*4.1. Plant Material, Medium Composition, Growth Conditions, and Bacterial Strain*

An in vitro-cultured plantlets system of *Pyrus communis* L. cv Dar Gazi was used to evaluate if the internal clock autonomously regulates the abundance of *PR1* and *PR10* transcripts. Plantlets of three different lines: *Dar Gazi-wt*, *Dar Gazi-phyB,* and *Dar Gazi-cry1* were submitted to different circadian experimental conditions and continuous BL, RL- and FRL conditions. Fluorescent WL was used as a control.

The two transgenic lines *Dar Gazi-phyB* and *Dar Gazi-cry1* were obtained starting from leaf explants of in vitro established cv *Dar Gazi-wt* co-cultivated for 20 min on MS liquid basal medium with two different *A. tumefaciens* strains (A, B) prepared as described below.

The disarmed *A. tumefaciens* (A) strain EHA 105, contained the helper plasmid pTiBo542 and the binary vector pROKB (kindly provided by Whitelam, Leicester University, England), harboring the *neomycin phosphotransferase II* (*nptII*) gene under the control of *nos* promoter and the *A. thaliana* cDNA *PHYB* gene under the control of the cauliflower mosaic virus 35S RNA (CaMV 35S) promoter. The disarmed *A. tumefaciens* (B) strain EHA 105, contained the helper plasmid pTiBo542 and the binary vector pBI12 (also provided by Whitelam), harboring the *neomycin phosphotransferase II* (*nptII*) gene under the control of *nos* promoter and *Lycopersicum esculentum* cDNA *CRY1* gene under the control of the CaMV 35S promoter. Vectors were introduced into EHA 105 using freeze-thaw transformation of *Agrobacterium* and *Escherichia coli* as described by [69]. For both transformation experiments *A. tumefaciens*, was cultured overnight at 28 ◦C on a shaker at 80 rpm in 10 mL liquid Luria-Bertani (LB) [70] medium, prepared with 1% LB containing Bacto-Tryptone, 0.5% Bacto-yeast extract, 1% NaCl, 0.1% glucose with 100 mg L−<sup>1</sup> of kanamycin added when the temperature arrived at 45◦ C for selecting bacteria carrying the binary plasmid. After 10 min of centrifugation at 3200× *g*, the pellet was resuspended in MS liquid medium with 3% (*w*/*v*) sucrose, and subsequent dilutions were done to reach a final concentration of around 0.3 (OD600).

After the co-cultivation, the leaf explants were transferred to a regeneration medium containing 100 mg L–1 acetosyringone (40-hydroxy-3,5-dimethoxyacetophenone) and incubated in a controlled environment chamber at 23 ◦C for two days. Cefotaxime (200 mg L−<sup>1</sup> ) was added to all media, to eliminate *Agrobacterium*. The transformed green shoots were picked out from callus tissue (assisted by a stereoscope) and moved to QL0 medium [71] with 10 mg L−<sup>1</sup> of kanamycin added (Figure S4a). The final selection was carried out onto

QL0 medium added with 100 mg L−<sup>1</sup> of kanamycin, subculturing every 8–10 days to select putative transgenic lines (Figure S4b).
