The GIGANTEA-LHY complex regulates citrus cold tolerance and participates in low-temperature-induced flowering

GIGANTEA (GI) is known to regulate multiple physiological processes, but its function in cold tolerance and low-temperature-induced flowering remains poorly understood in citrus. In this study, we found that between the two splice variants of the citrus GI homolog, CiGIα and CiGIβ, CiGIα expression was specifically induced by low temperature, whereas CiGIβ expression remained unchanged. Yeast one-hybrid assays demonstrated that the low-temperature-responsive bZIP transcription factor CiFDβ binds to the CiGI promoter and activates its expression. Further investigation revealed that cold stress upregulates CiFDβ expression, which in turn activates CiGI transcription and facilitates the CiGI-CiLHY complex formation. This complex then transmits the cold signal to CiFT, thereby participating in low-temperature-induced flowering. Functional analysis of transgenic citrus revealed that overexpression of CiGIα increased cold sensitivity, whereas suppression of CiGI enhanced cold tolerance in silenced citrus plants. Similarly, overexpression of CiLHY, an interactor of CiGI, also increased cold sensitivity, while its suppression improved cold tolerance. RNA-seq profiling of CiGI transgenic citrus lines identified differentially expressed genes related to hormone and sugar metabolism. Further experimental evidence showed that the CiGI-CiLHY complex binds to the promoter of 9-cis-epoxycarotenoid dioxygenase 3 (CiNCED3), repressing its transcription and lowering ABA levels. In addition, this complex also bound to the trehalase 1 (CiTRE1) promoter, activating its expression and reducing trehalose accumulation. Exogenous application of trehalose or ABA significantly enhanced the cold tolerance of CiGI and CiLHY transgenic citrus plants. These findings reveal a novel regulatory mechanism through which CiGI modulates cold stress responses by coordinately regulating ABA and trehalose metabolism.