O, JMJ14, miP1a, and miP1b in pink; putative interactors
O, JMJ14, miP1a, and miP1b in pink; putative interactors in gray. B, Venn diagram depicting the amount of proteins co-purified with FLAG-miP1a, P2X1 Receptor web FLAG-miP1b, FLAG-JMJ14, and FLAG-TPL. Nonspecific interactors identified in experiments with either WT plants or plants expressing FLAG-GFP have already been subtracted. C, Yeast-two-hybrid interactions were tested by transformations of empty vector or of fusions of miP1a, JMJ14, and TPL to the Gal4 activation domain (AD), and fusions of possible interactors for the Gal4 binding domain (BD). Shown would be the development of serial dilutions of co-transformants on nonselective (-LW) and selective (-LWH) SD medium. The latter medium was supplemented with five mM with the competitive HIS-inhibitor 3-aminotriazole (3-AT)exactly where expression from the KNAT1 promoter brought on quite early flowering, even inside the late flowering co mutant background (An et al., 2004). We noted that apart from CO, miP1a and miP1b (Graeff et al., 2016) showed robust expression inside the SAM. To investigate the spatial expression pattern of TPL and JMJ14 within the SAM, we obtained respective promoter-GUS reporter constructs that had been recently published (Cattaneo et al., 2019; Kuhn et al., 2020). JMJ14 and TPL showed really sturdy, ubiquitous GUS expression within the SAM and leaves, supporting the notion that these aspects are present in the SAM (Figure 6A). To assess if a potential JMJ14containing repressor complex would operate in the SAM, we crossed KNAT1::CO co-2 plants with jmj14-1 mutant plants. When grown under inductive long-day conditions, we located that WT plants flowered early compared to co-2 and KNAT1::CO co-2 plants, confirming earlier findings that expression of CO within the SAM will not be enough to induce flowering. However, we detected a very early flowering response when we introduced the KNAT1::CO transgene into the jmj14 mutant background (Figure 6, B and C). Also in mixture using a mutation in co, KNAT1::CO jmj14 co-mutant plants flowered incredibly early, supporting the idea that CO and JMJ14 are part of a repressor complex that acts within the SAM to repress FT expression. To independently determine that CO can induce FT expression in the shoot CK2 Biological Activity meristem when JMJ14 just isn’t active or present, we manually dissected shoot apices from Col-0 WT, jmj14-1, and KNAT1::CO jmj14-1 plants to establish abundances of CO and FT mRNAs. This analysis revealed that the levels of CO mRNA were comparable between Col-0 and jmj14-1 but enhanced in KNAT1::CO jmj14-1 (Figure 6D). This discovering confirms that KNAT1::CO jmj14-1 plants certainly exhibit ectopically elevated levels of CO inside the SAM, and that the early flowering phenotype of jmj14-1 single mutant plants isn’t a outcome of ectopic CO expression in the meristem. When the expression of FT was analyzed inside the same samples, we couldn’t detect any FT mRNA in the meristem from the WT plants. This is constant with prior findings that had shown expression of CO but not FT inside the SAM (An et al., 2004; Tsutsui and Higashiyama, 2017). Simply because we have been unable to detect FT within the meristem of WT plants, we normalized the information for the jmj14-1 mutant in which we had| PLANT PHYSIOLOGY 2021: 187; 187Rodrigues et al.Table 2 Interacting proteins identified by enrichment proteomicsAccession number At3g21890 At4g15248 At1g15750 At4g20400 At5g24930 At3g07650 At1g68190 At1g80490 At3g16830 At5g27030 At3g15880 At2g21060 At3g07050 At3g22231 At4g27890 At4g39100 At5g14530 At1g35580 At5g20830 At1g08420 At1g13870 At1g75600 At1g78370 At3g10480 At3g10490.