*2.1. Effect on the Monochromatic Light on the Growth of E. amylovora In Vitro*

To evaluate the effect of the monochromatic light on the cell growth of *E. amylovora* strain Ea273, the microorganism was cultivated in shake flasks under light and temperaturecontrolled conditions. All cultures were inoculated at the same initial optical density (OD600 = 0.1), with cells from precultures grown in the darkness and in the late exponential phase of growth to minimize the impact of the inoculum on the lag phase of growth. The results reported in Figure 1 indicate that the monochromatic light has had several effects on the growth of this microorganism. Significant differences were observed using OD<sup>600</sup> as an indirect measure of the cell growth and comparing the OD<sup>600</sup> values at the end of the growth (on, overnight; 18–24 h after the inoculum). Under the same cultivation conditions (inoculum, temperature, rotation speed, incubation time), the highest cell density (OD<sup>600</sup> = 4) was achieved for cultivating strain Ea273 under continuous RL (Figure 1). This value was approximately 1.4-fold higher than the one obtained in darkness and 1.2 and 1.3-times higher than the one obtained under FRL and BL. Under BL, the deceleration/decline phase started at least 3 h earlier (8 h after the inoculum), but no significant difference was observed compared to the OD<sup>600</sup> values at t = 11 and t = on (Figure 1). The

latent growth phase was shorter (approximatively 1 h) under FRL compared to darkness (Figure 1). The growth profile under RL showed a prolonged exponential phase (up to t = 8), a prolonged latent phase (up to t = 4), and a higher growth rate in the late phase of growth after the diauxic shift (between 10 and 11 h after the inoculum; Figure 1). These results indicate that monochromatic lighting can modulate the growth pattern of *E. amylovora* independently from the presence of the plant stimuli. growth phase was shorter (approximatively 1 h) under FRL compared to darkness (Figure 1). The growth profile under RL showed a prolonged exponential phase (up to t = 8), a prolonged latent phase (up to t = 4), and a higher growth rate in the late phase of growth after the diauxic shift (between 10 and 11 h after the inoculum; Figure 1). These results indicate that monochromatic lighting can modulate the growth pattern of *E. amylovora* independently from the presence of the plant stimuli.

1.3-times higher than the one obtained under FRL and BL. Under BL, the deceleration/decline phase started at least 3 h earlier (8 h after the inoculum), but no significant difference was observed compared to the OD<sup>600</sup> values at t = 11 and t = on (Figure 1). The latent

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**Figure 1.** The effect of monochromatic light on the growth of *E. amylovora* Ea273 in shake flask cultures. The data are representative of three independent experiments with three biological and two technical replicates. Error bars represent the SD. The number of asterisks adjacent to the symbols, at the same time point, indicates significant differences between different growth conditions (Student's *t*-test, *p* ≤ 0.05). **Figure 1.** The effect of monochromatic light on the growth of *E. amylovora* Ea273 in shake flask cultures. The data are representative of three independent experiments with three biological and two technical replicates. Error bars represent the SD. The number of asterisks adjacent to the symbols, at the same time point, indicates significant differences between different growth conditions (Student's *t*-test, *p* ≤ 0.05).

#### *2.2. E. amylovora Causes Tissue Necrosis in In Vitro Pear Dar Gazi Plantlets 2.2. E. amylovora Causes Tissue Necrosis in In Vitro Pear Dar Gazi Plantlets*

The in vitro grown Iranian pear cultivar Dar Gazi wild type (wt) inoculated with *E. amylovora*, showed oxidative stress in the central cylinder and the cortex of the basal portion of plantlets (Figure 2a). The stems resulted in characteristic signs of HR that cause rapid cell death in the vicinity of the infection point (Figure 2a). These observations are consistent with Electrical Conductivity (EC) measurements, where high values reflect the plasma membrane disruption. An ion leakage three-fold higher was detected in *Dar Gazi-*The in vitro grown Iranian pear cultivar Dar Gazi wild type (wt) inoculated with *E. amylovora*, showed oxidative stress in the central cylinder and the cortex of the basal portion of plantlets (Figure 2a). The stems resulted in characteristic signs of HR that cause rapid cell death in the vicinity of the infection point (Figure 2a). These observations are consistent with Electrical Conductivity (EC) measurements, where high values reflect the plasma membrane disruption. An ion leakage three-fold higher was detected in *Dar Gazi-wt* infected vs non-infected (Figure 2b).

*wt* infected vs non-infected (Figure 2b).

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**Figure 2.** Oxidative activity in inoculated pear plantlets. (**a**) A schematic representation of the oxidative activity in inoculated plantlets with necrotic sections in the stem (upper panel) and necrotic progression from the central cylinder to the cortex, indicated by yellow arrows in the leaf (lower panel); (**b**) Representative data of the electrolyte leakage of inoculated and non-inoculated *Dar Gazi-wt*. Electrolytic conductivity dramatically increased in plants after bacterial inoculation. Error bars represent the SD of three independent experiments, each with three biological replicates. Asterisks indicate significant differences of inoculated vs non-inoculated plants (Student's *t*-test, *p* ≤ 0.05). *2.3. Molecular Marker for E. amylovora Infection* **Figure 2.** Oxidative activity in inoculated pear plantlets. (**a**) A schematic representation of the oxidative activity in inoculated plantlets with necrotic sections in the stem (upper panel) and necrotic progression from the central cylinder to the cortex, indicated by yellow arrows in the leaf (lower panel); (**b**) Representative data of the electrolyte leakage of inoculated and non-inoculated *Dar Gazi-wt*. Electrolytic conductivity dramatically increased in plants after bacterial inoculation. Error bars represent the SD of three independent experiments, each with three biological replicates. Asterisks indicate significant differences of inoculated vs non-inoculated plants (Student's *t*-test, *p* ≤ 0.05).

#### A previous study demonstrated that the expression of the chloroplastic gene psbA in *2.3. Molecular Marker for E. amylovora Infection*

the pear cultivar Harrow Sweet is linked to the effects of *E. amylovora* infection [35]. Analysis of psbA expression in inoculated and not-inoculated pear Dar Gazi shoots revealed the presence of unexpected amplicons when we used, as a template, cDNA synthesized with a psbA-specific primer using mRNA extracted from inoculated plants (Figure 3a). The bacterial retrotranscript products were not detected when the cDNA was prepared from non-inoculated shoots or when the cDNA was synthesized using oligo d(T)8-12. Sequencing the PCR products indicated that all of the sequences belonged to *E. amylovora* (sequence identity > 99%). The 1056-bp amplicon, named *erw1*, contains gene sequences encoding: the C-terminal domain of a putative cyclopropane-fatty-acyl-phospholipid synthase (CFAS), an enzyme with synthase and methyltransferase activity involved in the fatty acid biosynthesis; the N-terminal domain of a predicted lipoprotein with an unknown function containing a DUF3833 domain. The 925-bp amplicon (*erw2*) contains the sequence encoding of the predicted Major Facilitator Superfamily (MFS) transporter. These transporters facilitate the transport across cytoplasmic or internal membranes and represent one of the two major classes of transport proteins involved in the protection against endogenous and exogenous toxic compounds in fungi [36]. The 519-bp amplicon (*erw3*) corresponds to the 3′-half of the *erw1* amplicon and contains the sequences encoding the DUF3833 domain-containing protein. The 384-bp amplicon (*erw4*) contains sequences encoding an MFS transporter of the sugar porter (SP) family, the most prominent family of MFS transporter [37]. Gene-specific primers were designed to amplify the same four genes: *erw1*, CFAS; *erw2*, MFS transporter; *erw3*/ *erw1*, DUF3833 protein; erw4, SP MFS transporter. Gene expression analysis was carried out on mRNA extracted from different sections of the asymptomatic pear plantlets (24 h after the infection). The qPCR results indicated that *erw2* was expressed in the basal section up to the middle section (4- 9 mm), while *erw1* was expressed in the low- and mid-section (Figure 3b). In contrast, the A previous study demonstrated that the expression of the chloroplastic gene psbA in the pear cultivar Harrow Sweet is linked to the effects of *E. amylovora* infection [35]. Analysis of psbA expression in inoculated and not-inoculated pear Dar Gazi shoots revealed the presence of unexpected amplicons when we used, as a template, cDNA synthesized with a psbA-specific primer using mRNA extracted from inoculated plants (Figure 3a). The bacterial retrotranscript products were not detected when the cDNA was prepared from non-inoculated shoots or when the cDNA was synthesized using oligo d(T)8-12. Sequencing the PCR products indicated that all of the sequences belonged to *E. amylovora* (sequence identity > 99%). The 1056-bp amplicon, named *erw1*, contains gene sequences encoding: the C-terminal domain of a putative cyclopropane-fatty-acyl-phospholipid synthase (CFAS), an enzyme with synthase and methyltransferase activity involved in the fatty acid biosynthesis; the N-terminal domain of a predicted lipoprotein with an unknown function containing a DUF3833 domain. The 925-bp amplicon (*erw2*) contains the sequence encoding of the predicted Major Facilitator Superfamily (MFS) transporter. These transporters facilitate the transport across cytoplasmic or internal membranes and represent one of the two major classes of transport proteins involved in the protection against endogenous and exogenous toxic compounds in fungi [36]. The 519-bp amplicon (*erw3*) corresponds to the 30 -half of the *erw1* amplicon and contains the sequences encoding the DUF3833 domain-containing protein. The 384-bp amplicon (*erw4*) contains sequences encoding an MFS transporter of the sugar porter (SP) family, the most prominent family of MFS transporter [37]. Gene-specific primers were designed to amplify the same four genes: *erw1*, CFAS; *erw2*, MFS transporter; *erw3*/ *erw1*, DUF3833 protein; erw4, SP MFS transporter. Gene expression analysis was carried out on mRNA extracted from different sections of the asymptomatic pear plantlets (24 h after the infection). The qPCR results indicated that *erw2* was expressed in the basal section up to the middle section (4-9 mm), while *erw1* was expressed in the low- and mid-section (Figure 3b). In contrast, the expression of *erw3* and *erw4* occurred mainly in the mid- and high-section of the plantlet (Figure 3b). This data indicates that, for improving the interaction with the different colonized tissues, *E.*

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*amylovora* modulates the expression of its genes during the internal movement through the vascular system. nized tissues, *E. amylovora* modulates the expression of its genes during the internal movement through the vascular system.

expression of *erw3* and *erw4* occurred mainly in the mid- and high-section of the plantlet (Figure 3b). This data indicates that, for improving the interaction with the different colo-

**Figure 3.** Identification of erw genes: (**a**) Electrophoretic profile of the retrotranscription products (*erw1-4*) obtained from the cDNA synthesized using a psbA gene-specific primer with mRNA from *E. amylovora*-infected plantlets, panel; (**b**) Spatial differential expression in pear tissue of the *E. amylovora* genes revealed using the *erw* genes-specific primers. **Figure 3.** Identification of erw genes: (**a**) Electrophoretic profile of the retrotranscription products (*erw1-4*) obtained from the cDNA synthesized using a psbA gene-specific primer with mRNA from *E. amylovora*-infected plantlets, panel; (**b**) Spatial differential expression in pear tissue of the *E. amylovora* genes revealed using the *erw* genes-specific primers.

#### *2.4. PR1 and PR10 Expression in Dar Gazi-wt 2.4. PR1 and PR10 Expression in Dar Gazi-wt*

To highlight the role of the internal clock in regulating the in vitro expression of *PR1* and *PR10* genes in Iranian pear cultivar *Dar Gazi-wt*, AtPHYB and LeCRY1 overexpressed lines; plantlets were initially exposed to a photoperiod of 16 h/8 h (light/darkness, Figure S1). In *Dar Gazi-wt*, the expression of the *PR1* gene was not oscillatory, keeping an almost constant level of expressed transcripts throughout the day. The level of *PR1* transcripts was, less than *PR10* transcripts during the day, irrespective of the lighting conditions. *PR10* showed an oscillatory state that would seem to be influenced by the circadian rhythm. The results reported in Figure 4 shows a peak expression after 2 h of exposure to darkness, a tendency to decrease after 6 h of darkness, a strong reduction in the first 2 h of exposure to light and faint up-regulation after 10 h of exposure to light, followed by and a subsequent down-regulation of expression (Figure 4). When plantlets were exposed to continuous light (Figure S2), the expression of the *PR1* was approximately doubled after 24 h (Figure 5a) while the expression of the *PR10*, To highlight the role of the internal clock in regulating the in vitro expression of *PR1* and *PR10* genes in Iranian pear cultivar *Dar Gazi-wt*, AtPHYB and LeCRY1 overexpressed lines; plantlets were initially exposed to a photoperiod of 16 h/8 h (light/darkness, Figure S1). In *Dar Gazi-wt*, the expression of the *PR1* gene was not oscillatory, keeping an almost constant level of expressed transcripts throughout the day. The level of *PR1* transcripts was, less than *PR10* transcripts during the day, irrespective of the lighting conditions. *PR10* showed an oscillatory state that would seem to be influenced by the circadian rhythm. The results reported in Figure 4 shows a peak expression after 2 h of exposure to darkness, a tendency to decrease after 6 h of darkness, a strong reduction in the first 2 h of exposure to light and faint up-regulation after 10 h of exposure to light, followed by and a subsequent down-regulation of expression (Figure 4). *Plants* **2021**, *10*, x FOR PEER REVIEW 22 of 23

**Figure 4.** *PR1* and *PR10* expression in *Dar Gazi-wt.* The results are presented after normalization with *ef1A*. The average was generated by two biological replicates run in triplicate. Error bars represent SD. Within the sampling time point, the asterisk indicated a statistically significant difference compared to the highest values of each gene (Student's *t*-test, *p* ≤ 0.05). The bars under the horizontal axis show the light and dark periods, respectively. **Figure 4.** *PR1* and *PR10* expression in *Dar Gazi-wt.* The results are presented after normalization with *ef1A*. The average was generated by two biological replicates run in triplicate. Error bars represent SD. Within the sampling time point, the asterisk indicated a statistically significant difference compared to the highest values of each gene (Student's *t*-test, *p* ≤ 0.05). The bars under the horizontal axis show the light and dark periods, respectively.

**Figure 5.** *PR1* and *PR10* expression in *Dar Gazi-wt* plantlets: (**a**) in 24 h continuous light (WL); (**b**) in 24 h continuous darkness. The results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. For each single gene expression pattern, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05).

*2.5. PR1 and PR10 Expression in CRY1 and PHYB Overexpressing Lines in WL*

Data for the *PR1* expression in the plantlets of *Dar Gazi-cry1* line indicate that the photoreceptor CRY1 plays a role in the regulatory system of this gene (Figures 6 and S3). Under darkness, in the plantlets of this line, the detected transcripts increased up to 3 times those detected in the plantlets of Dar Gazy-wt. Moreover, a semi-oscillatory rhythm would seem to be evocated by the increased presence of CRY1 in the plantlet tissues, strongly upregulating the expression of *PR1*. From these results, it was evident that BL plays a role as overexpressed CRY1 emphasizes this aspect. The role of RL turns out to be different than that of BL, as can be seen in the plantlets of the PHYB-overexpressing line (Figure 6). The peak expression of *PR1* during the darkness period was approximately 8 fold greater in the transformed lines relative to the wt-line, comparable to that detected in the plantlets of the *Dar Gazi-cry1*. During the light period, the behavior of gene expression

Uppercase and lowercase letters are referred to as *PR1* and *PR10*, respectively.

When plantlets were exposed to continuous light (Figure S2), the expression of the *PR1* was approximately doubled after 24 h (Figure 5a) while the expression of the *PR10*, instead, decreased to around zero. This behavior has prevented the peak of expression to be visible after 2 h of exposure to darkness, although a small peak after 10 h of light was observed. Therefore, the expression profile of *PR10* would seem to be independent of the internal clock since the course no longer follows the oscillations previously seen during alternating darkness and light. In fact, in the absence of environmental time cues, circadian rhythms should persist with a period close to 24 h. Under conditions of continuous darkness (Figure 5b and Figure S2), *PR10* expression was stimulated and showed an oscillatory profile that partially resembles what had been observed under photoperiodic conditions. Under continuous darkness, *PR1 r*emains at lower levels than *PR10*, showing the same expression behavior detected during constant light. **Figure 4.** *PR1* and *PR10* expression in *Dar Gazi-wt.* The results are presented after normalization with *ef1A*. The average was generated by two biological replicates run in triplicate. Error bars represent SD. Within the sampling time point, the asterisk indicated a statistically significant difference compared to the highest values of each gene (Student's *t*-test, *p* ≤ 0.05). The bars under the horizontal axis show the light and dark periods, respectively.

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**Figure 5.** *PR1* and *PR10* expression in *Dar Gazi-wt* plantlets: (**a**) in 24 h continuous light (WL); (**b**) in 24 h continuous darkness. The results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. For each single gene expression pattern, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). Uppercase and lowercase letters are referred to as *PR1* and *PR10*, respectively. **Figure 5.** *PR1* and *PR10* expression in *Dar Gazi-wt* plantlets: (**a**) in 24 h continuous light (WL); (**b**) in 24 h continuous darkness. The results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. For each single gene expression pattern, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p* ≤ 0.05). Uppercase and lowercase letters are referred to as *PR1* and *PR10*, respectively.

#### *2.5. PR1 and PR10 Expression in CRY1 and PHYB Overexpressing Lines in WL 2.5. PR1 and PR10 Expression in CRY1 and PHYB Overexpressing Lines in WL*

Data for the *PR1* expression in the plantlets of *Dar Gazi-cry1* line indicate that the photoreceptor CRY1 plays a role in the regulatory system of this gene (Figures 6 and S3). Under darkness, in the plantlets of this line, the detected transcripts increased up to 3 times those detected in the plantlets of Dar Gazy-wt. Moreover, a semi-oscillatory rhythm would seem to be evocated by the increased presence of CRY1 in the plantlet tissues, strongly upregulating the expression of *PR1*. From these results, it was evident that BL plays a role as overexpressed CRY1 emphasizes this aspect. The role of RL turns out to be different than that of BL, as can be seen in the plantlets of the PHYB-overexpressing line (Figure 6). The peak expression of *PR1* during the darkness period was approximately 8 fold greater in the transformed lines relative to the wt-line, comparable to that detected in the plantlets of the *Dar Gazi-cry1*. During the light period, the behavior of gene expression Data for the *PR1* expression in the plantlets of *Dar Gazi-cry1* line indicate that the photoreceptor CRY1 plays a role in the regulatory system of this gene (Figures 6 and S3). Under darkness, in the plantlets of this line, the detected transcripts increased up to 3 times those detected in the plantlets of Dar Gazy-wt. Moreover, a semi-oscillatory rhythm would seem to be evocated by the increased presence of CRY1 in the plantlet tissues, strongly upregulating the expression of *PR1*. From these results, it was evident that BL plays a role as overexpressed CRY1 emphasizes this aspect. The role of RL turns out to be different than that of BL, as can be seen in the plantlets of the PHYB-overexpressing line (Figure 6). The peak expression of *PR1* during the darkness period was approximately 8-fold greater in the transformed lines relative to the wt-line, comparable to that detected in the plantlets of the *Dar Gazi-cry1*. During the light period, the behavior of gene expression in the plantlets of *Dar Gazi-phyB* is similar to that seen in plantlets of the *Dar Gazi-wt*. During darkness, even in the plantlets of *Dar Gazi-phyB* the *PR1* transcripts level was significantly higher than in the plantlets of *Dar Gazi-wt* (Figure 6).

nificantly higher than in the plantlets of *Dar Gazi-wt* (Figure 6).

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in the plantlets of *Dar Gazi-phyB* is similar to that seen in plantlets of the *Dar Gazi-wt*. During darkness, even in the plantlets of *Dar Gazi-phyB* the *PR1* transcripts level was sig-

in the plantlets of *Dar Gazi-phyB* is similar to that seen in plantlets of the *Dar Gazi-wt*. During darkness, even in the plantlets of *Dar Gazi-phyB* the *PR1* transcripts level was sig-

**Figure 6.** *PR1* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). The bars under the horizontal axis show the light and dark periods. The analysis of *PR10* gene expression indicated that, in the plantlets of the three *Dar*  **Figure 6.** *PR1* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p* ≤ 0.05). The bars under the horizontal axis show the light and dark periods. **Figure 6.** *PR1* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). The bars under the horizontal axis show the light and dark periods.

*Gazi* lines, the overexpression of each photoreceptor gene drastically reduces the amount of transcript detected (Figures 7 and S3). Furthermore, the oscillatory rhythm detect in the plantlets of *Dar Gazi-wt* results was almost repressed. However, in the overexpressing of PHYB plantlets, expression was maintained in the first 2 h of darkness. The analysis of *PR10* gene expression indicated that, in the plantlets of the three *Dar Gazi* lines, the overexpression of each photoreceptor gene drastically reduces the amount of transcript detected (Figures 7 and S3). Furthermore, the oscillatory rhythm detect in the plantlets of *Dar Gazi-wt* results was almost repressed. However, in the overexpressing of PHYB plantlets, expression was maintained in the first 2 h of darkness. The analysis of *PR10* gene expression indicated that, in the plantlets of the three *Dar Gazi* lines, the overexpression of each photoreceptor gene drastically reduces the amount of transcript detected (Figures 7 and S3). Furthermore, the oscillatory rhythm detect in the plantlets of *Dar Gazi-wt* results was almost repressed. However, in the overexpressing of PHYB plantlets, expression was maintained in the first 2 h of darkness.

after normalization with *ef1A*. Data shown as the average of two biological replicates run in tripli-

Under continuous light, although the transcription rate of the *PR1* gene in the plantlets of the *Dar Gazi-cry1* was higher than that in plantlets of the *Dar Gazi-wt*, the behavior of transcription was different than under photoperiodic conditions (Figure 8a). A faint increase in the amount of transcript was detected after 24 h of exposure to continuous light. On the other hand, the *PR1* gene expression course in the plantlets of the *Dar GaziphyB*, under exposure to constant light, was similar to that detected under photoperiodic conditions (Figure 8a).

conditions (Figure 8a).

**Figure 8.** *PR1* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous light, panel; (**b**) in 24 h continuous darkness. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). **Figure 8.** *PR1* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous light, panel; (**b**) in 24 h continuous darkness. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point, values with different letters significantly differ according to the analysis of variance (ANOVA) and least significant difference (LSD) tests (*p* ≤ 0.05).

cally reduced in plantlets of both transgenic lines (Figure 9a). In the plantlets of the *Dar Gazi-wt*, an oscillatory behavior was detected, more pronounced in continuous darkness than continuous light (Figure 9b). The results suggest that the overexpression of the photoreceptors, irrespective of light conditions, strongly inhibits the expression of the *PR10* gene. The amount of transcript detected in the plantlets of the *Dar Gazi-wt* indicates that the physiological expression of photoreceptors could play a relevant role in permitting the oscillatory expression of the *PR10* gene. In conditions of continuous darkness, the transcription levels of the *PR1* gene were strongly increased in the tissue of *Dar Gazi-cry1* and *Dar Gazi-phyB*. In contrast, the level of transcript detected in the *Dar Gazi-wt* was very low but did not differ from that seen in continuous light and under photoperiodic conditions (Figure 8b). The highest amount of transcript in the *Dar Gazi-cry1* plantlets was found after 2 h of exposure to the darkness, thereafter, the amount of transcript decreased (Figure 8b). The highest amount of transcript in the plantlets *Dar Gazi-phyB* was found after 10 h of exposure to darkness, but after 24 h, the amount of transcript was the lowest (Figure 8b). Thus, the darkness condition induces always-high *PR1* gene transcription levels in the plantlets of the *Dar Gazi-cry1*. A similar trend was also observed in the plantlet of *Dar Gazi-phyB*, even if at a reduced level.

Under continuous light and darkness, the *PR10* gene expression level was dramati-

Under continuous light, although the transcription rate of the *PR1* gene in the plantlets of the *Dar Gazi-cry1* was higher than that in plantlets of the *Dar Gazi-wt*, the behavior of transcription was different than under photoperiodic conditions (Figure 8a). A faint increase in the amount of transcript was detected after 24 h of exposure to continuous light. On the other hand, the *PR1* gene expression course in the plantlets of the *Dar GaziphyB*, under exposure to constant light, was similar to that detected under photoperiodic

In conditions of continuous darkness, the transcription levels of the *PR1* gene were strongly increased in the tissue of *Dar Gazi-cry1* and *Dar Gazi-phyB*. In contrast, the level of transcript detected in the *Dar Gazi-wt* was very low but did not differ from that seen in continuous light and under photoperiodic conditions (Figure 8b). The highest amount of transcript in the *Dar Gazi-cry1* plantlets was found after 2 h of exposure to the darkness, thereafter, the amount of transcript decreased (Figure 8b). The highest amount of transcript in the plantlets *Dar Gazi-phyB* was found after 10 h of exposure to darkness, but after 24 h, the amount of transcript was the lowest (Figure 8b). Thus, the darkness condition induces always-high *PR1* gene transcription levels in the plantlets of the *Dar Gazi-cry1*. A similar trend was also observed in the plantlet of *Dar Gazi-phyB*, even if at a reduced level.

Under continuous light and darkness, the *PR10* gene expression level was dramatically reduced in plantlets of both transgenic lines (Figure 9a). In the plantlets of the *Dar Gazi-wt*, an oscillatory behavior was detected, more pronounced in continuous darkness than continuous light (Figure 9b). The results suggest that the overexpression of the photoreceptors, irrespective of light conditions, strongly inhibits the expression of the *PR10* gene. The amount of transcript detected in the plantlets of the *Dar Gazi-wt* indicates that the physiological expression of photoreceptors could play a relevant role in permitting the oscillatory expression of the *PR10* gene. *Plants* **2021**, *10*, x FOR PEER REVIEW 22 of 23

**Figure 9.** *PR10* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous light, panel; (**b**) in 24 h continuous darkness. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point values with different letters significantly differ according to the analysis of the variance (ANOVA) and least significant difference (LSD) tests (*p*  ≤  0.05). **Figure 9.** *PR10* expression in *Dar Gazi-wt*, *Dar Gazi-phyB*, and *Dar Gazi-cry1*: (**a**) in 24 h continuous light, panel; (**b**) in 24 h continuous darkness. Results are presented after normalization with *ef1A*. Data shown as the average of two biological replicates run in triplicate, with error bars representing SD. At each time point values with different letters significantly differ according to the analysis of the variance (ANOVA) and least significant difference (LSD) tests (*p* ≤ 0.05).

*2.6. PR1 and PR10 Expression in Dar Gazi-cry1 and Dar Gazi-phyB in RL, FRL, and BL*

Studying the role of photoreceptors in the regulation of the expression of the PRs, phytochrome has a pivotal role in regulating the internal clock and the perception of the photoperiod. The expression level of the *PR1* gene in plantlets *Dar Gazi-phyB* exposed to

*Dar Gazi-cry1* plantlets, the transcript level was constant, at a very low level of expression. Therefore, the photoconversion of phytochrome from the inactive (Pr) to the active form

As determined by exposing plantlets to continuous FRL (Figure 10b), the inactive form of phytochrome inhibits the expression of the *PR1* gene in *Dar Gazi-phyB* plantlets. The inactive form of phytochrome, and probably the amount of PHYB protein, either generates or allows an oscillatory behavior of the expression of the *PR1* gene in the plantlets of wt-line and the plantlets of *Dar Gazi-cry1*. The expression of *PR1* in *Dar Gazi-cry1* increases at a high level after 10 h of continuous FRL. Results, therefore, show that *PR1* expression was promoted by CRY1 activity the and the circadian rhythms are present

Under continuous BL conditions (Figure 10c), the highest level of *PR1* expression in *Dar Gazi-cry1* plantlets was reached after 6 h of exposure to light. An oscillatory behavior appeared in plantlets of *Dar Gazi-wt*, while in plantlets of *Dar Gazi-phyB* a very low ex-

pression rate without any oscillatory behavior was observed.

(Pfr) should play a permissive role (Figure 10a,b).

again.
