Light signaling project

phyB and HY5 are involved in the BLUE light mediated alleviation of dormancy of Arabidopsis seeds possibly via the modulation of expression of genes related to light, GA, and ABA

 

Stawska and Oracz, Int. J. Mol. Sci., 2019, 20, 5882; doi:10.3390/ijms20235882

 

Light is one of the most important environmental factors regulating seed germination. It is known that light inhibits seed germination of some monocotyledonous species and that it is mostly related to the blue wavelength of the spectrum received by cryptochromes (cry). Research has also found that the red light (R) stimulates germination of dicotyledonous seeds and that this reaction involves mainly phytochromes (phy). Surprisingly, up to date, the role and the mechanism of action of blue light (BL) in seed biology of dicot plants is still very poorly understood and some questions are unexplained, e.g., whether BL plays a role in regulation of dicot seeds dormancy and/or germination? If, so what particular elements of light signaling pathway are involved in modulation of this(ese) process(es)? Also, is the BL action in regulation of dicot seeds dormancy and/or germination maybe due to changes of expression of genes related to metabolism and/or signaling of two phytohormones controlling seed-related events, such as gibberellins (GA) and abscisic acid (ABA)? To answer these intriguing questions, the combination of biological, transcriptomic, and genetic approaches was performed in this particular study. The germination tests show that freshly harvested wild type (WT) Arabidopsis thaliana Col-0 seeds are dormant and do not germinate in darkness (at 25 ◦C), while nondormant (after-ripened) seeds germinate well in these conditions. It is also proven that dormancy of seeds of this species is released in the presence of white and/or BL (λ = 447 nm) when placed at 25 ◦C.

 

Figure 1. Characteristic of germination of wildtype (WT) Arabidopsis (Col-0) seeds: nondormant (a) and dormant (b,c) incubated on water in darkness or under blue (BL) (range of intensities from 20 to 180 µmol m−2 s −1 ) or white light (intensity = 180 µmol m−2 s −1 ) in a constant temperature of 25 ◦C: Experiments were conducted in three biological and two technical replicates (100 seeds per each replicate), * and ** indicate the significant differences for p ≤ 0.05 and p ≤ 0.01 respectively. Results are presented as mean ± SD. (Stawska and Oracz Int. J. Mol. Sci. 2019, 20, 5882; doi:10.3390/ijms20235882)

 

Presented here, novel results emphasize the role of BL in dormancy alleviation of dicot seeds, indicating that this wavelength of light spectrum received by phyB induces this process and that the sensitivity to this stimulus depends on the depth of seed dormancy.

Figure 2. The relative expressions of genes encoding photoreceptors CRY1, CRY2, and CRY3 (a) and PHYA and PHYB (b) in samples isolated from WT dormant Arabidopsis (Col-0) seeds incubated on water for 20, 36, and 48 h in different light conditions (BL and white light of intensity = 180 µmol m−2 s −1 , darkness) at a temperature of 25 °C: The transcript level was normalized to reference genes (ACT7, APC2, and HBT) and to the internal control, which was the expression value of CRY1 (Figure 2a) or PHYA (Figure 2b) obtained in darkness after 20 h of incubation. Three biological and two technical replicates were performed. The bars show the relative units ± SD. The effect of far red (FR) on the regulation of WT dormant Arabidopsis (Col-0) seeds germination by BL light (c). Seeds were incubated on water in constant temperature of 25 °C at various light conditions. The results are presented as germination % of WT dormant Arabidopsis (Col-0) seeds after 48, 72, and 168 h of incubation. For BLdependent germination assay, seeds were exposed to 5 min of FR light irradiation (intensity = 60 µmol m−2 s −1 ) after 12 h of pre-imbibition in the presence of BL (intensity = 180 µmol m−2 s −1 ). (d) The germination % of WT dormant seeds (in Col-0 background: WT Col–0, phyA, phyB, cry1, and cry3; in Ler-0 ecotype: WT Ler-0 and cry1cry2) after 168 h of incubation on water in the presence of constant BL and white light (intensity = 180 µmol m−2 s −1 ) or in darkness at temperature of 25 °C. Germination assays were performed in three biological and two technical replicates (100 seeds per each replicate). Results are presented as mean ± SD), ** indicates the significant differences for p ≤ 0.05 and p ≤ 0.01 respectively. Figure 2. The relative expressions of genes encoding photoreceptors CRY1, CRY2, and CRY3 (a) and PHYA and PHYB (b) in samples isolated from WT dormant Arabidopsis (Col-0) seeds incubated on water for 20, 36, and 48 h in different light conditions (BL and white light of intensity = 180 µmol m−2 s −1 , darkness) at a temperature of 25 ◦C: The transcript level was normalized to reference genes (ACT7, APC2, and HBT) and to the internal control, which was the expression value of CRY1 (Figure 2a) or PHYA (Figure 2b) obtained in darkness after 20 h of incubation. Three biological and two technical replicates were performed. The bars show the relative units ± SD. The effect of far red (FR) on the regulation of WT dormant Arabidopsis (Col-0) seeds germination by BL light (c). Seeds were incubated on water in constant temperature of 25 ◦C at various light conditions. The results are presented as germination % of WT dormant Arabidopsis (Col-0) seeds after 48, 72, and 168 h of incubation. For BL-dependent germination assay, seeds were exposed to 5 min of FR light irradiation (intensity = 60 µmol m−2 s −1 ) after 12 h of pre-imbibition in the presence of BL (intensity = 180 µmol m−2 s −1 ). (d) The germination % of WT dormant seeds (in Col-0 background: WT Col–0, phyA, phyB, cry1, and cry3; in Ler-0 ecotype: WT Ler-0 and cry1cry2) after 168 h of incubation on water in the presence of constant BL and white light (intensity = 180 µmol m−2 s −1 ) or in darkness at temperature of 25 ◦C. Germination assays were performed in three biological and two technical replicates (100 seeds per each replicate). Results are presented as mean ± SD), ** indicates the significant differences for p ≤ 0.05 and p ≤ 0.01 respectively. (Stawska and Oracz Int. J. Mol. Sci. 2019, 20, 5882; doi:10.3390/ijms20235882)

 

In addition, it is demonstrated that various elements of phy-mediated pathway can be used in response to the signal induced by BL in germinating dormant seeds of Arabidopsis. The quantitative real time PCR analysis supported by results of germination tests of WT, T-DNA insertion mutants (i.e., hy5, hfr1, and laf1) and overexpression transformants of Arabidopsis seeds (i.e., 35S:OE:HY5, 35S:OE:HYH, 35S:OE:HFR1, and 35S:OE:LAF1) revealed that the HY5 gene coding transcription factor is most probably responsible for the control of expression of genes involved in GA/ABA metabolism and/or signaling pathways during BL-dependent dormancy alleviation of Arabidopsis seeds, while biological functions of HYH and HFR1 are associated with regulation of germination. The model of BL action in regulation of dormancy alleviation and germination potential of Arabidopsis seeds is proposed.

The hypothetical model of BL action in regulation of dormancy alleviation and germination potential of dormant Arabidopsis seeds: The BL downregulates the expression of PHYA but not PHYB neither CRY1/2 photoreceptors. Decreased PHYA expression is associated with a possible low HY5 accumulation and/or its activity, leading to direct or indirect decrease in expression of genes encoding dormancy related genes. On the other hand, as the effect of BL illumination on the phyB and cry1/2 controlled by phyB, CIB1/5, and PHR2, the SUB1 and COP1 activity is suppressed, leading to HYH–- and HFR1-mediated activation of expression of specific genes involved in metabolism and signaling of GA and ABA, resulting in increase of germination potential. Dotted lines indicate hypothetical, possible interactions. Black arrows next to GA- and ABA-related genes indicate up- and downregulation (Stawska and Oracz Int. J. Mol. Sci. 2019, 20, 5882; doi:10.3390/ijms20235882).

 

Funding: This research was funded by the National Science Centre (Poland), grant SONATA2 number: 2011/03/D/NZ9/04059, and grant PRELUDIUM12 (number 2016/23/N/NZ3/02239).