Vernalizing cold is registered digitally at FLC

Abstract

A fundamental property of many organisms is an ability to sense, evaluate, and respond to environmental signals. In some situations, generation of an appropriate response requires long-term information storage. A classic example is vernalization, where plants quantitatively sense long-term cold and epigenetically store this cold-exposure information to regulate flowering time. In Arabidopsis thaliana, stable epigenetic memory of cold is digital: following long-term cold exposure, cells respond autonomously in an all-or-nothing fashion, with the fraction of cells that stably silence the floral repressor flowering locus C (FLC) increasing with the cold exposure duration. However, during cold exposure itself it is unknown whether vernalizing cold is registered at FLC in individual cells in an all-or-nothing (digital) manner or is continuously varying (analog). Using mathematical modeling, we found that analog registration of cold temperature is problematic due to impaired analog-to-digital conversion into stable memory. This disadvantage is particularly acute when responding to short cold periods, but is absent when cold temperatures are registered digitally at FLC. We tested this prediction experimentally, exposing plants to short periods of cold interrupted with even shorter warm breaks. For FLC expression, we found that the system responds similarly to both interrupted and uninterrupted cold, arguing for a digital mechanism integrating long-term temperature exposure.

Keywords: FLC; analog; digital; temperature; vernalization.

Fig. 1.

(A–C) Schematic plots of NR peak profile shown for three representative cells in the population for (A) analog, (B) digital class 1, and (C) digital class 2 models as a function of cold exposure duration.

Fig. 2.

(A) Experimentally measured H3K27me3 levels in NR (peak), and elsewhere across FLC (body), during and 7 d after cold for uninterrupted cold periods of 2 wk, 4 wk, 6 wk, and 8 wk. Also shown are simulated H3K27me3 levels for digital and analog models. (B) Experimentally measured levels of spliced FLC mRNA (expression) 7 d after cold relative to nonvernalized (NV), i.e., with no exposure to cold, values for uninterrupted cold periods of 2 wk, 4 wk, 6 wk, and 8 wk. Also shown are simulated relative mRNA levels for digital and analog models. Experimental error bars are SEM, with n = 5 for body H3K27me3 levels; n = 7, 7, 9, 5, and 8 for peak levels immediately after 0 wk, 2 wk, 4 wk, 6 wk, and 8 wk cold; n = 7, 8, 5, and 7 for peak levels following 2 wk, 4 wk, 6 wk, and 8 wk cold and a 7-d period of warm; and with n = 8, 13, 10, and 8 for relative expression 7 d after 2 wk, 4 wk, 6 wk, and 8 wk cold, respectively.

Fig. 3.

(A) Probability that the cell switches to the silenced state following cold as a function of population-averaged H3K27me3 NR peak level for digital and analog models. Plots start at the basal level of H3K27me3 present in all simulated nonvernalized cells in the nucleation region and end at maximum H3K27me3 nucleation level. (B) Experimental H3K27me3 NR peak profile as function of cold exposure duration (red dashed line and data points, as in Fig. 2). Solid blue line is simulated H3K27me3 NR peak profile, making the analog model as effective as the digital class 1 model.

Fig. 4.

(A) Postcold spliced mRNA relative to nonvernalized (NV) levels from analog (an) and digital models (both classes, d1 and d2), compared with experiments (ex). Uninterrupted cold is compared with (Left) one, (Center) two, and (Right) three interruptions; e.g., 2 × 7 d refers to two periods of 7 d cold duration, with a warm interruption in between, compared with 14 d of uninterrupted cold. Error bars are SEM, with n = 3 (Left) and n = 2 (Center and Right) for experiments and n = 10 for simulations. (B) Leaf number as a measurement of flowering time, for two and three interruption treatments. Box and whisker plots show median, 25th, and 75th percentiles and range of data excluding any outliers, which are represented as individual points; n = 12 for each set of measurements.

Fig. 5.

Conceptual model for switching in a symmetric bistable system. (A) Potential landscape showing two stable expression states (x = ±1), with switching threshold (x = 0). (B) Configuration before cold exposure, with distribution of states around the most stable value. (C) In the analog model, at end of the cold period, cells corresponding to the part of the distribution beyond threshold (dark red) “roll” down to the silenced state. (D) In the digital class 1 model, cells that have digitally nucleated (not nucleated) will be distributed around x = 1 (x = −1), respectively.