Understanding the Role of B-BOX Proteins in Temperature-mediated Regulation of Growth and Development in Arabidopsis thaliana

Malakar, Bidhan Chandra (2025) Understanding the Role of B-BOX Proteins in Temperature-mediated Regulation of Growth and Development in Arabidopsis thaliana. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (Bidhan Chandra Malakar (18RS109)) 18RS109.pdf - Submitted Version Restricted to Repository staff only Download (60MB)

Abstract

Thermomorphogenesis is an adaptive response in plants, enabling morphological and metabolic adjustments to fluctuating ambient temperatures. In Arabidopsis, an increase in ambient growth temperature by 5°C leads to massive changes in morphological features such as promotion in hypocotyl elongation, petiole hyponastic movement, early flowering, reduction in stomatal index, and decrease in seed set. The bHLH family of transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) plays a central role in promoting thermomorphogenesis, whose activity is negatively regulated by thermosensor EARLY FLOWERING 3 (ELF3) and PHYTOCHROME B (phyB). In response to warm temperatures, PIF4 transcript and protein levels increase to facilitate thermosensory growth. However, the regulatory mechanisms governing PIF4-mediated thermosensory growth remain elusive. Here, we demonstrate the role of three Group-IV, B-BOX proteins, BBX24, BBX25, and BBX21, in regulating thermomorphogenesis through the PIF4 pathway. We show that BBX24/BBX25 plays a positive regulatory role in warm-temperature-mediated hypocotyl growth, whereas BBX21 plays a negative regulatory function in controlling thermosensory hypocotyl growth. Analysis of bbx24 and bbx25 single mutants exhibits moderate insensitivity, but bbx24bbx25 double mutants showed strong insensitivity to warm temperature-mediated hypocotyl growth, which is due to a strong reduction in thermoresponsive gene expression. Warm temperatures have been found to upregulate gene expression and protein stability of BBX24/BBX25. Genetic and biochemical data revealed that BBX24/BBX25 functions through the PIF4 pathway, facilitating PIF4 protein accumulation in response to warm temperatures through direct physical interactions. BBX24/BBX25 gene expression follows the circadian pattern, where ELF3 suppresses their gene expression in the evening, in a temperaturedependent manner. BBX24/BBX25 physically interacts with ELF3 and interferes with its protein activity to indirectly help PIF4 induce elongation growth under warm temperatures. On the other hand, mutation in BBX21 (bbx21) results in a longer hypocotyl phenotype accompanied by upregulation in thermoresponsive growth hormone gene expression, whereas overexpression of BBX21 (BBX21-OE) results in an extremely short hypocotyl phenotype with dampened expression of temperatureresponsive genes. Genetic analysis reveals that BBX21 acts upstream of PIF4 to regulate warm-temperature-mediated hypocotyl growth. To repress excessive thermomorphogenesis, BBX21 inhibits PIF4 protein accumulation by repressing its transcript expression through directly binding to the PIF4 promoter. Furthermore, our genetic and biochemical data show that the short hypocotyl phenotype of the BBX21- OE line is dependent on ELF3 and phyB thermosensors. BBX21 enhances the ELF3 and phyB-mediated inhibition of PIF4 activity in SD and LD conditions, respectively, by enhancing their protein activity. Together, this study elucidates the critical role of BBX24, BBX25 and BBX21 in regulating thermomorphogenesis, providing new insights into the molecular mechanism of PIF4-mediated regulation of hypocotyl growth in response to warm temperatures.

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