Arabidopsis PII Proteins Form Characteristic Foci in Chloroplasts Indicating Novel Properties in Protein Interaction and Degradation

Abstract

The PII protein is an evolutionary, highly conserved regulatory protein found in both bacteria and higher plants. In bacteria, it modulates the activity of several enzymes, transporters, and regulatory factors by interacting with them and thereby regulating important metabolic hubs, such as carbon/nitrogen homeostasis. More than two decades ago, the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behavior of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies, we found that AtPII interacts in chloroplasts with itself as well as with known interactors such as N-acetyl-L-glutamate kinase (NAGK) in dot-like aggregates, which we named PII foci. In these novel protein aggregates, AtPII also interacts with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal component of AtPII is crucial for the formation of PII foci. Altogether, the discovery and description of PII foci indicate a novel mode of interaction between PII proteins and other proteins in plants. These findings may represent a new starting point for the elucidation of physiological functions of PII proteins in plants.

Keywords: BiFC; FRET/FLIM; PII foci; cpUPR; plant PII protein; plastidic protein degradation; protein–protein interaction.

Figure 1

AtPII aggregates in focal structures in chloroplasts. (A) AtPII-GFP (green) under the control of p35S (p35S CaMV::AtPII_cDNA-GFP) and co-expressed with mCherry-tagged transit peptide of tobacco Rubisco (CD3-999 pt-rk [24]; magenta) localizes to plastids in transiently transformed N. benthamiana 2 days after infiltration. (B) Genomic AtPII-GFP (green) expressed under the control of the endogenous PII promoter (pAtPII::AtPII_genomic-GFP) and co-expressed with mCherry-tagged transit peptide of tobacco Rubisco (CD3-999 pt-rk; magenta) localizes to plastids in transiently transformed N. benthamiana 2 days after infiltration. (C) Genomic AtPII-GFP (green) under the control of endogenous pAtPII (pAtPII::AtPII_genomic-GFP) localizes to plastids (magenta) in stably transformed A. thaliana. In each row, the GFP fluorescence is shown first, the mCherry fluorescence is shown second, and the merge of both pictures is shown last. White arrows mark exemplarily AtPII aggregates.

Figure 2

PII foci formation in Arabidopsis thaliana under different temperature and light regimes. Expression of genomic AtPII-GFP under the control of the pUBQ10 promotor (pUBQ10::AtPII_cDNA-GFP) was analyzed in 6-day-old seedlings. Seedlings were incubated for 24 h for temperature treatment in the dark at (A) RT, (B) 8 °C, (C) 37 °C, and for light treatment in (D) blue light, (E) green light, (F) red light, and (G) far red light. Seedlings were fixed after incubation. In each row, the GFP fluorescence is shown first, chlorophyll autofluorescence is shown second, and the merge of both pictures is shown last. White arrows: PII foci. Scale bar: 10 µm.

Figure 3

BiFC analysis of AtPII with itself and known interactors in PII foci. AtPII-nYFP was co-expressed with AtPII-cYFP (A), AtNAGK-cYFP (B), and AtBCCP1-cYFP (C), respectively, under the control of p35S promotor using 2in1-BiFC vectors. Images were taken 3 days after transient transformation of N. benthamiana leaves. In each row, the YFP fluorescence (yellow) is shown first, chlorophyll autofluorescence (blue) is shown second, free RFP fluorescence as expression control (magenta) is shown third, and the merge of all pictures is shown last. Scale bar: 10 µm.

Figure 4

FRET/FLIM analysis of AtPII interaction with candidate proteins in PII foci. AtPII-GFP was co-expressed with AtPII-mCherry (A), AtNAGK-mCherry (B), and AtBCCP1-mCherry (C), respectively, under the control of p35S promotor in N. benthamiana using 2in1 FRET vectors. Images were taken 2 days after transient transformation of N. benthamiana. In each row, the fluorescence of GFP is shown first, mCherry (magenta) is shown second, the brightfield image is shown third, and the merge of both fluorescence pictures is shown is shown last. Scale bar: 10 µm. (D) FLIM analyses of fluorescent co-localising signals in (A–C). Student’s t-test used for calculation of significance. Data points marked with an “x” represent statistical outliers of measurement. *** p < 0.001.

Figure 5

Appearance of PII foci over time. Overlay images of time series of AtPII-GFP (green) and AtNAGK-mCherry (magenta), both expressed under the control of p35S, 2 days after transient transformation of N. benthamiana. White arrows mark exemplarily PII foci observable over the whole time range. Scale bar: 10 µm.

Figure 6

AtPII is found in different plastidial aggregates. AtPII-GFP was co-expressed with AtRBCS3B-mCherry (A), AtDXR-mCherry (B), and AtDXS-mCherry (C), respectively, under the control of p35S in N. benthamiana. In each row, the GFP fluorescence is shown first, the mCherry fluorescence is shown second, and the merge of both fluorescence images with the brightfield image as background is shown last. White arrows mark exemplarily AtPII aggregates in chloroplasts (dark and round structures in the brightfield image), orange arrows indicate extraplastidic vesicle-like structures. (D) FLIM analyses of fluorescent co-localizing signals in (A–C) together with AtPII-GFP/AtNAGK-mCherry as positive control. Student’s t-test used for calculation of significance. Data points marked with an “x” represent statistical outliers of measurement. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

Truncation of the C-terminus of AtPII leads to different aggregation behavior. AtPII-GFP or AtPIIΔCT15-GFP were co-expressed either with AtPII-mCherry ((A,D), respectively), AtNAGK-mCherry ((B,E), respectively) or AtRBCS3B- mCherry ((C,F), respectively), under the control of p35S in N. benthamiana. Each of these pictures shows the merge of GFP and mCherry fluorescence with the brightfield image as background. Size bars show 10µm except for (D) and (F) where they indicate 22 µm. In G-I, the average total number (Sum) and the number of small and large co-localizing fluorescent signals per chloroplast for the co-expressions of AtPII-GFP/AtPII_ΔCT15-GFP + AtPII-mCherry (G), + AtNAGK-mCherry (H), and AtRBCS3B-mCherry (I) are provided (n = 25). Student’s t-test used for calculation of significance. *** p < 0.001.