*3.1. Regulation of PR Genes by Circadian Rhythms and Photoreceptors*

This research shows that the expression of the *PR10* gene is partially regulated by the internal biological clock, while photoreceptors mainly control the *PR1* gene. The expression of the *PR10* gene in plantlets exposed to continuous RL and FRL under a 16/8 h photoperiod (Figure 7) maintained an oscillatory pattern, which appeared to be controlled by the circadian rhythm (Figure 11a,b). In contrast, the transcript of the *PR1* gene appeared to be independent of the oscillator and dependent on the photoreceptor's activities. These agree with the observations by Genoud et al. [5] on the effect of single and multiple nil mutants in the light perception (*phyA* and *phyB*) and the light-signal processing (*psi2*, phytochrome signaling) on the interaction between *A. thaliana* and the pathogen *Pseudomonas syringae* pv. *tomato* single and multiple mutants' nil in light perception (*PHYA* and *PHYB*) and light-signal processing (*psi2*, phytochrome signaling).

In these mutants, the growth of an incompatible bacterial strain of this pathogen was enhanced in the double mutant *phyAphyB* and decreased in the *psi2* mutant under darkness and dim light conditions [20]. The last mutant increased the light signal transduction regulated by PHYA and PHYB [39]. Similarly, the results of this work demonstrated that the overexpression of *PHYB* and *CRY*1 is associated with an upregulation of the Dar Gazi *PR1* (Figures 6 and 8).

Salicylic acid (SA) induces pathogen-related gene expression and accumulation of related proteins, and its production also depends on the light regime [40]. Phytochromes are required for the expression of the PR1 protein [19]. In *Dar Gazi-phyB*, the expression of *PR1* was dependent on the phytochrome. When the plantlets were exposed to continuous RL an up-regulation of *PR1* was observed (Figure 10a); in contrast, in plantlets exposed to continuous FRL, the expression of this gene was inhibited (Figure 10b). When the plantlets were exposed to FRL and BL, a co-participation of the cryptochrome into the regulation system was also observed (Figure 10b,c).

Although in this study, free SA was not measured, it is known that the SA levels oscillate throughout the day in a circadian rhythm [41], so a fine coordinated regulation of *PR1* gene between this hormone and photoreceptors pathways could be strongly hypothesized, and it will be the challenge for the further investigation. It has been shown that transcription of *PR* genes during plant defense involves a key transcriptional regulator of SA signaling known as Nonexpressor of Pathogenesis-related protein 1 (NPR1). The inactive NPR1 oligomers monomerize in the cytosol after an SA-induced change of the cell's redox state, and a circadian oscillation occurs, peaking at night [42]. The state of monomers allows NPR1 to be translocated to the nucleus where they interact with TIMING OF CAB2 EXPRESSION 1 (TOC1), an evening circadian clock gene, and TGACG-BINDING FACTORs (TGAs), leading to the expression of defense-related genes involved in the set-up of plant immune defense, including *PR* genes [42–45]. The oscillatory rhythms of TOC1 mRNA expression were associated with parallel oscillations in histone acetylation [46]. NPR1 forms an activator complex with histone acetyltransferases (HATs) HAT1 and HAT5. Through NPR1–TGA interaction, the complex is recruited to chromatin finally relaxing genomic DNA and facilitating *PR*s transcription activation [47].

The obtained data suggest that the *PR*s clock-associated regulation is co-regulated by the photoreceptors phytochrome and cryptochrome, maybe functionally as elements of regulator-Zeitlupe systems. In fact, in overexpressing *PHYB* gene plantlets, a circadian oscillation is observed when exposed to a continuous RL (Figures 9 and 10). The results indicate that this behavior is red/far-red reversible. When plantlets are exposed to FRL and BL, a co-participation of cryptochrome into the regulation system was also observed.

In overexpressing *phyA* cherry plants, Cirvilleri et al. [48] concluded that the induction of *PR*s gene is strictly dependent on light quantity and quality, inducing plant resistance to *Pseudomonas syringae* pv. *mors-prunorum*. Therefore, the relationship between biological clock and overexpression of *PHYB* and *CRY1* was tested under different light qualities to unravel their role on *PR*s gene expression pear cv Dar Gazi.

In a previous study, a possible link between *PR1* and light was postulated [40]. However, until now there has been a gap of knowledge around the role of the internal clock in the *PR1* regulome. The effects of many biotic and abiotic stress, including pathogen infection, salt tolerance, UV irradiation, and ozone stress, have been investigated in *PR10* gene expression [49]. These stresses have been shown to activate *PR10* gene expression, suggesting their importance during plant defense responses. Plant hormones and related signaling molecules have been reported to regulate *PR10* gene expression, including jasmonic acid, salicylic acid, abscisic acid [50], kinetin, and auxin [51].
