水稻擬磷酸轉運蛋白對於初生根之向地性探討

Abstract

水稻擬磷酸轉運蛋白 (pseudo-histidine phosphotransfer proteins, OsPHPs) 在細胞分裂素訊號傳遞中扮演負向調控的角色，但其在水稻初生根向地性反應中的功能尚不明確。本研究首先建立系統性分析根向地性反應的流程，並透過根生長方向圖及根彎曲程度指標，量化水稻初生根對重力刺激的外表型變化。結合外源性細胞分裂素 (trans-zeatin)、生長素 (IAA、IBA) 處理與基因型比較，深入探討 php1/2/3 三重突變株在多層次訊號整合中的調控作用。表型分析結果顯示，php1/2/3 三重突變株對重力刺激反應速度顯著加快以及根彎曲程度下降，證實 OsPHP1/2/3 具有調節根向地性反應的能力。分子層級證據進一步顯示，OsPHP1/2/3 缺失導致細胞分裂素訊號負回饋失衡，使 Type-A OsRR2 表現更敏感，同時調控部分 OsPINs 生長素極性運輸基因的表現，進而影響生長素極性運輸與梯度建立。此外，php1/2/3 突變株初生根的活性氧 (ROS) 累積能力也明顯降低，進一步影響根部的彎曲調節。綜合上述，OsPHP1/2/3 作為細胞分裂素訊號傳遞途徑的負回饋因子，協同整合細胞分裂素、生長素及 ROS 累積之多層面網絡，確保水稻初生根對重力訊號作出合理的向地性反應，並維持根系適應環境及生長方向調整的能力。本研究成果不僅揭示 OsPHP1/2/3 的相關調控機制，亦為作物根生長方向適應能力與分子育種提供了理論依據。

Pseudo-histidine phosphotransfer proteins (OsPHPs) in rice act as negative regulators within the cytokinin signaling pathway, yet their roles in primary root gravitropic responses remain largely unexplored. In this study, we developed a systematic workflow for analyzing root gravitropism, quantifying phenotypic changes in rice primary roots in response to gravistimulation using vectorial changes of root growth and root bending level parameters. By integrating exogenous cytokinin (trans-zeatin) and auxin (IAA, IBA) treatments with genotype comparisons, we investigated the regulatory roles of OsPHP1/2/3 through analysis of a php1/2/3 triple mutant in the multi-layered integration of signaling networks. Phenotypic analyses showed that the php1/2/3 triple mutant responded significantly more rapidly to gravitstimulation and displayed reduced root curvature, demonstrating that OsPHP1/2/3 modulate primary root gravitropic responses. At the molecular level, loss of OsPHP1/2/3 disrupted negative feedback within cytokinin signaling, resulting in heightened sensitivity of the Type-A response regulator OsRR2, as well as altered expression of specific OsPINs auxin transport genes, thereby affecting auxin polar transport and gradient formation. Furthermore, the capacity for reactive oxygen species (ROS) accumulation in the primary roots of the php1/2/3 mutant was significantly reduced, further influencing root bending regulation. Collectively, OsPHP1/2/3 function as negative feedback regulators in cytokinin signaling, integratively coordinating cytokinin, auxin, and ROS accumulation in a multilayered network to ensure appropriate gravitropic responses of rice primary roots and maintain the adaptability and directional growth of the root system. This study not only elucidates the regulatory mechanisms of OsPHP1/2/3 but also provides a theoretical foundation for improving crop root orientation and molecular breeding strategies.