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

Silicon (Si) is abundant in soils and taken up by plant roots as non-charged silicic acid, with accumulation ranging from 0.1% to 10% of shoot dry weight depending on species. High Si accumulation is characteristic of many primitive land plants and several angiosperms, notably grasses (Gramineae) and sedges (Cyperaceae), and confers multifaceted protection against abiotic and biotic stresses. Rice (Oryza sativa) is a high Si accumulator (up to ~10% shoot DW), where Si deposition supports yield stability by mitigating damage from pathogens, pests, drought, salinity, metal toxicity, lodging, and nutrient imbalances. In rice roots, Si uptake is mediated by the influx transporter Lsi1 and the efflux transporter Lsi2, polarly localized to distal and proximal sides of exodermal and endodermal cells, respectively, enabling symplast–apoplast cycling and delivery to the stele. After uptake, Si is rapidly loaded into the xylem primarily via Lsi2 and Lsi3 (a pericycle-localized Lsi2 homolog), transported as silicic acid, and unloaded in shoots by Lsi6 (an Lsi1 homolog) at xylem parenchyma cells. In leaves, silicic acid concentrates with transpiration and polymerizes into amorphous silica, forming silica cells/bodies and silicified bulliform cells, and also deposits beneath the cuticle to form cuticle–silica double layers. Although cell-type-specific Si deposition is well documented, its molecular control remained unclear. This study identifies and functionally characterizes SIET4 (Silicon Efflux Transporter 4) as a key transporter required for proper, cell-specific Si deposition in rice leaves and, consequently, for healthy growth in the presence of Si.

Prior work established the cooperative Si transport system in rice: Lsi1 (influx) and Lsi2 (efflux) in root exodermis/endodermis enable uptake and radial transfer; Lsi3 (Lsi2 homolog) mediates efficient xylem loading in pericycle; Lsi6 (Lsi1 homolog) unloads Si from xylem in leaf sheaths/blades with polar localization. These transporters’ expression, localization, and polarity underlie interspecific differences in Si accumulation. Si deposition forms silica cells/bulliform cells and cuticle–silica double layers and has been linked to stress resilience. Overexpression of Lsi1 in non-accumulator Arabidopsis increases Si uptake but causes ectopic Si deposition, necrotic lesions, and growth inhibition when Si is present, underscoring the importance of proper deposition. Phylogenetically, Lsi2/SIET-like transporters occur broadly across land plants (except gymnosperms and Hepatopsida), suggesting an early acquisition in terrestrial adaptation, with functional diversification into Lsi2/Lsi3-like and SIET-like subgroups in graminaceous plants.

• Plant materials and growth: Wild-type rice (cv. Nipponbare) and two independent SIET4 knockout lines (siet4-1, siet4-2; T3/T4) were grown in greenhouse conditions (25–30 °C). Soil culture used field soil (Si in soil solution 0.6–0.8 mM); hydroponics used half-strength Kimura B with 0 or 1 mM silicic acid; solutions changed every 2 days. Dose-response used river sand pots with graded Water Silica applications to modulate solution Si.
• CRISPR/Cas9 gene knockout: Two ORF targets upstream of PAM were selected for SIET4; constructs (pU6gRNA and pZDgRNA_Cas9ver.2_HPT) transformed into rice callus (Agrobacterium-mediated). Mutants genotyped by PCR and sequencing; two independent lines without Cas9 (1-bp insertions in exon 1 or 2) selected.
• Phenotyping: Growth monitored to maturation; biomass (dry/fresh weight) recorded. SPAD (chlorophyll) measured on fully expanded youngest leaves. Visual leaf symptom documentation under +/- Si.
• Elemental analysis: Si in tissues determined after HNO3-H2O2-HF microwave digestion via molybdenum blue colorimetry; xylem sap Si collected after decapitation and measured similarly. Germanium (Ge) analog uptake assessed (5 µM GeO2) with ICP-MS; other mineral elements also quantified. Short-term Si uptake (0.5 mM silicic acid) measured by depletion assays, normalizing for water loss and tissue FW.
• Si deposition mapping: LA-ICP-MS on cross-sections of leaf blades after 6 days in 1 mM Si to visualize spatial Si distribution. SEM-EDX (surface mode, 5 kV) quantified Si/C ratios on leaf surfaces after 4, 8, 24 h exposure to 1 mM Si.
• Transport assay: Full-length SIET4 and Lsi2 expressed in E. coli (β-pET-28a(+)-β), purified, and reconstituted into proteoliposomes. Si transport measured by ICP-MS under pH gradients (outside 7.5 vs inside 6.0), with/without CCCP (2 µM) or Ge (2 mM); time-course and dose-response performed at pH 6.0 or 7.5.
• Gene expression: Organ- and leaf-age-dependent SIET4 expression assessed by qRT-PCR (Histone H3/Actin/Ubiquitin controls). Si responsiveness tested after 1–2 days of 1 mM Si.
• Localization: Tissue/cellular localization via immunostaining with SIET4-specific antibody on cross-sections and longitudinal sections of leaf blade/sheath in young and mature leaves under +/- Si. Subcellular localization by double staining with ER marker (HDEL) and DAPI; confocal microscopy used.
• Transcriptomics: RNA-seq (DNBSEQ-G400FAST, 2×150 bp; 30–40M reads/sample; n=3) on leaf blades of WT and siet4-1 treated +/- 1 mM Si for 24 h. Reads mapped to IRGSP-1.0; differential expression called (>2-fold, FDR<0.05). GO enrichment via PANTHER 17.0.
• Phylogenetics: Lsi2/SIET homologs from 55 species retrieved (BLAST), aligned (ClustalW), and analyzed by Maximum Likelihood (MEGA X) with 1000 bootstraps.
• Statistics: Student’s t test or ANOVA with Tukey’s test (BellCurve for Excel).

• SIET4 is essential for rice growth in the presence of Si: SIET4 knockout lines (siet4-1, siet4-2) displayed severe growth inhibition and death in soil and hydroponics with 1 mM Si, but grew similarly to WT without Si. In hydroponics with Si, mutant root and shoot dry weights were <10% of WT; mutants failed to set seed. Time-course with 1 mM Si showed mutant fresh weight at day 45 was ~22% of WT. Leaf symptoms under +Si included white spots, chlorosis, twisting; SPAD decreased by ~25% in mutants vs WT under +Si, with no difference under -Si.
• SIET4 affects Si deposition, not uptake: Shoot Si concentrations were similar between WT and mutants in soil and hydroponics with Si; xylem sap Si and short-term uptake did not differ, nor did Ge accumulation or other minerals, indicating SIET4 is not required for uptake or xylem loading.
• Aberrant Si deposition in mutants: LA-ICP-MS showed WT Si localized to apoplastic regions at the leaf surface and bulliform cells, avoiding mesophyll. Mutants had reduced surface Si and abnormal mesophyll Si deposition. SEM-EDX quantified significantly lower Si/C ratios on mutant leaf surfaces after 4–24 h in Si compared to WT.
• SIET4 is a proton-gradient-driven Si efflux transporter: Proteoliposomes with SIET4 transported Si under an outward pH gradient (outside 7.5 vs inside 6.0), similar to Lsi2; activity was abolished without pH gradient, reduced by CCCP, and inhibited by 2 mM Ge. Transport increased over time (saturating ~2 min) and with external Si concentration; higher at pH 7.5 than 6.0 across concentrations.
• Expression and localization: SIET4 is expressed in all organs, higher in leaf sheath/blade at vegetative stage and in nodes, rachis, peduncle, spikelet, and husk at reproductive stage. Expression in shoots was constitutive and not Si-responsive. Immunostaining localized SIET4 to epidermal cells and cells neighboring bulliform cells in young and mature leaves, with polar localization toward the distal side and facing bulliform cells; localization unaffected by Si supply. Subcellular localization was mainly plasma membrane with partial ER overlap.
• Transcriptomic changes indicate stress responses in mutants: After 24 h +Si, WT had 297 up- and 251 down-regulated genes, whereas siet4-1 had 1250 up- and 793 down-regulated genes. Mutant-specific upregulated genes were enriched in stress-related GO terms (response to chitin, osmotic stress, wounding, fungus, regulation of stress responses), while downregulated genes were enriched for metal ion homeostasis, ROS metabolism, and cellular detoxification, consistent with Si mis-deposition triggering broad stress pathways.
• Evolutionary context: Lsi2/SIET homologs are present in most land plants (monocots, dicots, Polypodiophyta, Lycopodiophyta, Sphenopsida, Bryophyta) but absent in gymnosperms, Hepatopsida, and algae. Two subgroups emerged: Lsi2-like (including Lsi2/Lsi3 and homologs in non-angiosperm lineages) and SIET-like (including SIET3–5 in monocots and all dicot homologs). Both subgroups co-occur only in graminaceous plants, suggesting functional diversification supporting high, properly localized Si accumulation.

The study addresses how rice achieves cell-specific Si deposition necessary for leveraging Si’s protective benefits without incurring toxicity. SIET4 is demonstrated to export silicic acid from specific leaf cells (epidermal and cells adjacent to bulliform cells) into the apoplastic space for proper deposition at the leaf surface and bulliform cells. Loss of SIET4 does not impair total Si uptake or xylem transport but disrupts deposition, causing silicic acid to accumulate within mesophyll cells where polymerization and associated metabolic disturbances likely ensue. This mis-localization triggers widespread biotic and abiotic stress-like transcriptional programs and results in severe growth inhibition and death in the presence of Si. The findings reconcile previous observations that ectopic Si accumulation/deposition can be deleterious (e.g., Lsi1 overexpression in Arabidopsis) and highlight the necessity of spatially controlled efflux in Si-rich environments. Phylogenetic analyses suggest that diversification of the Lsi2/SIET family in grasses allowed specialized roles: Lsi2/Lsi3 for root uptake and long-distance transport, and SIETs (including SIET4) for precise accumulation/deposition in shoots. Thus, SIET4 is a critical component of the Si utilization network enabling healthy growth on land.

This work identifies SIET4 as a plasma membrane Si efflux transporter with polar localization in leaf epidermal and bulliform-adjacent cells, required for exporting Si to the apoplast for proper, cell-specific deposition. SIET4 knockout causes aberrant mesophyll Si accumulation, extensive stress-related transcriptional changes, and plant death under Si presence, while not affecting overall Si uptake or xylem loading. The study refines the model of Si transport in rice by adding a deposition-specific efflux step and suggests functional specialization within the Lsi2/SIET family in grasses. Future directions include testing whether analogous deposition-specific efflux mechanisms operate across diverse land plants, dissecting regulatory factors governing SIET4 polarity and expression, and exploring the roles of other SIET family members in tissue-specific Si deposition.

The study focuses on rice and does not experimentally assess SIET4 homolog function in other species; whether deposition-specific Si efflux is a common strategy in land plants remains to be investigated. While localization and transport assays support SIET4’s role, the precise molecular regulators of its polar localization and the downstream cellular consequences of mesophyll Si accumulation were not elucidated.