MdBT2 mediates the degradation of MdRGL3a. Supplemental Physique S8. (Xu et?al., 2014; Daviere and Achard, 2016). For example, DELLA proteins directly interact with PHYTOCHROME INTERACTING FACTORS (PIFs), JA ZIM-domain 1 (JAZ1), ETHYLENE INSENSITIVE3 (EIN3), and class I TCP to inhibit their binding to promoters of target genes, thus regulating hypocotyl elongation, root growth, apical hook formation, and herb height, respectively (de Lucas et?al., AICAR phosphate 2008; Feng et?al., 2008; Hou et?al., 2010; An et?al., 2012; Daviere et?al., 2014). Moreover, GA-DELLA pathway has also been identified to be involved in nitrate-mediated anthocyanin biosynthesis and flowering time (Zhang et?al., 2017; Gras et?al., 2018). However, the exact molecular mechanism by which nitrate regulates herb growth and development via the crosstalk between nitrate and GA signaling pathways is largely unknown. More recently, a BTB/TAZ subfamily scaffold protein BT2 was identified as a central hub of the predicted NUE gene network (Araus et?al., 2016). Its transcription is usually induced by nitrate, and overexpression of partially rescues growth defects due to a decrease in the activity of nitrate-responsive gene NODULE INCEPTION-LIKE PROTEIN6 (and apple genomes have five BTB/TAZ proteins. Members of the BT family participate in different signaling pathways that regulate herb growth and development. AtBT2 induces telomerase activity in mature leaves and participates AICAR phosphate in gametophyte development; it also responds to multiple environmental and hormonal signals, such as sugar and nutrient status, wounding and hydrogen peroxide stress, circadian regulation, ABA, and auxin (Ren et?al., 2007; Mandadi et?al., 2009; Robert et?al., 2009). In apple, MdBT2 participates in Fe homeostasis, anthocyanin biosynthesis, cold stress, and leaf senescence by modulating the protein abundances of MdbHLH104, MdMYB1, MdMYB23, and MdbHLH93, respectively (Zhao et?al., 2016; An et?al., 2018, 2019; Wang et?al., 2018). In this study, we found that MdBT2 is usually involved in nitrate-induced growth of apple plants, and that MdBT2 exercised its role in regulating herb growth by directly interacting with the DELLA protein MdRGL3a, thus promoting its ubiquitination and degradation in response to nitrate. Our results further elucidate the molecular mechanisms underlying herb growth regulation by nitrate. Results Nitrate promotes herb growth partially via a GA-associated pathway To assess the effect of nitrate on herb growth, apple AICAR phosphate plantlets were produced in pots filled with inert substrate. They were fertilized weekly with the Hoagland nutrient solutions without nitrogen or supplemented with nitrate at a concentration range of 0C15 mM. After a 45-d growth period, increased height and biomass of plants were observed with increasing nitrate concentration, whereas the fertilizer solutions made up of no or lower nitrate (0.5 mM) significantly limited herb growth (Determine?1, ACC). To examine if GA is usually involved in the nitrate-induced herb growth, GA3 or GA biosynthesis inhibitor paclobutrazol (PAC) was ADFP exogenously applied to plantlets treated with 0.5 mM KNO3 or 5 mM KNO3. As a result, exogenous application of 100 M GA3 partially restored herb height and biomass under nitrate-deficient conditions, resulting in comparable growth to plants treated with 5 mM KNO3 (Physique?1, DCF). In addition, 10 M PAC noticeably inhibited nitrate-induced herb growth with a roughly 50% decrease in the herb height and 80% decrease in biomass (Physique?1, DCF), suggesting that GA may participate in nitrate-induced herb growth. Open in a separate window Physique 1 Nitrate promotes herb growth partially via a GA-associated pathway. (A) Phenotype of 4-week-old apple plantlets grown AICAR phosphate in vermiculite and fertilized weekly with N-free Hoagland AICAR phosphate nutrient solution made up of either 0, 0.5, 2, 5, 10, or 15 mM KNO3 (KCl was added to maintain the same K concentration) for 45 d. Scale bar represents 2 cm. (B, C) Analysis of herb height (B) and dry weight (C) of apple plants shown in (A). (D) Phenotype of apple plantlets under 0.5 mM KNO3 growth conditions with or without 100 M GA3 treatments or under 5 mM KNO3 growth conditions with or without 10 M PAC treatments for 45 d. Scale bar represents 2 cm. (E, F) Analysis of herb height (E) and dry weight (F) of apple plants shown in (D). Biomass was measured as dry weight per herb. In (B, C, E, F), error bars represent SD of.