以生理與轉錄體學分析探究 L-3,4-二羥基苯丙氨酸對水稻鎘累積之效應

Abstract

鎘（Cadmium, Cd）污染已成為全球農業生產與糧食安全的重要挑戰。水稻（Oryza sativa L.）為世界主要糧食作物之一，容易吸收並將鎘累積於稻米中，進而增加人體暴露風險。近年研究指出，天然存在之非蛋白質胺基酸 L-3,4-二羥基苯丙氨酸（L-DOPA）可活化 Arabidopsis thaliana 的缺鐵反應並提升其鎘耐受性，但其在水稻中的生理與分子作用機制仍未完全釐清。因此，本研究利用生理分析、元素定量分析、即時定量反轉錄聚合酶鏈反應（RT-qPCR）及轉錄體分析，探討外源性 L-DOPA 對水稻鐵恆定（iron homeostasis）與鎘累積之影響。

本研究以日本稻 (Oryza sativa ssp. japonica cv. Kitaake) 為材料，在半濃度 Kimura B 水耕培養液中培養幼苗，分別施用不同濃度之 L-DOPA（100、250 與 500 μM）、10 μM CdCl₂ 及其組合處理。評估植株生長、葉綠素含量、生物量、發育歷程及元素累積情形，並利用 RT-qPCR 與 RNA 定序分析探討相關基因表現與分子調控機制。

研究結果顯示，L-DOPA 對水稻生長具有明顯的濃度依賴性。在非逆境條件下，高濃度 L-DOPA 顯著抑制地上部及根部生長，但可提升相對葉綠素含量；在鎘逆境下，低濃度 L-DOPA 則可部分緩解鎘造成之根系生長抑制、生物量下降及發育延遲，而高濃度則表現出明顯毒害效應。長期栽培試驗亦顯示，中等濃度 L-DOPA 可促進正常條件下早期生殖發育，而低濃度 L-DOPA 可部分改善鎘處理下之發育延遲。此外，RT-qPCR 與轉錄體分析結果顯示，L-DOPA 可調控多個與鐵恆定及金屬運輸相關之基因表現，顯示其可透過調節鐵相關訊息傳遞與轉錄網絡影響水稻對鎘逆境之適應能力。

綜合而言，本研究證實 L-DOPA 為一種具濃度依賴性的植物調控分子。在適當濃度下，可部分減輕鎘毒害並調節鐵恆定及相關轉錄調控網絡。本研究不僅深化了鐵營養與鎘逆境交互作用之理解，也為未來利用天然植物訊號分子降低稻米鎘累積、提升污染土壤中水稻安全生產提供重要理論基礎。

Cadmium (Cd) contamination in agricultural soils poses a serious threat to crop productivity and food safety because rice (Oryza sativa L.), one of the world's major staple crops, readily accumulates Cd in edible grains. Recent studies have demonstrated that L-3,4-dihydroxyphenylalanine (L-DOPA), a naturally occurring non-proteinogenic amino acid, activates iron (Fe)-deficiency responses and enhances Cd tolerance in Arabidopsis thaliana. However, whether a similar mechanism operates in rice remains largely unknown. This study investigated the physiological and molecular effects of exogenous L-DOPA on Fe homeostasis and Cd accumulation in rice through physiological characterization, elemental analysis, reverse transcription quantitative PCR (RT-qPCR), and transcriptomic analysis.

Rice seedlings (Oryza sativa ssp. japonica cv. Kitaake) were grown hydroponically in half-strength Kimura B nutrient solution and treated with L-DOPA (100, 250, or 500 μM), CdCl₂ (10 μM), or their combinations. Plant growth, chlorophyll content, biomass accumulation, developmental progression, and elemental concentrations were evaluated. Gene expression analyses were performed using RT-qPCR and RNA sequencing to elucidate the molecular responses associated with L-DOPA and Cd treatments.

L-DOPA exhibited concentration-dependent effects on rice growth. Under non-stress conditions, high L-DOPA concentrations inhibited shoot and root growth while simultaneously increasing relative chlorophyll content. Under Cd stress, however, low L-DOPA concentrations partially alleviated Cd-induced reductions in root growth, biomass accumulation, and developmental delay, whereas higher concentrations exerted phytotoxic effects. Long-term experiments further demonstrated that moderate L-DOPA concentrations accelerated early reproductive development under normal conditions, while low L-DOPA partially mitigated Cd-induced developmental delays under hydroponic culture. Transcriptomic and RT-qPCR analyses further revealed that L-DOPA modulated the expression of genes associated with Fe homeostasis and metal transport, suggesting extensive transcriptional reprogramming in response to combined L-DOPA and Cd treatments. Together with elemental analyses, these findings indicate that L-DOPA influences physiological adaptation to Cd stress through modulation of Fe-related regulatory pathways.

Overall, this study demonstrates that L-DOPA functions as a concentration-dependent regulator of rice growth and Cd responses. At appropriate concentrations, L-DOPA partially alleviates Cd toxicity while modulating Fe homeostasis and associated transcriptional networks. These findings provide new insights into the molecular interactions between Fe nutrition and Cd stress and establish a foundation for developing environmentally compatible strategies to reduce Cd accumulation and improve rice production in contaminated agricultural systems.