Proline accumulation is regulated by transcription factors associated with phosphate starvation

Proline accumulation in plants is a well-documented physiological response to osmotic stress caused by drought or salinity. In Arabidopsis thaliana the stress and ABA-induced Delta1-PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 (P5CS1) gene was previously shown to control proline biosynthesis in such adverse conditions. To identify regulatory factors which control the transcription of P5CS1, yeast one hybrid (Y1H) screens were performed with a genomic fragment of P5CS1, containing 1.2 kB promoter and 0.8 kB transcribed regions. The transcription factors PHOSPHATE STARVATION RESPONSE 1 (PHR1) and PHR1-LIKE 1 (PHL1) were identified to bind to P5CS1 sequences in the first intron, which carry a conserved PHR1-binding site (P1BS) motif. PHR1 and PHL1 binding to P1BS was confirmed by Y1H, electrophoretic mobility assay (EMSA) and chromatin immune precipitation (ChIP). Phosphate starvation led to gradual increase in proline content in wild type Arabidopsis plants as well as transcriptional activation of P5CS1 and PROLINE DEHYDROGENASE 2 (PDH2) genes. Induction of P5CS1 transcription and proline accumulation during phosphate deficiency was considerably reduced by phr1 and phl1 mutations and was impaired in the ABA deficient aba2-3 and ABA insensitive abi4-1 mutants. Growth and viability of phr1phl1 double mutant was reduced in phosphate-depleted medium, it was slightly diminished in the aba2-3 mutant, suggesting that ABA mediates growth retardation in such stress. Our results reveal a previously unknown link between proline metabolism and phosphate nutrition, and show that proline biosynthesis is target of crosstalk between ABA signaling and regulation of phosphate homeostasis through PHR1 and PHL1-mediated transcriptional activation of the P5CS1 gene