over 100 years of intensive mining, roasting and industrial activities have extensively damaged local ecosystems. Terrestrial plant communities in the Greater Sudbury area have been and continue to be impacted not only by metals in soil, but also by soil erosion, low nutrient levels, lack of soil organic matter, and / or low soil pH (Warren et al., 2015; Meyer-Jacob et al., 2020). Eriophorum vaginatum thriving in the contaminated wetlands suggests that this species possesses an innate resistance to local pollutants.

So far very little is known about how Al-resistant E. vaginatum ameliorates the toxic effects of this metal persistent in polluted environments, including where exactly Al is located in the plant body. While the signs and effects of Al toxicity on plants were already widely described (Hussain et al., 2018; Liu et al., 2018), the accurate tracing of the metal within the tissues for a period of time has remained problematic, mainly because there is no useful radioisotope of Al. Classic stains and probes like morin or haematoxylin seem to be not sensitive enough to detect trace amounts of Al in plant tissues (Qian et al., 2016; Böhlenius et al., 2018). This has limited accurate observations of the metal distribution at cell and tissue level for a long time. Precise observation of Al present within plant tissues has been revolutionized recently with the use of the highly sensitive fluorescence dye, lumogallion. Lumogallion forms a strong, fluorescent complex with both bound and free Al ions, which can be qualitatively detected with a confocal laser microscope using an excitation wavelength of 488 nm and an emission ranging from 520 to 580 nm (Riaz et al., 2018a, 2019; Silva et al., 2000).

The objectives of this study were to visualize the distribution of Al within leaves, corms and roots of E. vaginatum by using lumogallion staining and confocal laser microscope to understand the existing mechanisms of resistance used by this species in response to Al toxicity. As a secondary objective, the effect of Al accumulation on root growth inhibition and recovery of E. vaginatum was determined.