The LNK Gene Family: At the Crossroad between Light Signaling and the Circadian Clock

Abstract

Light signaling pathways interact with the circadian clock to help organisms synchronize physiological and developmental processes to periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Members of the family of NIGHT LIGHT⁻INDUCIBLE AND CLOCK-REGULATED (LNK) genes play key roles linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana. Particularly, LNK1 and LNK2 were shown to control circadian rhythms, photomorphogenic responses, and photoperiod-dependent flowering time. Here we analyze the role of the four members of the LNK family in Arabidopsis in these processes. We found that depletion of the closely related LNK3 and LNK4 in a lnk1;lnk2 mutant background affects circadian rhythms, but not other clock-regulated processes such as flowering time and seedling photomorphogenesis. Nevertheless, plants defective in all LNK genes (lnkQ quadruple mutants) display developmental alterations that lead to increased rosette size, biomass, and enhanced phototropic responses. Our work indicates that members of the LNK family have both distinctive and partially overlapping functions, and are an essential link to orchestrate light-regulated developmental processes.

Keywords: NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED; circadian activators; circadian clock; light signaling; phototropism.

Figure 1

The NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED (LNK) family exhibits partial redundancy in the control of circadian rhythms. (a) Circadian rhythms of leaf movement in continuous light (LL), after entrainment under long-day conditions. (b) CCA1::LUC activity measured for 5 days in LL, after entrainment under long-day conditions. Bioluminescence was recorded every 2 h over 6 days. Periods of circadian rhythms in leaf movement and CCA1::LUC activity were estimated with BRASS 3.0 software (Biological Rhythms Analysis Software System). Error bars indicate standard error of the mean (SEM). Student’s t-test was performed between mutants and wild-type (WT) (*: significantly different, p ≤ 0.05; **: significantly different, p ≤ 0.01; ***: significantly different, p ≤ 0.001; ns: not significant).

Figure 2

LNK1 and LNK2, but not LNK3 or LNK4, play a role in the photoperiodic control of flowering. Flowering time measured as the number of rosette leaves at bolting in (a) long days (LD; 16:8), (b) 12 h light: 12 h dark cycles (LD; 12:12), and in (c) short days (SD; 8:16). Error bars indicate SEM. Student’s t-test was performed between mutants and WT (*: significantly different, p ≤ 0.05; **: significantly different, p ≤ 0.01; ***: significantly different, p ≤ 0.001; ns: not significant).

Figure 3

Role of the LNK family in the control of photomorphogenesis. Hypocotyls of WT and LNK mutants grown under different wavelengths and photoperiods; for continuous red and blue light measurements are expressed relative to the dark control; for white light treatments absolute values are displayed. (a) Continuous red light (10 μmol·m⁻² s⁻¹). (b) Continuous blue light (2 μmol·m⁻² s⁻¹). (c) Short-day photoperiod in white light (1 μmol·m⁻² s⁻¹; SD; 8:16). (d) 12 h light:12 h dark cycles in white light (1 μmol·m⁻² s⁻¹; LD; 12:12). (e) Long-day photoperiod in white light (1 μmol·m⁻² s⁻¹; LD; 16:8). (f) Continuous white light (1 μmol·m⁻² s⁻¹). Error bars indicate SEM. Student’s t-test was performed between mutants and WT (*: significantly different, p ≤ 0.05; **: significantly different, p ≤ 0.01; ***: significantly different, p ≤ 0.001; ns: not significant).

Figure 4

LNK family members regulate the expression of central morning and evening clock genes. Mean expression levels were measured using quantitative PCR (qPCR) for the evening genes (a) TOC1 and (b) PRR5, as well as for the morning genes (c) CCA1 and (d) LHY. Four biological replicates were used for measuring mRNA abundance. The analysis was conducted after pooling samples collected every 4-h across a time-course series, in order to evaluate difference in absolute mRNA levels among genotypes, rather than differences resulting from alterations in the timing of gene expression. Error bars indicate SEM. Student’s t-test was performed between mutants and WT (*: significantly different, p ≤ 0.05; **: significantly different, p ≤ 0.01; ***: significantly different, p ≤ 0.001; ns: not significant).

Figure 5

LNK family members act as growth regulators. To assess the role of the LNK family in the control of growth in the vegetative stage, morphological traits of plants grown in short-day conditions were measured. (a) Rosette phenotype of 10-week old WT and lnk mutant plants grown in a short-day photoperiod. (b) Rosette perimeter measured after bolting. (c) Dry weight of 10-week old flowered adult plants, inflorescence stems removed. For leaf morphology characterization, plants were grown under long days, and at the 12th-leaf stage, the 8th leaf was cut and measured. (d) Representative 8th leaves of WT and lnk mutant plants. (e) Blade to whole leaf length ratio. (f) Petiole length in centimeters. Error bars indicate SEM. Student’s t-test was performed between mutant and WT plants (*: significantly different, p ≤ 0.05; **: significantly different, p ≤ 0.01; ***: significantly different, p ≤ 0.001; ns: not significant).

Figure 6

lnkQ mutants displayed normal shade avoidance but enhanced phototropic responses. Shade avoidance responses in WT plants and in lnk mutants. (a) Blade to whole leaf length ratio of the eighth leaf was determined in plants grown in long days and treated or not (control) with a far-red pulse of fifteen minutes after lights went off (end-of-day far-red treatment). (b) Hypocotyl length of seedlings grown in white light or white light with an end-of-day far-red treatment. (c) For the analysis of leaf angles in adult plants, six-week old plants grown in a short-day photoperiod were photographed from the side. The angle between two opposing leaves closest to the light source was measured. (d) The proportion of leaf blades perpendicular to the light source was determined in plants grown in long days with vertical illumination until the first pair of leaves appeared, and then exposed to a lateral light source until the plants developed 8–10 leaves. Error bars indicate SEM. Student’s t-test was performed between mutants and WT (*: significantly different, p ≤ 0.05; **: significantly different, p ≤ 0.01; ***: significantly different, p ≤ 0.001; ns: not significant).