Abstract
Aluminum (Al) toxicity in acid soils significantly affects plant growth, agricultural productivity and ecosystem health. Here we investigated plant Al tolerance from evolutionary physiological, molecular, and ecological perspectives. Genetic similarity and phylogenetic analysis of Al tolerance-associated gene families showed that many of these were conserved from streptophyte algae to angiosperms, indicating land plants have evolved gradually in adaptation to Al-rich acid soil during plant terrestrialization. In particular, vacuolar phosphate transporter SPX-major facility superfamily (SPX-MFS) and inorganic phosphate (Pi) transporter 1 subfamily (PHT1s) of streptophyte algae showed higher genetic similarity to land plants than chlorophyte algae. PHT1 subfamily exhibited a significant expand during the evolution from streptophyte algae to liverworts and then to eudicots. Moreover, we identified an Al-tolerant Tibetan wild barley accession XZ29, showing high levels of Phosphorus(P)-containing glycolytic intermediates under Al stress. We found a new Al-tolerance mechanism that Al-induced Pi efflux from root elongation zone to chelate rhizosphere Al3+ and immobilization of Al with P reduce Al accumulation in barley root cells. These results indicated that Tibetan wild barley has evolved unique P transport and metabolism for the adaptation to harsh conditions in eastern and southeastern Tibet where acid soils contain high P.
Key words: acid soil, gene family evolution, Hordeum spontaneum , phylogenetic analysis, phosphate metabolism, phosphate transporters 1. Introduction
Aluminum (Al) is the most abundant metal in Earth’s crust and Al toxicity is a major factor limiting crop production especially in acid soil, which cover almost 40% of the world’s arable land (Kochian, Pineros, Liu, & Magalhaes, 2015). Phosphorus (P) is an important macro-nutrition for all living organisms, and functions as a substrate in many metabolic processes such as glycolysis. P deficiency in soil causes significant stress to plants and limiting global food production (Runge-Metzger, 1995). Inorganic phosphate (Pi) can bind with Al to format Al-P complex (Zheng et al., 2005). P availability is dramatically reduced in acid soils because it forms sparingly soluble complexes with Al, thus P deficiency and Al toxicity usually coexist in acid soils (Kochian, Hoekenga, & Pineros, 2004). Soil P and Al have significant ecological and evolutionary implication for the evolution of plant nutrient uptake and crop domestication for the adaptation to acid soils (Liang et al., 2013).
Despite the importance of crop Al tolerance for sustainable food production and human health, there is limited investigation on the evolution of plant Al tolerance. Streptophyte algae, the sister lineage of land plants, have gradually evolved and equipped with new innovations in gene families for the adaptation of green plants to terrestrial habitats, including Al-rich acidic soils (Taylor et al., 2000; Nishiyama et al. 2018; Leebens-Mack et al. 2019; Zhao et al. 2019; Wang et al., 2020). Herburger, Remias, & Holzinger (2016) reported that a streptophyte alga Zygogonium ericetorum (Zygnematophyceae, Charophyta) has evolved the capability of Al tolerance by binding Al with pectin-rich parental cell wall matrix, thereby reducing Al accumulation in unpigmented filaments. It is thus suggested that acquisition of Al tolerance by modification of cell wall components (Yang et al., 2011; Zhu et al., 2012) may be originated from streptophyte algae. On the contrary, Al-induced organic acids secretion was mostly reported in seed plants, but not in lower plants (Yang, Fan, & Zheng, 2019) while the vital roles of P in plant nutrition and stress tolerance have been conserved across green plants (Lambers et al., 2015; Prodhan, Finnegan, & Lambers, 2019). Moreover, key P metabolism, Pi transporters SPX-MFSs (Syg1/Pho81/XPR1 with Major Facilitator Superfamily) and PHT1s (phosphate transporter 1 family) have been implicated to function in plants Al tolerance (Cardoso, Pinto, & Paiva, 2019; Rae, Cybinski, Jarmey, & Smith, 2003; Wang et al., 2012; Wang et al., 2015). However, the evolutionary origin of P metabolism and Pi transporters and their roles in Al tolerance is still elusive.
Micro-molar concentrations of Al3+ can rapidly inhibit root elongation, leading to reduction of water and nutrient uptake, and biomass production and yield reduction (Jones, Blancaflor, Kochian, & Gilroy, 2006). Some of the key mechanisms conferring this tolerance include the Al-induced release of organic anions from roots (Ma, Ryan, & Delhaize, 2001; Sasaki et al., 2004; Wang et al., 2007), the modification of cell wall chemistry to reduce accumulation in the apoplast (Yang et al., 2011; Zhu et al., 2012), and phytohormone mediated inhibition of root growth under Al stress (Yang et al., 2014, Yang et al., 2017). Application of phosphate alleviated Al toxicity by reducing Al3+ availability and stimulating Al tolerance genes (Jiang, Tang, Zheng, Lie, & Chen, 2009; Liang et al., 2013; Sun, Shen, Zhao, Chen, & Dong, 2008). Immobilization of Al by P in the cell wall conferred higher Al tolerance in buckwheat (Zheng et al., 2005). Although the interactions between P and Al in plants have been found (Chen et al., 2012; Du et al., 2009; Liang et al., 2013), the underlying mechanisms are still not fully understood.
The center of barley origin and domestication is in the Fertile Crescent (Nevo, Baum, Beiles, & Johnson, 1998; Dai et al., 2012), where soils are largely saline with higher pH (IGBP-DIS, 1998). Wild barley in Tibet (Hordeum spontaneum ) is well adapted to the harsh conditions of the Tibetan plateau, where large areas of the soil is acidic and contains high P content (IGBP-DIS, 1998; Wang, Yang, & Ma, 2008). Tibetan wild barley is one of the progenitors of the modern cultivated barley, possessing a wider genetic diversity and a larger variation to abiotic stress tolerance than cultivated barley (Dai et al., 2012; Dai et al., 2014; Wu et al., 2013; Feng et al., 2020). By screening more than one hundred wild barley genotypes, we identified accessions with similar tolerance to Al toxicity as Dayton, a well-known Al-tolerant cultivar (Cai et al., 2013; Dai et al., 2011). Two novel loci bpb-9458 (Chromosome 2H) and bpb-8524 (Chromosome 7H) were linked with Al tolerance in Tibetan wild barley (Cai et al., 2013). These results suggested that the mechanisms controlling Al tolerance are likely to be different between Tibetan wild barley and modern barley cultivars.
It was thus hypothesized that unique P metabolism and transport facilitate Tibetan wild barley’s adaptation to low pH and high P soil during the evolution. In this study, we have combined evolutionary bioinformatics (Zhao et al., 2019), metabolomics (Urano, Kurihara, Seki, & Shinozaki, 2010), confocal microscopy (Klug, Specht, & Horst, 2011), secondary ion mass spectrometry (SIMS) (Kopittke et al., 2015), and electrophysiology (Newman, 2001) to investigate the Al tolerance mechanisms in Tibetan wild barley. We found that most of Al tolerance-related genes, including genes encoding Pi transporters and glycolysis, were conserved from streptophyte algae to angiosperms. Tibetan wild barley has developed an Al tolerance mechanism based on P-related metabolites and gene expression and phosphate efflux from the root apices to chelate toxic Al3+.