High concentrations of Zn can be harmful to plants. Zn toxicity inhibits root growth and affects nutrient absorption in plants (dos Santos et al. 2020). Our results showed that NHE were dwarfed with short roots under high Zn stress (Fig. 1a–b), which was consistent with the phenotype of plants after Zn poisoning. However, in HE roots under high-Zn treatment for 7 d, the total biomass, total root length, surface area, volume, number of lateral roots, and length of lateral roots increased. Our results indicated that high-Zn treatment promoted the growth of HE of S. alfredii roots and shoots.
Previous studies have also reported that high Zn levels can increase the biomass of HE shoots, especially by promoting the rapid growth of new shoots (Long et al. 2006). Li et al. reported that high Zn concentrations were beneficial for HE growth, and 500 µM Zn induced a significant increase in root biomass (Li et al. 2008). This is a very interesting phenomenon, indicating that HE not only has a high tolerance to Zn but may also have high demands for Zn. It has been suggested that S. alfredii has evolved root foraging mechanisms associated with greater Zn requirements (Liu et al. 2010).
High Zn promotes HE growth through transport, compartmentalization, and regulation of auxin anabolism
Nano-XRF analysis showed that Zn was located in the cortical intercellular space of HE root cells to avoid excess Zn toxicity, whereas in NHE, Zn was scattered in the root cells (Fig. 5). The data indicate that more Zn was retained in NHE root cells, whereas HE transported Zn to the shoots. Previous studies have shown that HE root cells induce cell wall methylation and hemicellulose modification under Zn stress (Guo et al. 2020; Guo et al. 2020), which promotes root cell wall analysis and xylem transport of Zn to the shoots quickly and then participates in phloem remobilization transport (Lu et al. 2013). Previously, it has been observed that the epidermal layers serve as important storage sites for the accumulation of Zn in the HE of S. alfredii (Tian et al. 2009). These results are consistent with those of Dinh et al. who reported the Zn hyperaccumulator Noccaea caerulescens (Dinh et al. 2015). However, in NHE, a large amount of Zn is adsorbed on the cell wall or stored in vacuoles (Yang et al. 2006) and is not transported to the shoots. Plants can weaken the toxicity of heavy metals by accumulating heavy metals in their cell walls (Wang & Liu 2013).
Zn is an essential substance in the tryptophan-dependent auxin biosynthesis pathway (Wang et al. 2012), and auxin plays an important role in the development of lateral roots, affecting various stages of lateral root development (Motte et al. 2019). Our data showed that the auxin content of HE roots increased and the auxin oxidase activity decreased rapidly reducing the degradation of auxin under high-Zn treatment, while NHE roots exhibited the opposite effects. The effect of high Zn concentrations on cytokinin in HE and NHE roots was consistent with that of auxin (Fig. 7). Thus, the high Zn content in HE can effectively promote plant growth and root development by regulating the anabolism of auxin and cytokinin. A recent study found that excess Zn increases the expression of auxin biosynthesis-related genes and results in auxin accumulation in the root tips (Zhang et al. 2018). Studies have found that auxin may also affect the process of Zn absorption by plants. Zn deficiency can increase the expression of the Zn transport-related genes OsIRT1, OsZIP4, and OsZIP1 in rice roots; however, these genes were significantly downregulated after blocking the auxin signal, indicating that auxin is involved in the absorption and transport of Zn in rice when Zn is deficient (Begum et al. 2016).
In summary as to how Zn promotes the growth of HE plants, HE reduces the inhibitory effect of excessive Zn accumulation on root cells through efficient transport to the shoots and root cell apoplast compartments. In contrast, Zn accumulation in HE roots regulates the anabolism of growth hormones and effectively promotes lateral root germination and root growth.
HE of S. alfredii suffers adversity under low Zn conditions while it prospers under high Zn conditions
KEGG analysis of HE root cells revealed that the upregulated DEGs were mainly enriched in the regulation of carbohydrate metabolism, nitrogen metabolism and carbon fixation, and tryptophan biosynthesis. The predicted functions of the upregulated DEGs were related to germin-like proteins (GLPs), auxin response factor (ARF), tryptophan (Trp) metabolism, sugar transport proteins, ammonium (AMT) and urea transporters (UAT) (Fig. 10). Various germin-like proteins are known to be involved in the growth and development of root tissues (Dunwell et al. 2008). Overexpression of Arabidopsis Plasmodesmata germin-like proteins reduces primary root growth but enhances lateral root growth by regulating the phloem-mediated allocation of resources between the primary and lateral root meristems (Ham et al. 2012). Auxin biosynthesis via the tryptophan aminotransferase of arabidopsis (TAA)/YUCCA (YUC) route involving tryptophan aminotransferases and YUC flavin-dependent monooxygenases that produce the auxin indole-3-acetic acid (IAA) from tryptophan is currently the most researched auxin biosynthetic pathway (Poulet & Kriechbaumer 2017). Tryptophan aminotransferases (TAA1/TARs) catalyze the first step in the conversion of L-tryptophan to indole-3-pyruvic acid (Kakei et al. 2017). The auxin response factor (ARF) family functions by binding to auxin response elements (AuxREs) on the promoters of auxin response genes, activating or repressing them, leading to plant growth (Guilfoyle & Hagen 2007). Sugars are essential carbohydrates generated by photosynthesis in source leaves and are then transferred through the phloem to sink organs such as seeds, fruits, stems, tubers, and swollen taproots (Wen et al. 2022). Sugar-transport proteins play a crucial role in the cell-to-cell and long-distance distribution of sugars in plants (Williams et al. 2000). Ammonium transporters can transport ammonium from the roots to the shoots to mediate the acquisition of ammonium nitrogen from the soil, which is essential to meet the nitrogen demand of plants (Hao et al. 2020).
The downregulated DEGs were mainly involved in the regulation of oxidative phosphorylation in energy metabolism, steroid and fatty acid biosynthesis in lipid metabolism, and linoleic acid metabolism. These metabolic processes are associated with plant stress responses. Under Zn-deficient conditions, resistant plants can reduce this damage via lipid remodeling (Ruelland et al. 2015). Fatty acids play an important role in plant plasma membrane stability, stress resistance, tissue senescence (Rontani et al. 2005), and seed development (Smith et al. 2003). The predicted functions of the downregulated DEGs were also related to plant stress responses. The wall-associated kinase (WAK) gene family, a receptor-like kinase gene family in plants, plays important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance in Arabidopsis thaliana (Zhang et al. 2005). Polyamines not only play roles in plant growth and development but also in adaptation to environmental stresses. Previous studies have shown that maize polyamine oxidase (PAO) plays a role in cell wall maturation and root differentiation by producing H2O2 (Cona et al. 2005; Rea et al. 2004). Gomez-Jimenez et al. reported that PAO and DAO have significant functions in olive fruit abscission zone (AZ) development by providing apoplastic H2O2 for cell wall strengthening and lignosuberization events, and that a peroxidase substrate is provided in these cells throughout AZ development (Gomez-Jimenez et al. 2010). Plant MAPK cascades regulate numerous processes, including various environmental stress responses, hormones, cell division, and developmental processes. Jasmonic acid (JA) and its methyl ester (MeJA) are plant signaling compounds involved in regulating stress responses and development. Several studies have revealed that some MAPK cascades are involved in the wound/JA signaling pathway (Kumar et al. 2013). Serine/arginine-rich (SR) proteins are conserved RNA-binding proteins that play a major role in RNA metabolism. Stress signals control both the phosphorylation status and subcellular localization of plant SR proteins, indicating potential mechanistic links between abiotic stress and the regulation of alternative splicing (Duque 2011).
This indicates that there are two reasons for the growth-promoting effects of high Zn levels on HE. Upregulated key genes, such as auxin synthesis, carbohydrate synthesis and metabolism, and amino acid metabolism, provide an energy basis for the growth and development of roots. In contrast, the downregulated genes were related to Zn deficiency stress, implying that high Zn levels alleviated this stress in HE roots. This indicates that HE suffers adversity under low-Zn conditions and exhibits a stress response, whereas high Zn provides conditions for HE to prosper (Fig. 11).
In conclusion, this study highlights the positive effect of high Zn concentrations on the growth of the Zn/Cd hyperaccumulator Sedum alfredii Hance. At a 250 µM Zn level, HE root biomass and Zn concentration in roots increased while those in NHE were reduced. Nano-XRF analysis showed that Zn was located in the cortical intercellular space in HE roots, whereas Zn was scattered in the root cells of NHE. The auxin content in HE roots was increased and the auxin oxidase activity decreased after exposure to 100 µM Zn, while NHE exhibited the opposite effects. To adapt to high Zn conditions, HE cells upregulated genes related to carbohydrate metabolism, nitrogen metabolism, carbon fixation, and tryptophan biosynthesis, and downregulated genes related to lipid metabolism and linoleic acid metabolism. We further screened key gene resources, laying a foundation for the molecular mechanism underlying the positive effect of high Zn conditions on HE growth. This study provides a theoretical reference for the optimization of phytoremediation technologies for heavy metal-contaminated soil.