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. 2023 Sep 19;120(39):e2304513120. doi: 10.1073/pnas.2304513120

Fig. 2.

Fig. 2.

Nitrate increases PIF4 abundance in hypocotyl and cotyledon cells to promote hypocotyl growth. Seedlings transferred from 0.5 mM to 5.0 mM nitrate 1 h after the beginning of the photoperiod of day 4 are compared to the controls that remained on 0.5 mM nitrate. (A–E) Hypocotyl growth rate in seedlings of the wild type, pif1, pif3, pif4, pif5, pif7 single and multiple mutants (A), arf6, arf7, and arf8 single and multiple mutants [B and C, in C, treatments and measurements conducted a day later for all genotypes because these double mutants have elevated growth at earlier stages (44)], rga gai and rga-∆17 mutants (E) 0-9 h after the nitrate upshift. (F–H) Nuclear fluorescence driven by the pPIF4:PIF4-GFP, pBES1:BES1-GFP, pHY5:HY5-YFP, pARF6:ARF6-GFP, and pARF7:ARF7-VENUS in hypocotyl cells (F), p35S:PIF4-GFP in hypocotyl cells (G, including the chl1-5 background) or pPIF4:PIF4-GFP in cotyledon cells (H), measured by confocal microscopy 6 h after the nitrate upshift (2 h included for pPIF4:PIF4-GFP; red shows chlorophyll fluorescence). (I–L) Bioluminescence driven by pPIF4:PIF4-LUC (I, J, and L, including the chl1-5 background) or pPIF4:LUC (K) during the photoperiod of day 4 (I and K) or 1.5 h after the nitrate upshift (J and L). (M and N) PIF4-HA (M) and PIF4-GFP (N) protein blots of entire seedlings harvested 1.5 h after the application of nitrate (for quantification see SI Appendix, Fig. S6). (O) Addition of 5 µM Picloram bypasses the requirement of cotyledons for the growth promotion of Col-0 but not pif4 hypocotyls. (P) Repositioning Col-0 but not pif4 cotyledons partially restores the elongation response of Col-0 hypocotyls without cotyledons. Data are means ± SE and individual values (omitted in kinetics data). Asterisks indicate the significance of the effect of nitrate upshift in Bonferroni tests following significant interaction (int, A–E, G, L, O, and P), the main effect of nitrate upshift if the interaction is not significant (F, PIF4) or in Student’s t tests (F, I–K, M, and N): *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.