4.1 Physiological response of I. tectorum to Cr stress
Chromium hardly participates in metabolic function of plants, but it is potentially toxic and can adversely affect its morphophysiological properties and antioxidant defense mechanisms (Shahid et al., 2017). In the control group, Cr content in iris roots was significantly lower than that in leaves (Figure 1b and c). Because roots tend to Cr accumulation and transport as a result of preferential contact to heavy metals when plants grow in Cr-contaminated soil (Zong et al., 2020). After exogenous addition of Cr, the content of Cr in the roots and leaves of iris was significantly higher than that in the control group (Figure 1b and c). This is because wetland plants can effectively purify the Cr-contaminated soil and fix Cr in their own cell walls and vacuoles, thus reducing the toxicity of Cr (Wang et al., 2021). Moreover, previous studies have shown that hemicellulose (HC-1) in plant cell walls binds heavy metals to the greatest extent under heavy metal stress, thus reducing the toxicity of heavy metals to plants. (Yuan et al., 2022).
Under Cr pollution, the significant increase in SOM (Figure 1a) could be attributed to the following three reasons: 1) Cr would compete with plants for sulfate channels, thus affecting the absorption of water and nutrients by plants (Islam et al., 2016). 2) Damage to plasma membrane integrity, resulting in damage to root tip cells and more organic matter transport to the soil (Singh et al., 2022). 3) It may also be related to dead bacteria in the soil, which is due to the inanimate bacterial biomass accumulating in the soil over time. Meanwhile, Cr stress-sensitive bacterial communities provides the soil with a large amount of organic matter (Sokol et al., 2022). From the above results, we can see that Cr stress has a certain amount of influence on plants and rhizosphere bacterial communities, but iris can still enrich Cr in large quantities in extreme environments. Therefore, it can be seen that iris has its own advantages in Cr removal.
4.2 Effects of Cr stress on soil bacterial community structure
Under Cr stress, it is very important to study the complex bacterial community structure and species diversity in plant rhizosphere soil for understanding the regulation mechanism of the ecosystem itself (Vijay et al., 2017, Yu et al., 2021). In this study, exogenously added Cr reduced the α diversity index (Sobs, Shannon and Ace index) of bacterial community in rhizosphere of iris iris (Figure 2), because high concentration of Cr(VI) inhibited the nitrification and denitrification of bacterial community. This reduced the diversity and richness of bacterial communities (Sun et al., 2019). Moreover, under the action of reductase or reductive substances, Cr(VI) in cells will produce a large number of reactive oxygen species (ROS) in the process of reduction to Cr(III), which will cause DNA damage after binding with DNA, resulting in cell deformation, genetic variation and even death (Guo et al., 2020). This thus negatively affected the diversity and abundance of bacterial communities. In addition, the analysis of PCoA and NMDS showed that Cr stress significantly changed the spatial structure of bacterial community in the rhizosphere soil of iris (Figure 3a and b), which has also been confirmed in previous studies, such as Windmill Grass (Wang et al., 2021) and rice (Huang et al., 2021). In addition, PLS-DA (Figure 3 c) and Veen (Figure S3 a and b) analysis again proved that exogenous chromium supplementation significantly affected the structural composition and species number of bacterial communities. As can be seen from the above results, the addition of exogenous Cr significantly reduces the diversity of the rhizosphere bacterial community of iris and changes the structure of the rhizosphere bacterial community, causing damage to the microecology of I. tectorum.
4.3 Responses of rhizosphere bacteria to Cr stress and different cultivation patterns
Bioreremediation relies on the universality and diversity of bacterial communities, and responds to Cr stress through its own REDOX system, extracellular adsorption and efflux mechanisms (Guo et al., 2020, Joutey et al., 2015). In this study, it was found that Proteobacteria, Actinobacteria and Chloroflexi were significantly more abundant in the control group than in the blank group (CK) (Figure 4), which may be because root exudates (polysaccharides, phenolic compounds, flavonoids and organic acids), It provides more nutrients for the survival of the bacterial community (Figure 6), thus promoting the growth of bacteria (Winkel-Shirley et al., 2002). Under Cr stress, compared with the control group, the growth of Proteobacteria was not inhibited, but showed an upward trend (Figure 4). This is because Proteobacteria are Gram-negative bacteria, and the gram-negative cell envelope is composed of outer membrane (containing anion lipopolysaccharide, phospholipid and outer membrane protein) and peptidoglycan, which plays a key role in heavy metal binding (He et al., 2020). Moreover, bacteria possess polysaccharide slime layers,which readily offer amino, carboxyl, phophate and sulphate groups for metals binding (Munees et al., 2021). Compared with the control group, the growth of Actinobacteria, Chloroflexi, Acidobacteria and Gemmatimonadetes were restricted under Cr stress (Figure 4). Although these four dominant bacteria can alleviate heavy metal stress through strong secondary metabolism, more metabolic functions and self-regulation of energy supply (Sharma et al., 2021a). Actinobacteria as a typical Gram-positive bacterium exhibits remarkable Cr resistance, which plays a direct role in the reduction and removal of Cr (Ramesh et al., 2010).
Through species analysis between groups, it can be seen that Actinobacteria is the dominant species in sole-Cultivated pattern, but its abundance is lower than that of the control group. For two-cultivated and three-cultivated patterns, the abundance of Gammaproteobacteria, Gemmatimonadetes, Alphaproteobacteria and Actinobacteria the abundance was not only the highest, but also significantly higher than that of the control group. (Figure S5). This may be due to the redistribution of bacterial communities in the soil caused by human disturbances (i.e., different cultivation patterns) or root exudates of wetland plants (Figure 6, Li et al., 2019). The symbiosis network diagram showed that although the bacterial community structure among all samples is similar, the correlation between dominant bacterial communities is weak in sole-cultivated pattern and three-cultivated pattern, and the symbiosis pattern is relatively scattered (Figure 5a and c). This may be caused by the sole-cultivated pattern, the plant itself and its rhizosphere bacterial community have a weak ability to cope with chromium stress. In the three-cultivated pattern, a large number of plant roots and their exudates compete for soil microbial resources. This results in the dispersion of rhizosphere bacterial community, which makes the symbiotic relationship between bacteria weaker. In the two-cultivated pattern, the correlation between dominant species is significantly enhanced, the relationship between species is more close, and the symbiotic system is highly concentrated (Figure 5b). It mentioned in the study that environmental changes played a dominant role in the formation of plant rhizosphere bacterial communities (Hyeseon et al., 2022, Zhou et al., 2022).