All Arabidopsis lines used in this work are of the Columbia (Col) accession. The ppk123, ppk124 and pif1345 mutants are gifts from Dr Peter H. Quail. The ppk single mutants, ppk1 (GABI_756G08), ppk2 (SALK_026482), ppk3 (SALK_000758) and ppk4 (SALK_017102C), were used for triple mutants preparation. To prepare PPK artificial microRNA (amiRNA) knock-down lines, the amiRNAs targeting PPKs were designed online new-lightsusing the WMD3 Web microRNA Designer50. The gene-silencing efficiency of the amiRNA candidates was tested in plants (described in ‘Tobacco transient expression’), the most effective microRNAs were selected for each PPK gene. The amiRNA precursor was derived from pRS300 using overlapping PCR. The amiRNA targeting PPK1 and PPK4 were cloned into Ti plasmid (pDT1B). The amiRNAs targeting PPK2 and PPK3 were cloned into another Ti plasmid (pDT1H). Ti plasmid pDT1B (Basta) and pDT1H (hygromycin) were modified from pCambia3301, which possess two expression cassettes. The recombinant plasmid, pDT1B-amiPPK1-amiPPK4 and pDT1H-amiPPK2-amiPPK3, were co-transformed into the Arabidopsis (Col) using the standard floral-dip method51. The transgenic T1 populations were screened on MS agar media containing 25?mg?l?1 Hygromycin and 25?mg?l?1 Basta. To generate GFP-PPKs lines, the new-lights coding sequences of PPKs were PCR-amplified and cloned into a Ti plasmid (pFGFP) modified from pCambia3301 by the In-Fusion cloning method, resulting in expression of GFP-PPKs driven by the ACTIN2 promoter (pACT2::GFP-PPKs). Ti plasmids expressing recombinant proteins were introduced into rdr6-11 allele, which suppresses gene silencing, by the floral-dip method51. The transgenic T1 populations were screened on compound soil sub-irrigated with the Basta solution. Plants were grown in walk-in growth chambers at 22?°C, 65% relative humidity under cool white fluorescent tubes. Long-day (LD) photoperiod is defined as 16?h light/8?h dark. Light-emitting diode was used to obtain monochromatic blue light (peak 450?nm; half-bandwidth of 20?nm), red light (peak 660?nm; half-bandwidth of 20?nm) or far-red light (peak 730?nm; half-bandwidth of 20?nm).
Yeast two-hybrid assays

The prey vector pGADT7 expressing PPK or PPKC fused to the GAL4 activation domain and the Bait vector pBridge expressing CRY2 fused to the GAL4 DNA binding domain were used. The pairs of plasmids were co-transformed into yeast strain AH109. Colonies were selected on plates (SD-LW). After culture in SD-LW medium for 16?h (Dark), transformants were subcultured into fresh SD-LW medium and kept in dark or irradiated with blue light (30?mol m?2 s?1). β-galactosidase activity was measured using chlorophenol red-β-D-galactopyranoside as substrate and Miller Units were calculated according to the Clontech Yeast Protocols Handbook.
Tobacco transient expression

To test the gene-silencing efficiency of those designed amiRNAs, the Ti plasmids expressing 4 × Myc-PPKs was co-transformed with the plasmids expressing the corresponding amiRNA or the empty plasmid into Nicotiana benthamiana leaves. The efficiency of amiRNA was evaluated by examination of the PPK expressed from the epitope-tagged PPK-expressing plasmids co-transfected with the amiRNA-expressing plasmids, using immunoblot.