A silicon transporter gene required for healthy growth of rice on land

Silicon (Si) is a beneficial element for many plants, particularly rice (*Oryza sativa*), where it enhances resistance to various stresses and contributes significantly to yield. Rice accumulates Si in its shoots, reaching up to 10% dry weight. This accumulation is mediated by a complex system of transporters: Lsi1 (influx) and Lsi2 (efflux) in roots, and Lsi6 (unloading from xylem) in shoots. Si is taken up as silicic acid, moves through the xylem, and is deposited as amorphous silica (phytoliths) in specific cells, primarily epidermal cells and cells surrounding bulliform cells. The precise molecular mechanisms governing this cell-specific deposition, however, remain unclear. This study aimed to identify and characterize a gene responsible for the proper accumulation and cell-specific deposition of Si in rice leaves and its contribution to overall plant health and growth.

Extensive research has explored the role of Si in plant biology. Its accumulation varies widely across plant species, with high levels observed in some primitive land plants and grasses. The benefits of Si accumulation include protection against various biotic and abiotic stresses, such as pathogens, pests, drought, salinity, metal toxicity, lodging, and nutrient imbalances. Previous studies identified Si transporters in rice and other species. Lsi1 and Lsi2 in rice roots mediate Si influx and efflux, respectively, facilitating Si transport from the root apoplast to the xylem. Lsi3, a homolog of Lsi2, also plays a role in xylem loading. Lsi6, localized in leaf sheaths and blades, is involved in Si unloading from the xylem. However, the mechanisms underlying cell-specific Si deposition in leaves remain largely unknown. The present study builds upon this existing knowledge to pinpoint a specific transporter responsible for this vital process in rice.

The researchers generated knockout lines of *SIET4*, a homolog of *Lsi2*, in rice using the CRISPR/Cas9 system. Two independent mutants, *siet4-1* and *siet4-2*, were characterized. The effects of Si on growth were assessed by comparing the mutants to wild-type (WT) rice in soil and hydroponic conditions, both with and without Si supplementation. Si accumulation in shoots and roots was determined using colorimetric methods. Si deposition patterns were visualized using Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) and Scanning Electron Microscopy-Energy Dispersive X-ray Spectrometry (SEM-EDX). The transport activity of SIET4 was investigated using the proteoliposome method. The expression pattern of *SIET4* was determined through quantitative real-time PCR (qRT-PCR) across various tissues and developmental stages and in response to Si supply. Immunostaining with a specific antibody was employed to study the tissue and cellular localization of SIET4. Finally, transcriptomic analysis (RNA-seq) compared gene expression profiles in WT and *siet4-1* mutants under Si-treated and untreated conditions. Phylogenetic analysis of Lsi2/SIET-like proteins across various plant species was also performed.

Knockout of *SIET4* resulted in severely inhibited growth and eventual death of rice plants when grown in the presence of Si in soil or hydroponic solution. Surprisingly, the mutants grew similarly to WT plants in the absence of Si. Si accumulation in the shoots of *siet4* mutants was similar to that of WT plants, indicating that *SIET4* does not directly affect Si uptake. However, the *siet4* mutants exhibited an altered Si deposition pattern, with a decrease in Si accumulation on the leaf surface and abnormal deposition in mesophyll cells. In WT, Si is predominantly accumulated in the apoplast of leaf surface and at bulliform cells (motor cells). Functional assays using proteoliposomes confirmed that SIET4 is an efflux transporter of Si, similar to Lsi2. *SIET4* showed constitutive expression across various organs and growth stages, with higher expression in leaf sheaths and blades. Immunostaining revealed that SIET4 is polarly localized at the distal side of epidermal cells and cells surrounding bulliform cells. Transcriptomic analysis revealed that Si treatment caused a dramatic upregulation of hundreds of stress-responsive genes in the *siet4* mutant compared to WT, suggesting that the abnormal Si deposition in mesophyll cells induces various stress responses, leading to plant death. Phylogenetic analysis indicated that Lsi2/SIET-like transporters are present in most land plants, except gymnosperms and Hepatopsida, suggesting their importance in Si adaptation in the terrestrial environment. Graminaceous plants possess both *Lsi2*-like and *SIET*-like subgroups.

This study provides compelling evidence that SIET4 is essential for healthy rice growth in Si-containing environments. Its role as an efflux transporter directing Si to the apoplast of specific leaf cells is crucial for preventing Si toxicity. The abnormal Si accumulation in mesophyll cells of *siet4* mutants triggers a cascade of stress responses, ultimately leading to plant death. The findings highlight the importance of precise Si deposition for efficient Si utilization and avoidance of toxicity. The results also suggest a functional differentiation of Lsi2/SIET transporters in graminaceous plants, with Lsi2/Lsi3 involved in Si uptake and long-distance transport, and SIETs in Si accumulation and deposition. The distinct presence of both subgroups only in graminaceous plants suggests a specialized mechanism for high Si accumulation in these species. This study deepens our understanding of Si transport and deposition in rice and highlights the importance of precise regulation for optimal plant growth and stress tolerance.

The research definitively establishes SIET4's crucial role in healthy rice growth by facilitating efficient silicon export and deposition in leaves. The findings underscore the importance of precise silicon localization and underscore the need for further exploration of Si transport mechanisms in other land plants, especially to determine whether this specific mechanism is conserved.

While this study provides strong evidence for SIET4's function, further research could explore the precise molecular interactions between SIET4 and other Si transport proteins. Additional studies could investigate the downstream effects of abnormal Si deposition in mesophyll cells at a more detailed level. The study focused primarily on rice; therefore, further investigations are warranted to determine the extent to which this specific mechanism is conserved across different plant species.