These authors contributed equally.
The plastid redox insensitive 2 mutant of Arabidopsis is impaired in PEP activity and high light-dependent plastid redox signalling to the nucleus
Version of Record online: 28 DEC 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 70, Issue 2, pages 279–291, April 2012
How to Cite
Kindgren, P., Kremnev, D., Blanco, N. E., de Dios Barajas López, J., Fernández, A. P., Tellgren-Roth, C., Small, I. and Strand, Å. (2012), The plastid redox insensitive 2 mutant of Arabidopsis is impaired in PEP activity and high light-dependent plastid redox signalling to the nucleus. The Plant Journal, 70: 279–291. doi: 10.1111/j.1365-313X.2011.04865.x
- Issue online: 3 APR 2012
- Version of Record online: 28 DEC 2011
- Received 21 October 2011; revised 21 November 2011; accepted 22 November 2011; published online 28 December 2011.
Figure S1. Chl a fluorescence photochemical quenching coefficients, qP and qL, light response curve for wild type. The measurements were performed at 20°C and the initial Fv/Fm measured in the leaves was 0.83 ± 0.02. Each point represents mean of 4 independent biological replicates.
Figure S2. Expression pattern of PRIN2 (At1g10522). (a) Tissue expression. (b) Expression in photoperiodic conditions. (c) Expression of PRIN2 following 3 h HL exposure (1000 μmol photons m−2 sec −1). Relative expression was calculated using RCE1 (At4g36800) as reference gene. Data represents mean ± CI 95% from at least 3 independent biological replicates.
Figure S3. Phenotype and TEM images of the prin2 mutants. Seedlings were grown for 7 day or 4 weeks in 150 μmol photons m−2 sec −1. (a, e, i) Transmission electron microscopy (TEM) pictures of 7 day old seedlings. Bar represents 1 μm. (b, f, j) Visible phenotype. (c, g, k) Transmission electron microscopy (TEM) pictures of 4 weeks old rosette plants. Bar represents 1 μm. (d, h, l) Visible phenotype.
Figure S4.ELIP1 (At3g22840) and APX2 (APX1b, At3g09640) expression in prin2-1 and wild type following exposure to HL. Ten-day-old seedlings grown in control conditions (CL; 150 μmol photons m−2 sec −1) and exposed to 3 h HL treatment (1000 μmol photons m−2 sec −1). Relative expression was calculated using RCE1 (At4g36800) as reference gene. Data represents mean (±CI 95%) from at least 3 independent biological replicates.
Figure S5. Subcellular localization of RIN2. PRIN2:YFP fusion protein is localized to the chloroplast and co-localizes with PTAC12:CFP. The PRIN2:YFP and PTAC12:CFP constructs were transformed into Arabidopsis protoplasts and visualized using confocal microscopy. Auto fluorescence from chlorophyll can be seen in red. Bar represents 10 μm.
Figure S6. Expression levels of photosynthesis genes encoded in the plastid (psaA, AtCg00350; psbA, AtCg00020; rpoB, AtCg00190) and the nucleus (LHCB1.1, At1g29920; LHCB2.4, At3g27690; RBCS, At1g67090) and in 4-week-old rosette plants (a-b) grown in CL and (c) exposed to HL. Relative expression was calculated using RCE1 (At4g36800) as reference gene. Data represents mean (±CI 95%) from at least 3 independent biological replicates.
Figure S7. Plastid gene expression in the gun1 mutant and the prin2-2 mutant.
Seven-day-old seedlings were grown in 150 μmol photons m−2 sec −1 and sampled. Using real time PCR, 78 chloroplast genes were investigated for its expression. The genes were classified by their chloroplastic function. All expression data represent mean ±CI 95% from at least 3 independent biological replicates. Grey bars represent prin2-2, and dark grey bars gun1-1. All expression data is compared to wild type levels.
Figure S8. Expression levels of LHCB1.1 (At1g29920) and LHCB2.4 (At3g27690) in mutants with impaired PEP activity. Ten-day-old seedlings were in control conditions (CL; 150 μmol photons m−2 sec −1). Expression levels in the mutants were compared to wild type and relative expression was calculated using RCE1 (At4g36800) as reference gene. Data represents mean (±CI 95%) from at least 3 independent biological replicates.
Figure S9. Changes in chlorophyll fluorescence emission by treatment with PET inhibitors. Traces of chlorophyll fluorescence recorded in 10–12 days-old control seedlings treated with 100 mm DBMIB (yellow trace), 50 mm DCMU (red trace) or non-treated (black trace). The inset is a magnification of the traces at beginning of AL illumination. Black arrows indicate AL saturating pulses. Black and white boxes in the inset indicate transition dark-light in the measurement.
Figure S10. Full-length PRIN2 protein does not directly interact with TRXz and FLN1 in Yeast Two-Hybrid assay. (a) Schematic diagram of the proteins cloned into pLexA bait and pGAD-HA prey vectors. NMY51 yeast cells co-transformed with pLexA p53+pACT LargeT are provided as a positive control; cells co-transformed with pLexA TrxZ+pGAD-HA and pLexA RIN2+pGAD-HA serve as negative controls for physical interaction. (b) The growth of NMY51 yeast cells co-transformed with corresponding prey and bait constructs on the selective media SD –Trp-Leu-His supplemented with 7,5mM 3-AT. FLN1 and TRXz directly interact as reported in Arsova et al., 2010. PRIN2 does not interact with FLN1 and TRXz. (c) β-galactosidase agarose overlay assay was done on the co-transformants streaked on SD –Trp-Leu-His media supplemented with 7,5mM 3-AT.
Table S1. Expression raw data for plastid encoded genes in the prin2 alleles.
Table S2. Genes co-expressed with PRIN2 (At1g10522). ATTED-II generated co-expression network of PRIN2 (At1g10522). MR reflects gene co-expression and the coefficient is calculated by taking a geometric average of the Pearson’s correlation coefficient (PCC) from gene A to gene B and that of gene B to gene A (Obayashi, Kinoshita 2009).
Table S3. Primers used for RT-PCR in this study.
Table S4. Mutants used in this study and primers used to identify the T-DNA insertion lines.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
|TPJ_4865_sm_FigS1.tif||798K||Supporting info item|
|TPJ_4865_sm_FigS10.tiff||3342K||Supporting info item|
|TPJ_4865_sm_FigS2.tif||2243K||Supporting info item|
|TPJ_4865_sm_FigS3.tiff||8229K||Supporting info item|
|TPJ_4865_sm_FigS4.tif||1098K||Supporting info item|
|TPJ_4865_sm_FigS5.tiff||2244K||Supporting info item|
|TPJ_4865_sm_FigS6.tif||4428K||Supporting info item|
|TPJ_4865_sm_FigS7.tif||986K||Supporting info item|
|TPJ_4865_sm_FigS8.tif||2007K||Supporting info item|
|TPJ_4865_sm_FigS9.tif||842K||Supporting info item|
|TPJ_4865_sm_SupportingInformationLegends.doc||92K||Supporting info item|
|TPJ_4865_sm_TableS1.xls||29K||Supporting info item|
|TPJ_4865_sm_TableS2.xls||25K||Supporting info item|
|TPJ_4865_sm_TableS3.doc||48K||Supporting info item|
|TPJ_4865_sm_TableS4.doc||43K||Supporting info item|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.