Ubiquitin ligase switch in plant photomorphogenesis: A hypothesis

Abstract

The E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC1) plays a key role in the repression of the plant photomorphogenic development in darkness. In the presence of light, COP1 is inactivated by a mechanism which is not completely understood. This leads to accumulation of COP1's target transcription factors, which initiates photomorphogenesis, resulting in dramatic changes of the seedling's physiology. Here we use a mathematical model to explore the possible mechanism of COP1 modulation upon dark/light transition in Arabidopsis thaliana based upon data for two COP1 target proteins: HY5 and HFR1, which play critical roles in photomorphogenesis. The main reactions in our model are the inactivation of COP1 by a proposed photoreceptor-related inhibitor I and interactions between COP1 and a CUL4 (CULLIN4)-based ligase. For building and verification of the model, we used the available published and our new data on the kinetics of HY5 and HFR1 together with the data on COP1 abundance. HY5 has been shown to accumulate at a slower rate than HFR1. To describe the observed differences in the timecourses of the "slow" target HY5 and the "fast" target HFR1, we hypothesize a switch between the activities of COP1 and CUL4 ligases upon dark/light transition, with COP1 being active mostly in darkness and CUL4 in light. The model predicts a bi-phasic kinetics of COP1 activity upon the exposure of plants to light, with its restoration after the initial decline and the following slow depletion of the total COP1 content. CUL4 activity is predicted to increase in the presence of light. We propose that the ubiquitin ligase switch is important for the complex regulation of multiple transcription factors during plants development. In addition, this provides a new mechanism for sensing the duration of light period, which is important for seasonal changes in plant development.

Fig. 7

The reaction scheme of the full model in SBGN format. The scheme was draw using Edinburgh Pathway Editor (EPE), which is freely available from http://sourceforge.net.

Fig. 8

The phase diagrams of the full system under various light conditions. The panels correspond to the constant light (A), constant darkness (B) and diurnal 12 L:12 D light/dark cycles (C). The direction of the trajectories are shown by arrows, black points correspond to steady states and limit cycle is shown by gray line. The simulations were done in MATLAB.

Fig. 9

Relative changes in the kinetics of the model after dark-to-light transition for 10% increase of each parameter of the COP1/CUL4 module. The following kinetic characteristics are shown: The value of the first sharp fall in COP1 activity (COP1min) and its time (COP1t); values of the maximum of HFR1 and HY5 proteins (HFR1max and HY5max); steady state value of the total COP1 content (COP1tot).

Fig. 10

Dynamics of the model after dark-to light transition (at time 0) upon variation of the rate constant of COP1 inactivation by light (k₀). Parameter k₀ was increased/decreased by order of 2 from its optimal value (k₀⁎, marked by thick lines). A, B, C panels show the kinetics of HFR1, HY5 proteins and COP1 activity, respectively.

Fig. 1

Timecourse of HY5 protein and mRNA upon dark-to-light transition. Seedlings were grown for 4 days in darkness and transferred to constant light at time 0. A: A western blot of protein extracts from wt and HY5-overexpressor line, probed with anti-HY5. B: A representative anti-HY5 western blot from wt seedlings, which were grown in darkness for 4 days and then transferred to light at time 0. Protein extracts were done at indicated time points. Tubulin protein was used as a loading control. The experiments were repeated three times with similar results. C: Quantification of the western blot, shown in B. Prior to quantification, the quantitative linear range of detection was determined by a series of dilutions on Western blots as described previously (Khanna et al., 2007). D: HY5 expression was analyzed by real-time PCR after dark-to light transition (see Experimental Methods in Appendix).

Fig. 2

Schematic description of the mathematical models of the ligase switch. Light inputs are shown by yellow flashes, which stimulate HY5 expression and inactivation of COP1 activity through the activation of inhibitor I₀ in complex with protein P. Activated inhibitor is denoted as I. Arrows without starting or terminal substances correspond to production/translation or degradation of the corresponding proteins. The targeted degradation of HY5 and HFR1 proteins by the active ligases is shown by red dotted lines. A: Simple model (Scheme 1) with only one-sided negative regulation of CUL4 by active COP1 (blue dotted connection). B: Full model (Scheme 2) with two-sided mutual negative regulation of COP1 and CUL4 (blue lines). The dissociation of the COP1-inhibitor complexes and targeted degradation of the free COP1 (COP1_f) by CUL4 are included. Negative regulation of HY5 expression by COP1 is shown (green line). The accelerated inactivation of CUL4 ligase in darkness is shown by the black flash. The detailed scheme of the full model in SBGN format is given in Fig. 7 of the Appendix.

Fig. 3

The simulated kinetics of the simple model (Scheme 1) upon dark-to-light transition. The activities of COP1 and CUL4 ligases are shown by green and magenta lines, respectively. The kinetics of HFR1 and HY5 proteins and HY5 mRNA are shown by blue, black and red lines, respectively. Experimental points for HFR1 protein (blue) are taken from (Duek 04) and for HY5 protein (black)—from this paper (Fig. 1C). The simulation was run starting from initial conditions, which correspond to the steady state of the system in darkness: c_COP1i=0.2; c_COP1a=0.3; c_P=1; c_CULi=0.594; c_CULa=0.406; $c_{HY5}^m=0.167$; c_HY5=0.167; c_HFR1=0.183.

Fig. 4

The simulated kinetics of the full model (Scheme 2) upon dark-to-light transition. A: The activities of COP1 and CUL4 ligases are shown by green and magenta lines, respectively. The kinetics of HFR1 and HY5 proteins and HY5 mRNA are shown by blue, black and red lines, respectively. Experimental data points—as in Fig. 3. B: The kinetics of different forms of COP1 (red) and CUL4 (blue): Active forms are shown by dotted lines, inactive forms—by dashed lines, free COP1—by dashed-dotted line, total content—by solid lines. The simulation was run starting from initial conditions, which correspond to the steady state of the system in darkness: c_COP1i=0; c_COP1a=0.737; c_I=0; c_P=1; c_COP1f=0.28; c_CULi=0.911; c_CULa=0.089; $c_{HY5}^m=0.081$; c_HY5=0.266; c_HFR1=0.1.

Fig. 5

The simulated kinetics of the full model upon light-to-dark transitions. A: The activities of COP1 (green) and CUL (magenta) ligases and HFR1 protein kinetics (blue) after the transition of dark-grown plants, which were exposed to 2h of light, back to darkness. The data for HFR1 protein was taken from (Duek et al., 2004). The initial conditions, which correspond to the state of the system after 2 h of light, were c_COP1i=0.549; c_COP1a=0.086; c_I=0.358; c_P=0.018; c_COP1f=0.033; c_CULi=0.055; c_CULa=0.945; $c_{HY5}^m=0.946$; c_HY5=0.814; c_HFR1=1. B: The kinetics of HY5 protein (black) after the transition of the light-grown plants to darkness. The activities of CUL4 and COP1 ligases are shown by magenta and green solid lines, respectively. The total CUL4 and COP1 contents are shown by dashed line. The simulation was run starting from initial conditions, which correspond to the steady state of the system in the presence of light: c_COP1i=0; c_COP1a=0.217; c_I=0; c_P=0; c_COP1f=0.028; c_CULi=0.098; c_CULa=0.902; $c_{HY5}^m=0.789$; c_HY5=1.005; c_HFR1=0.341.

Fig. 6

Simulated kinetics of the model components under various photoperiods. The simulations were initially run for 4 days under each photoperiod to entrain the system, so that only 5th and 6th days are shown. A, B: Solid, dashed and dashed-dotted lines correspond to 6 L:18 D, 12 L:12 D and 18 L:6 D light–dark cycles, respectively. Blue, black, green and magenta colors show the kinetics of HFR1, HY5 proteins and the activity of COP1 and CUL4 respectively. C: Simulated kinetics of the hypothetical COP1 (blue) and CUL4 (red) substrates under 12L:12D. HFR1 equation was used for COP1 substrate with the following parameter values: p₅=0.28 h⁻¹; h₇=2 h⁻¹. HY5 protein equation was used for CUL4 substrate with constant expression level p₄ and the following parameter values: p₄=0.22 h⁻¹; h₄=1 h⁻¹; h₅=0. The rest of the parameters are shown in Table 2 of the Appendix.