Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence

Senescence is a highly regulated process driven by developmental age and environmental factors. Although leaf senescence is accelerated by nitrogen (N) deficiency, the underlying physiological and molecular mechanisms are largely unknown. Here, we reveal that BBX14, a previously uncharacterized BBX-type transcription factor in Arabidopsis, is crucial for N starvation-induced leaf senescence. We find that inhibiting BBX14 by artificial miRNA (amiRNA) accelerates senescence during N starvation and in darkness, while BBX14 overexpression (BBX14-OX) delays it, identifying BBX14 as a negative regulator of N starvation- and dark-induced senescence. During N starvation, nitrate and amino acids like glutamic acid, glutamine, aspartic acid, and asparagine were highly retained in BBX14-OX leaves compared to the wild type. Transcriptome analysis showed a large number of senescence-associated genes (SAGs) to be differentially expressed between BBX14-OX and wild-type plants, including ETHYLENE INSENSITIVE3 (EIN3) which regulates N signaling and leaf senescence. Chromatin immunoprecipitation (ChIP) showed that BBX14 directly regulates EIN3 transcription. Furthermore, we revealed the upstream transcriptional cascade of BBX14. By yeast one-hybrid screen and ChIP, we found that MYB44, a stress-responsive MYB transcription factor, directly binds to the promoter of BBX14 and activates its expression. In addition, Phytochrome Interacting Factor 4 (PIF4) binds to the promoter of BBX14 to repress BBX14 transcription. Thus, BBX14 functions as a negative regulator of N starvation-induced senescence through EIN3 and is directly regulated by PIF4 and MYB44.