Abstract: Plants are highly susceptible to abiotic stresses, particularly heat stress during the reproductive stage. However, the specific molecular mechanisms underlying this sensitivity remain largely unknown. In the current study, we demonstrate that the Nuclear Transcription Factor, X-box Binding Protein 1-Like 1 (NFXL1), directly regulates the expression of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2A (DREB2A), which is crucial for reproductive thermotolerance in Arabidopsis. NFXL1 is upregulated by heat stress, and its mutation leads to a reduction in silique length (seed number) under heat stress conditions. RNA-Seq analysis reveals that NFXL1 has a global impact on the expression of heat stress responsive genes, including DREB2A, Heat Shock Factor A3 (HSFA3) and Heat Shock Protein 17.6 (HSP17.6) in flower buds. Interestingly, NFXL1 is enriched in the promoter region of DREB2A, but not of either HSFA3 or HSP17.6. Further experiments using electrophoretic mobility shift assay have confirmed that NFXL1 directly binds to the DNA fragment derived from the DREB2A promoter. Moreover, effector–reporter assays have shown that NFXL1 activates the DREB2A promoter. The DREB2A mutants are also heat stress sensitive at the reproductive stage, and DEREB2A is epistatic to NFXL1 in regulating thermotolerance in flower buds. It is known that HSFA3, a direct target of DREB2A, regulates the expression of heat shock proteins genes under heat stress conditions. Thus, our findings establish NFXL1 as a critical upstream regulator of DREB2A in the transcriptional cassette responsible for heat stress responses required for reproductive thermotolerance in Arabidopsis.

Key words: Arabidopsis, DREB2A, heat stress, NFXL1, reproductive, thermotolerance