Time-resolved interaction proteomics of the GIGANTEA protein under diurnal cycles in Arabidopsis

Abstract

The plant-specific protein GIGANTEA (GI) controls many developmental and physiological processes, mediating rhythmic post-translational regulation. GI physically binds several proteins implicated in the circadian clock, photoperiodic flowering, and abiotic stress responses. To understand GI's multifaceted function, we aimed to comprehensively and quantitatively identify potential interactors of GI in a time-specific manner, using proteomics on Arabidopsis plants expressing epitope-tagged GI. We detected previously identified (in)direct interactors of GI, as well as proteins implicated in protein folding, or degradation, and a previously uncharacterized transcription factor, CYCLING DOF FACTOR6 (CDF6). We verified CDF6's direct interaction with GI, and ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LIGHT KELCH PROTEIN 2 proteins, and demonstrated its involvement in photoperiodic flowering. Extending interaction proteomics to time series provides a data resource of candidate protein targets for GI's post-translational control.

Keywords: Arabidopsis thaliana; affinity purification; circadian rhythms; flowering time; quantitative mass spectrometry.

Figure 1

Evaluation of the GI‐3F6H complementation line. (A,C,D) mRNA expression was tested in samples of Arabidopsis plants of the Col‐0 WT (black open squares), gi‐2 mutant (blue crosses) and gi‐2 plants constitutively expressing the GI‐3F6H fusion protein (red filled circles) under long‐day conditions. qPCR assays detected GI (A), CO (C) or FT (D) mRNA. Data are means of biological triplicates, normalized to an IPP2 internal control; error bar, SEM. (B) Rosette leaf number was measured (data in bold) at flowering time in GI‐3F6H, with WT and parental gi‐2 mutant controls, under long‐day conditions. The flowering experiment was simulated using the FMv2 (pale colors). Data are averages of 16 plants; error bar, SEM. (E) GI transcription in the FMv2 was adjusted to match the GI mRNA profile of GI‐3F6H in (A); predicted expression profiles of CO and FT are shown (as in C, D). (F) Overview of proteomics studies using GI‐3F6H.

Figure 2

GI‐TAP time series. (A) Samples from GI‐3F6H transgenic plants and WT control plants grown in short‐day conditions were harvested at the indicated time points and replication. (B) Workflow for protein extraction, TAP using FLAG and His tags, and peptide preparation for MS. (C) Workflow for label‐free, quantitative data analysis, statistical and bioinformatics tests. (D) Heat map of protein abundance over time for 88 proteins with significant enrichment of at least two‐fold (excluding proteins that bind to GFP or are only found in inaccessible compartments). (E) Distribution of peak times, for 16 proteins shown in (D) with significant change over the GI‐3F6H time course (ANOVA q < 0.05 or JTK_CYCLE q < 0.05). (F) PCA separates GI‐TAP time points (grouped by number and contour, with replicate letter, all quantified proteins used) over components 1 and 2 (PC1, PC2). Note 31 h time point replicates 7 h.

Figure 3

Diel profiles of immunoprecipitated GI and interacting proteins. Immunoprecipitated protein abundance of (A) GI and direct and indirect interactors detected in the time series study, along with (B) HSP90 and candidate direct or indirect interactors. Multiple gene identifiers indicate related proteins were not distinguished by the three CUL or five HSP90 peptides detected. GI‐3F6H samples, markers; error bar, SEM. Average of WT control, horizontal line, ± SEM, dashed line. Time (T); Significance of enrichment and temporal change are shown, as q‐values of t‐test comparing GI‐TAP peak to WT (Enrich q) and of JTK_CYCLE within the GI TAP‐MS time series (Ch q). Biological replicates in (A and B):: 5, except for ZT 19 (four replicates) and ZT 31 (two replicates). (C) Simulation of the GI and ZTL protein time series, using the model matched to GI:3F6H RNA data (Fig. 1E) under short‐day conditions, closely matches observations (panel A).

Figure 4

CDF6 interacts with GI and functions in photoperiodic flowering. (A) GI‐interaction profile of CDF6 in the time series study is similar to CDF3 (Fig. 3A). n = 5 (except ZT19: n = 4) (B) Yeast two‐hybrid assays validate interaction of CDF6 with full‐length GI, N‐ and C‐terminal domains of GI, as well as ZTL, FKF1 and LKP2. AD, activation domain; DBD, DNA binding domain. (C) Circadian expression profile of CDF6 mRNA, in WT plants 3 days after transfer to constant light, n = 3. (D) CDF6 over‐expression delays flowering of transgenic SUC2:HA‐CDF6 lines more under long days, compared to WT control, than under short days. Each transgenic line differed significantly from WT, t‐test P < 0.0001, except #8 in SD, not significant. n ≥ 16 (E) mRNA expression profiles of CDF6, CO and FT were tested by qPCR in WT and the overexpressor lines, confirming that CDF6 suppresses evening CO and FT expression. n = 3.