1. PTOX Mediates Novel Pathways of Electron Transport in Etioplasts of Arabidopsis 
Sekhar Kambakam, Ujjal Bhattacharjee, Jacob Petrich, Steve Rodermel 
Molecular Plant 
Volume 9, Issue 9, Pages (September 2016)
DOI: /j.molp
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2. Figure 1 Phytoene Accumulation.
Phytoene contents were measured by HPLC analysis of cotyledons (200 mg) from seedlings grown in the dark for 3 days. Samples included wild-type (WT), im, and WT germinated on norflurazon (0.1 μM) (WTNF). A phytoene standard prepared from light-grown, NF-treated leaves served as a control (SD) (see Methods); 20 pmol of this standard was used for analysis. For simplicity, only the peaks for phytoene (P) and phytoene isomers (PI) are shown. P and PI were detected at 296 nm (retention time of 20–28 min).
Molecular Plant 2016 9, DOI: ( /j.molp )
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3. Figure 2 immutans (im) Greening.
(A and B) Wild-type (WT) and im seeds were germinated on MS medium in the dark for 3 days at 22°C (etiolated seedlings). For greening, the plates were moved to the light (∼50 μmol photons m−2 s−1) at 22°C for up to 48 h. Greening was monitored by accumulation of total carotenoids (A) and chlorophylls (B) on a fresh weight (FW) basis (μg g−1). Values represent the mean ± SE from three independent biological replicates.
(C) Cotyledons from WT and im seedlings were examined by confocal microscopy during greening (0, 3, 6, and 24 h). Red represents chlorophyll autofluorescence (AF). The TL lane is transmitted light and shows the outline of the cotyledons used in the AF lanes. Scale bars represent 100 μm.
(D) Transmission electron micrographs of etioplasts from WT and im seedlings. P represents prolamellar bodies, PGs plastoglobules, and S starch granules.
Molecular Plant 2016 9, DOI: ( /j.molp )
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4. Figure 3
Redox Status of Etioplasts from Single, Double, and Triple Mutants.
(A) Levels of phytoene in cotyledons from 3-day-old, etiolated wild-type (WT), single mutants (im, crr2-2 and pgr5), double mutants (im crr2-2 and im pgr5), and the triple mutant (im crr2-2 pgr5). Phytoene levels were measured by HPLC (as described in Figure 2). Absolute phytoene amounts (in pmol per mg FW) were determined by comparing peak values to the phytoene standard (described in Methods). Values represent mean ± SE from three independent biological replicates. *P < 0.01, significant difference in comparison with im. N.D., not detectable.
(B) Total carotenoid levels were measured in cotyledons from the same samples as in (A). Values represent mean ± SE from three independent biological replicates. *P < 0.01 and **P < 0.05, significant differences in comparison with im.
(C) Ratio of NADPH/NADP+ in 3-day-old cotyledons from dark-grown WT, im, and crr2-2. Values represent mean ± SE from three independent biological replicates. *P < 0.01, significant difference in comparison with WT.
(D) Transcript levels of AOX1a in 3-day-old cotyledons from the same samples as in (C). AOX1a transcript abundance was quantified using qRT–PCR. Values represent mean ± SE from three independent biological replicates. *P < 0.01, significant difference in comparison with WT.
Molecular Plant 2016 9, DOI: ( /j.molp )
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5. Figure 4 Chlorophyll Biosynthesis in im, flu, and im flu Etioplasts.
(A) Pchlide contents in cotyledons from dark-grown wild-type (WT), im, flu, and im flu seedlings were determined by spectrofluorometry. Excitation at 440 nm produces an emission peak at 632 nm that corresponds to Pchlide. Bars to the right of the chart show quantification of the major peak for each sample (mean ± SD from three independent biological replicates). *P < 0.01, significant difference in comparison with WT.
(B) Rates of ALA synthesis were determined in cotyledons from etiolated WT, im, flu, and im flu. ALA production was measured spectrophotometrically at 553 nm, following a 12-h incubation of etiolated seedlings in a solution containing 50 mM levulinic acid. Values represent the mean ± SD from three independent biological replicates. *P < 0.01, significant difference in comparison with WT.
(C) Rates of Pchlide synthesis were determined in etiolated WT and im seedlings. Pchlide amounts were measured (as in A) at various times after incubation of dark-grown seedlings in 10 mM ALA. Values are mean ± SD from three independent biological replicates.
(D) Effects of ALA feeding on greening. WT and im seeds were germinated on MS medium (±0.01 mM ALA) for 3 days in the dark, then shifted to the light for 2 days (growth conditions are as in Figure 1). Total chlorophyll contents were measured at different times after illumination. Values are mean ± SD from three independent biological replicates.
Molecular Plant 2016 9, DOI: ( /j.molp )
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6. Figure 5 Transcript Profiling of Etiolated and Greening im.
RNAs were isolated from etiolated (0 h) and greening (2 h and 6 h) im and wild-type (WT) seedlings, and RNA-seq analyses were conducted as described in Methods. Responsive genes were defined as those with P ≤ 0.05 and at least a two-fold change in expression in im versus WT.
(A and B) Distribution of responsive genes according to (A) the number of repressed versus induced at each time point and (B) their degree of responsiveness (two-fold or more). Induced genes are above the x axis, while repressed genes are below the x axis.
(C) Functional classification of genes using the Arabidopsis MIPS classification scheme and gene ontology searches (
Molecular Plant 2016 9, DOI: ( /j.molp )
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7. Figure 6 Heat Map of Responsive Genes in Etiolated and Greening im.
The heat map shows responsive genes from the RNA-seq that play a role in plastid metabolism and differentiation. Red, induced in im; green, repressed in im; black, similar levels in im and wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
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8. Figure 7 qRT–PCR Validation of RNA-Seq Data.
Patterns of gene expression from the RNA-seq experiments were validated for representative genes by qRT–PCR. These included PhANGs (LHCB, RBCS); genes for two mitochondrial alternative oxidase isoforms (AOX1a and AOX1d); key regulatory genes for the biosynthesis of chlorophylls (HEMA1, PORA, PORB, and PORC) and carotenoids (PSY); and genes for three transcription factors that play a major role in chloroplast biogenesis (GLK1, GLK2, and ABI4). Values are mean ± SD from three independent biological replicates. WT, wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
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9. Figure 8 Altered Plastid Redox Rescues im.
(A) Relative Pchlide contents were determined as in Figure 4A. Samples included cotyledons from 3-day-old dark-grown wild-type (WT), im, im pgr5, im crr2-2, and im crr2-2 pgr5. Bars to the right show quantification of main peak amounts (mean ± SD from three independent biological replicates).
(B) Total chlorophyll contents during greening of the seedlings in (A). Seeds were germinated on MS medium for 3 days in the dark, then shifted to the light (∼50 μmol photons m−2 s−1) to initiate greening. Values represent mean ± SE from three independent biological replicates.
(C) Relative expression of HEMA1, GLK2, and PSY in dark-grown seedlings of WT, im, and im crr2-2 pgr5 measured by qRT–PCR (as in Figure 7). Values are mean ± SD from three independent experiments.
Molecular Plant 2016 9, DOI: ( /j.molp )
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10. Figure 9 A Model: PTOX-Mediated Electron Transport in Etioplasts.
The plastoquinone (PQ) pool is reduced by three different electron transport pathways: (1) the desaturation reactions of carotenoid biosynthesis mediated by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS); (2) and (3) redox pathways mediated by NDH and PGR5. We assume that PGRL1 and NDH have ferredoxin-plastoquinone reductase (FQR) activity, as in chloroplasts, and that they transfer electrons to the PQ pool from reduced ferredoxin (FD); transfer via PGR5 would occur in a PGR5-dependent manner. We also assume that NAP(P)H is the ultimate source of reducing power in etioplasts, and that reduced Fd is produced by a PGRL1-bound Fd reductase (FNR); FNR reduces ferredoxin using NAD(P)H as a cofactor. Electrons would be transferred from the PQ pool to molecular oxygen via PTOX in etioplasts. The unknown nature of electron transfer beyond Cyt b6/f is indicated by a question mark. We propose that NDH and the Cyt b6/f complex have proton-pumping activities (shown by the green arrows) that produce a gradient which allows for the chemiosmotic synthesis of ATP, using an ATP synthase with a modified γ subunit (product of the ATPC2 gene). In addition to serving as a terminal oxidase, PTOX might directly or indirectly (via the PQ pool) be the source of plastid signals to regulate nuclear gene expression (retrograde signaling) (thick blue arrow). Etioplast membranes contain abundant ternary complexes (protochlorophyllide oxidoreductase [POR]-Pchlide-NADPH) that are involved in the early stages of chlorophyll biosynthesis, during the conversion of etioplasts to chloroplasts. See text for references and further details.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions