A red/blue optoswitch for temporal control of chloroplast transcription and biogenesis in Arabidopsis

Domain: chemistry
Published: 2026-02-20
Editorial outcome: minor_revision

Abstract
Photosynthesis genes in plant chloroplasts are transcribed by the plastid-encoded RNA polymerase called PEP. Consequently, PEP-deficient mutants cannot generate a photosynthetic apparatus and develop non-viable albino seedlings. Inducible complementation of such mutants thus could provide interesting insights in PEP action and chloroplast biogenesis. Here we show the effects of photo-inducible complementation in the albino Arabidopsis mutant pap7-1 using a red/blue optoswitch with monochromatic LEDs. Expression of a blue-light-induced PAP7 construct that is silent under red light reconstitutes PEP at any time point of pap7-1 development resulting in proper chloroplast biogenesis that rescues the non-viable mutant. Induction of chloroplast biogenesis, however, can only occur in very young leaf tissues indicating the existence of a cell-autonomous, biogenic coupling between cell and organelle development. We further uncover that initial PEP formation and function is independent of photosynthesis. The optoswitch termed blue-light-valved biogenesis opens experimental avenues to study non-viable plant mutants.

Introduction
Chloroplasts are special organelles in cells of plants and algae where photosynthesis takes place. Their ability to harvest energy from sun light and to convert it into chemical energy is essential for the biosphere of planet Earth1,2. Vascular plants inherit chloroplasts to the next generation as undifferentiated proplastids in seeds. These are devoid of photosynthesis and need to build the apparatus after germination. In addition, if germination occurs underground plastids develop into non-photosynthetic etioplasts that rapidly develop into chloroplasts as soon as the seedling reaches light3. The initial establishment of photosynthesis in cotyledons requires the simultaneous generation of the thylakoid membrane system, the photosynthetic apparatus and the connected enzymatic pathways that ultimately produce the carbohydrates that drive plant energy metabolism4. Detailed knowledge of photosynthetic gene regulation is essential for engineering crop improvement and global food security5,6.

A large part of the photosynthetic apparatus is encoded by the plastid genome, the plastome7. These genes are transcribed mainly by the plastid-encoded RNA polymerase (PEP) that is comprised of five prokaryotic RPO core subunits encoded in the plastome and 16 PEP-associated proteins (PAPs) encoded in the nucleus. The RPO subunits are remnant of the evolutionary origin of plastids and trace back to the endosymbiosis of a cyanobacteria-like ancestor, while PAPs evolved later likely during the terrestrialization event8,9. Recently, 3D cryo-EM structures of the PEP complex have been reported which revealed the overall arrangement and the structural details of the PEP subunits10–13. In a nutshell, the RPO core largely retained the ancient bacterial crab claw architecture including all essential sites involved in transcription, while the PAPs are arranged in a belt around this core generating a starfish-like shape. The structural information has advanced our understanding of the PEP complex and provides hypotheses for potential PAP functions. It, however, does not provide proof for the functional roles of PAPs during chloroplast transcription arguing for additional experimental approaches using pap mutants of Arabidopsis14.

Homozygous inactivation mutants of PAPs all exhibit severe chlorotic phenotypes. Plastids in these mutants do not develop a thylakoid membrane system for photosynthesis15–21 and the seedlings, therefore, are non-viable without external carbon supply. PAPs thus are essential for both PEP function and the initiation of chloroplast biogenesis22. These pap mutants generally produce no or only few seeds and must be maintained in a heterozygous state that segregates in the off-spring15,23,24 (see Supplementary Fig. 1a for pap7-1) impeding the sampling of homozygous plant material and the application of classical methods used in physiology or genetics. To overcome these constraints we aimed to develop a switchable complementation system allowing us to restore the PEP complex on demand by selectively activating the expression of a given pap gene construct in its corresponding mutant background. This would allow growth of homozygous mutants in a complemented state until flower and seed production, but also to switch off the construct in the next generation in order to obtain non-complemented mutant off-spring that does not segregate anymore. This would provide (i) a source for homogeneous albino material for molecular studies of chloroplast biogenesis and (ii) a tool to study potential interactions of PEP with other processes.

Temporally controlled expression of a defined pap gene construct could be done with chemically induced systems; however, the chemical inductors can cause technical limitations due to low tissue diffusion rates, potential metabolic or toxic side effects or slow removal in shut-off switches25,26. Technological alternatives with high spatio-temporal resolution and precision could be optogenetic tools that have been already established in many variations for prokaryotic, fungal or animal model systems. In plants widespread application of optogenetics, however, is lagging behind because of the organisms´ energetic dependency on light, the lack of required co-factors, or the interference of endogenous photomorphogenic photoreceptors eventually causing imprecise or constitutive expression of optogenetic tools27,28. Recent studies reported the engineering of sophisticated actuator modules that overcome some of these issues and function even in white light. In the plant-useable light switch element (PULSE) system two combined photoswitches control a synthetic promoter that remains silent in blue light but becomes activated under red light. Because of the blue light portion of white light vegetative growth becomes possible and the gene cassette is expressed only in monochromatic red light29. Another system called Highlighter is comprised of a photoswitchable cyanobacterial CcaS-CcaR system that has been reingeneered for the use in plants. The designed CcaS actuator efficiently photoswitches with the plant endogenous phytochromobilin chromophore allowing for repression of target gene expression in blue light or blue light enriched white light while becoming active in other light regimes26.

In this study we aimed to establish a tool for photo-inducible complementation (PINC) of the non-viable albino pap mutants, which should provide precise, rapid and reversible activation of the complementation construct by a simple change in growth light colour of the seedlings. Because of the albino phenotype of the pap mutants the properties of the applied optoswitch should (i) allow for a light induced germination of seeds without activation of the complementation construct and (ii) enable a positive selection of complemented individuals upon light induction. Active expression or repression of the photo-induced gene cassette in white light, therefore, was not our prime interest. As regulatory elements we used the blue-light-responsive elements (BLREs) of the chalcone synthase promoter that are specifically recognized by the plant-internal cryptochrome system30. The basic idea of this approach was that the BLREs cloned in front of a complementation construct would activate the gene cassette by monochromatic blue-light illumination (Bon) but would keep it silent in monochromatic red light (Roff), a condition that enables light induced germination. We expected to monitor the functionality of the system by the greening of complemented plants in Bon conditions since the expression of the complementation construct would restore the PEP complex that subsequently would initiate chloroplast biogenesis in the albino mutant background (Supplementary Fig. 1b). This optoswitch therefore was termed blue-light-valved biogenesis (BVB)(Fig. 1a).

Here we show the application of our strategy to the well characterized albino pap7-1 mutant of Arabidopsis thaliana16,31. We demonstrate the successful establishment and application of the BVB optoswitch in pap7-1 enabling us to control PEP formation by blue-light induced PAP7 complementation. We show that this can be done in a reversible and repeatable manner. By testing temporal requirements of the BVB optoswitch, we observe that initiation of chloroplast biogenesis must occur before a specific time point during leaf development. We further use the BVB optoswitch in pap7-1 to test whether PEP formation and activity depends on photosynthetic activity since several PEP subunits posses predicted domains with potential redox-related functions. The results indicate that PEP formation and principal function are largely independent from photosynthesis which contrasts some working hypotheses for redox-related PAP functions. Our results demonstrate high functionality of the optoswitch, providing an easy approach for spatio-temporal and qualitative analysis of PEP and its properties. The PINC approach using the red/blue optoswitch as tool further represents an attractive system for the study of other viable and non-viable plant mutants with a wide range of potential biotechnological applications.

Results
A red/blue optoswitch for controlled complementation of PEP
For the establishment of the PINC approach, we used temperature-controlled growth chambers equipped with narrow band-width light-emitting diodes (LEDs) providing high spectral selectivity (Supplementary Fig. 1c). Wild type (wt) seedlings grown under red or blue light developed normal green phenotypes both on agar plates or soil and displayed highly functional photosynthesis, while pap7-1 mutants remained albino under all illumination conditions (Supplementary Figs. 1d,e and 2a) as originally observed16,31. To establish the PINC approach we cloned one to four repeats of a 123 base pair (bp) DNA fragment containing the blue-light responsive ACE and MRE elements (BLREs) of the Arabidopsis thaliana chalcone synthase (AtCHS) promoter30 in front of a full-length PAP7 gene construct containing the N-terminal chloroplast transit peptide and a C-terminally fused GFP as reporter (Fig. 1a) and stably transformed these constructs into heterozygous pap7-1 mutants. Subsequently, we selected individuals in the off-spring that demonstrated an albino phenotype in Roff, but a green phenotype in Bon and genotyped them. We then isolated lines being homozygous for both the mutant allele and the optogenetic construct. While transformation of constructs with a single BLRE cassette did not yield green plants (Supplementary Fig. 2b), experiments done with constructs containing two, three or four BLRE repeats were successful (Supplementary Fig. 2c,d). In Bon, representative lines with two (BVB04), three (BVB09) and four (BVB12) BLRE repeats developed green cotyledons (Fig. 1b, Supplementary Fig. 2c), with PAP7 expression of approx. 70–90% of wt levels (Fig. 1c) and green follow-up leaves after 14 days of cultivation (Fig. 1d, Supplementary Fig. 2c). In older plants we observed a promoter dosage effect with increasing PAP7 construct expression the more BLRE repeats were present (Fig. 1e). We further tested the photosynthetic capacity (determined by chlorophyll (Chl) fluorescence parameter Fv/Fm) in BVB lines grown in either Roff or Bon conditions (Fig. 1f,g) and determined also the accumulation of photosynthetic pigments (Fig. 1h). In Roff conditions all lines were unable to become photoautotrophic while in Bon conditions the BVB lines developed green photosynthetic leaves. Again we observed a promoter-dosage effect with respect to development of green tissues, photosynthetic capacity and pigment accumulation (Fig. 1f–h). Constructs with four BLRE repeats performed best in Chl fluorescence measurements, but appeared to be slightly leaky under Roff since we detected trace amounts of chlorophyll in such lines (Fig. 1h, Supplementary Table 1). Although the Fv/Fm values did not indicate photosynthetic capacity of these plants, we decided to perform all subsequent analyses with lines carrying just three BLRE repeats (i.e. BVB09) since these exhibited complete tightness in Roff. By this we aimed to avoid any ambiguities arising from an incomplete albinism in BVB12 that could potentially compromise our conclusions in further experiments. In turn, we regarded a complementation degree of around 80% as sufficiently complete to obtain conclusive results for the induction of PEP and chloroplast biogenesis.

Switchability and expression properties of the BVB optoswitch
Seeds of the BVB09 line were germinated and grown in Roff for 7, 14, or 21 days before being shifted to Bon. Resulting plantlets produced white cotyledons as well as albino follow-up leaves if cultivated in Roff for long enough time. Upon shift to Bon, all newly formed leaves of BVB09 plantlets became green regardless of pre-cultivation time in Roff indicating successful functional complementation at all time points (Fig. 2a-c). BVB09 plants exhibited retarded growth in Roff when compared to wt (Fig. 2d) which corresponds to the growth characteristics of the original pap7-1 line (Supplementary Fig. 1d). All green leaves that developed after the shift to Bon were photosynthetically competent (Fig. 2d,e). Thus, the activation capability of the optoswitch is independent of seedling age and not restricted to cotyledons, where highest PEP activity typically is found in wt plants8,9. We further performed light cycling experiments with alternating Roff and Bon conditions in a 7-day-rhythm (Fig. 2f,g). These cycles generated plants with white, green and mixed-coloured leaves within one and the same rosette. Mixed-coloured leaves often exhibited white blades with green tips and edges in variable degrees of expansion (e.g. leaves 3a and 3b in Fig. 2f). The BVB optoswitch thus produces green and white tissues on demand even in older stages of plant development. Its switchability was functional regardless of the starting condition, i.e. it worked both in Roff-Bon-Roff-Bon and in Bon-Roff-Bon-Roff cycles. Since the BVB09 seedlings grew faster in Bon, full synchronicity between light shifts and speed of leaf development was difficult to achieve and we, therefore, were unable to generate plants with perfectly alternating green or white leaves.

In order to understand the action kinetics of the BVB optoswitch we determined its expression dynamics in response to light shifts (Fig. 2h). BVB09 and wt seeds were grown for 7 days in Roff conditions and the PAP7 expression level was determined by qRT-PCR. Subsequently, the seedlings were shifted to Bon for 24 h followed by a shift back to Roff for another 48 h. Multiple samples were taken along this time course to determine activation and deactivation dynamics of the optoswitch. Induction of the construct raised PAP7 expression from zero to wt levels within 30 min and reached a largely stable maximum level after 4 h with more than 2 times the expression level of wt. The shut-off was rapid (reaching less than 10% of maximal level within 1 h and almost zero after 4 h). These data indicate high tightness and rapid activation and deactivation behaviour of the optoswitch. The formation of leaves with mixed green and white areas, therefore, are neither caused by low temporal dynamics or an intrinsic leakiness of the system, but must be caused by constraints in the developmental programmes of the cotyledons and follow-up leaves.

In sum, the results demonstrate that (i) the optoswitch can be activated independent of the age of the plant and (ii) that repeated activation/deactivation cycles are possible. However, only newly emerging tissues became green while older white tissues did not suggesting the existence of a specific cell age threshold for initiation of chloroplast biogenesis within the leaf development programme. In cells younger than this threshold (e.g. in the shoot apical meristem (SAM)), PEP induction can initiate chloroplast biogenesis, while in cells that have already passed it, an induction of PEP has no effect anymore and the cells remain white even in extended Bon conditions. This cell age threshold, thus, represents a point-of-no-return in leaf cell development with respect to chloroplast formation.

Temporal constraints of chloroplast biogenesis
We aimed to determine more precisely the observed cell age threshold by performing growth experminents with increasing pre-cultivation times in Roff before a switch to Bon (Fig. 3a). When the BVB09 line was pre-cultivated for four to seven days in Roff the cotyledons remained albino after the shift to Bon. They, however, could green after pre-cultivation of only one to two days in Roff with an intermediate state after three days, where cotyledons developed patchy green and white leaf areas (Fig. 3b, Supplementary Fig. 3). Follow-up leaves frequently developed green tips and edges in experiments with repetitive light shifts (Fig. 2e, leaves 3a and 3b; further examples in Supplementary Fig. 3). These patterns appear to be different from those observed in the cotyledons and are most likely generated by the shift to Roff during their development. Cells that developed in leaf primordia in Bon became green, while cells that developed after the switch to Roff became white and largely remained white even after a subsequent switch back to Bon. This strongly suggests that the point-of-no-return acts cell-autonomously coupling the initiation of chloroplast formation to a specific juvenile age of individual cells rather than to the tissue or the plant.

Since chloroplast formation is integrated into the photomorphogenesis programme, we wondered what would happen if we perform the same experiment with a pre-cultivation in the dark instead of red light. The BVB09 line was morphologically indistinguishable from wt plants either in the dark or in blue light (Fig. 3c), but in contrast to wt it did not express PAP7 in the dark (Fig. 3d). Further, while wt seedlings did green regardless of the length of the dark period before the shift to Bon (Fig. 3e), BVB09 seedlings were unable to respond to Bon after three days pre-cultivation in the dark. Only follow-up leaves turned green when Bon conditions last for 14 days (Fig. 3f). The point-of-no-return thus is not light-dependent, suggesting a fixed check-point in cell development that is important for the acquisition of competence to initiate chloroplast biogenesis in the light.

As alternative hypothesis one could argue that the lack of chloroplast biogenesis in older non-greening cotyledon cells could be simply caused by a developmental block or down-regulation of BVB optoswitch activity at day three and later. In order to test this we performed control experiments in which we determined the gene expression of the PAP7 gene construct in BVB09 seedlings at days two, three, four and five after germination, before and two days after the shift to Bon. We observed high induction of the construct at all these tested time points (Supplementary Fig. 3g,h). This result excludes the possibility of a time- or development-dependent deactivation of the BVB optoswitch at this critical time point and provides additional support for our hypothesis of a cell-age-dependent block of chloroplast formation in albino leaf cells (Supplementary Fig. 3).

PEP formation and initial activity do not depend on photosynthesis
The PEP subunits PAP6, PAP10, PAP13 and PAP15 were proposed to mediate photosynthetic redox signals towards chloroplast transcription, affecting both assembly and activity of the complex18,32. The BVB optoswitch provides a suitable tool to prove these proposals by combining the induction of PEP formation with a pre-treatment of seedlings with 3-(3´,4´-dichlorophenyl) 1,1´-dimethylurea (DCMU), a membrane-permeable, highly stable inhibitor of photosystem II that blocks photosynthetic electron transport33. We grew wt and BVB09 plants seven days in Roff, applied the herbicide and put the seedlings for seven days to Bon (Fig. 4). Non-treated 14-day-old controls demonstrated high photosynthetic capacity in wt (both in Roff and Bon) and BVB09 (only in Bon) (Fig. 4, first two top rows). The Roff-Bon shifted control plants demonstrated high Fv/Fm values in all wt leaves and in the Bon-induced leaves of BVB09 (Fig. 4, third row), while Roff–grown BVB09 lines remained photosynthetically incompetent. Herbicide treatment before the shift to Bon, however, blocked photosynthesis in all plants (Fig. 4, bottom two rows). Despite the DCMU-treatment, the BVB09 lines started to develop green follow-up leaves when put to Bon (Fig. 4, bottom two rows) suggesting that PEP is active in these tissues.

To prove this we isolated these new green leaf tissues by micro-dissection. In blue-native polyacrylamide gel electrophoresis (BN-PAGE) and subsequent immuno-blot analyses we analyzed the formation of PEP and of major photosynthesis complexes in these greening tissues of the BVB09 lines and in wt as control (Fig. 5). RubisCO, the CO2-fixing enzyme of photosynthesis, photosystems I and II (PSI and PSII) (Fig. 5a) and PEP (Fig. 5b) accumulated to wt-levels in the Bon-induced BVB09 tissues, but not or only in trace amounts in Roff conditions. The DCMU treatment did not hinder the observed PEP complex formation, rather it appeared to promote it, especially in BVB09 (Fig. 5b).

We further tested the transcriptional activity of this PEP complex, and also that of nuclear RNA polymerase (RNAP) II, in the newly formed green leaves of DCMU-treated BVB09 seedlings by determining the transcript accumulation of GFP (as marker for transgene expression) and selected nuclear and plastid encoded genes for plastid proteins (Fig. 6, Supplementary Fig. 4). Overall BVB09 seedlings grown 14 days in Roff exhibited very similar expression characteristics as the pap7-1 mutant. Growth of 14 days in Bon resulted in a strong promotion of most plastid and nuclear encoded genes in BVB09 reaching expression levels comparable to the wt control under the same condition. We could not find any inhibitory effect of the DCMU treatment on the accumulation of PEP- or RNAPII-transcribed genes in green BVB09 tissues and only minor effects on a minority of genes in WT seedlings. This is notably true for PAP15/PRIN2 (Fig. 6b), which is proposed to mediate PEP formation in response to photosynthetic redox signals32, but also for GUN1 (Fig. 6c) encoding a component proposed to be central in retrograde signalling during chloroplast biogenesis34,35. However, we observed a slight promotion of rpoB transcripts in BVB09 (Fig. 6e) that is consistent with the PEP immunoblot data (Fig. 5b). The rpo genes are transcribed by a second phage-type nuclear encoded RNA polymerase (NEP)36, suggesting a potential connection between photosynthesis and NEP activity. In the case of the rbcL gene (encoding the large RubisCO subunit) we could not observe any DCMU effect on the transcript accumulation, however in the BN-PAGE analyses DCMU negatively affected the accumulation of the full RubisCO complex suggesting a selective inhibitory effect on protein translation (compare Fig. 5a and Fig. 6g). These results were confirmed by testing the expression of additional genes including Lhcb2.2, Lhcb2.3, accD, petD, ycf2.1, atpE, clpP, psaJ, psbB and psbN (Supplementary Fig. 4). Besides the gene specific expression control, we observed that transcript accumulation of plastid genes in BVB09 under Roff conditions equals that of the pap7-1 mutant indicating the tightness of the BVB optoswitch. The results further demonstrate an increase in PEP-dependent gene expression in Bon conditions (including an increase after a shift) confirming the data obtained for PAP7 expression under the same conditions (compare Figs. 2, 3). In sum, the data indicate that control of PAP7 expression by the BVB optoswitch results in a corresponding regulation of PEP-dependent transcripts and that inhibition of photosynthetic electron flow has no negative effects on PEP formation and its gene expression activity.

Methods
Plant material
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild type control in this study. The well-characterized PAP7/pTAC14 (AT4g20130) inactivation mutant line SAIL_566_F_06 served as the genetic background for the blue light-inducible complementation lines.

Plasmid construction
All cloned plasmids used in this study are listed in the Supplemental material (Table S2). Constructs were PCR-amplified from Arabidopsis thaliana Col-0 cDNA or genomic DNA using primers synthesized by Eurofins Genomics (listed in Supplemental Data). PCR was carried out using Phusion DNA Polymerase (New England Biolabs) and purified with the NucleoSpin PCR Clean-up Kit (Macherey-Nagel). Golden Gate modular cloning57 was used for assembly, employing the type IIS restriction enzymes BpiI and BsaI (New England Biolabs), following the Cut-Ligation protocol as described58. Ligation products were transformed into chemically competent E. coli (DH5α strain). Resulting colonies were screened via PCR using Taq DNA Polymerase (Thermo Fisher Scientific), and positive clones were subjected to plasmid purification using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). All constructs were confirmed by Sanger sequencing (SeqLab).

Growth and Illumination Conditions
Seeds were surface sterilized by sequential washes in 70% ethanol (twice), followed by a final wash in 90% ethanol. Sterilized seeds were sown on ½ Murashige and Skoog (MS) medium (Duchefa), supplemented with 1% (w/v) sucrose (Roth) and 0.8% (w/v) phytoagar (Duchefa), and stratified for 2 days at 4°C. Seedlings were grown under controlled conditions in LED-equipped growth chambers (Polyklima) under a 10/14 h light/dark photoperiod at 22/18°C day/night temperatures with a photon flux density of 100 μmol m⁻² s⁻¹. For experiments requiring constant illumination, plants were grown under the same spectral and intensity conditions at a constant 22°C. For seed production, plants were transferred to soil after a minimum of two weeks on plates and grown under a 16/8 h light/dark cycle with 100 μmol m⁻² s⁻¹ light intensity. The spectral characteristics of the LED panels (measured with LI-180; LI-COR) are provided in Supplementary Fig. 1.

Generation of stably transformed mutant lines
Heterozygous A. thaliana SAIL_566_F_06 mutants were grown for 5 weeks under white light. The presence of the heterozygous T-DNA insertion was confirmed by PCR-based genotyping using Edwards buffer59 and specific primers (Supplemental Data). Plants were then shifted to long-day conditions, and immature flower buds were transformed using the floral dip method with Agrobacterium tumefaciens strain GV3101 pMP90, following a previously described protocol60. T₁ transformants were selected by growth on ½ MS medium supplemented with 50 μg mL⁻¹ kanamycin for 7 days under red light, followed by continued growth in blue light. After 4 weeks, all green seedlings were genotyped to confirm both construct insertion and homozygosity for pap7 gene inactivation. T₂ seeds were screened by growing seedlings in red light for 7 days, during which albino seedlings were marked. Plates were then shifted to blue light, and after another 7 days, the previously marked seedlings were assessed for greening. All selected transformants were genotyped, and lines showing a 3:1 segregation ratio (green: albino) were retained. Lines that were not homozygous for the pap7 gene in T₂-evident by the presence of green seedlings already under red light-underwent an additional selection round in the T₃ generation. This process was repeated until homozygosity for both T-DNA insertions (construct and pap7 inactivation) was confirmed. These verified lines were used in all further experiments (see Supplementary Fig. 2).

RNA isolation and quantitative reverse transcription PCR
Samples were harvested, immediately frozen in liquid nitrogen, and ground to a fine powder. Total RNA was extracted using the TRIzol method (Invitrogen), following the manufacturer’s protocol. RNA purity was assessed by measuring absorbance at 260 nm and 280 nm, with a ratio of ~2.0 considered indicative of pure RNA. RNA concentration was calculated accordingly, and 1 μg of total RNA was treated with DNase I (Thermo Fisher Scientific) to eliminate genomic DNA contamination. cDNA synthesis was performed using MMLV Reverse Transcriptase (New England Biolabs) and a mixture of random nonamer primers and oligo(dT)18-primers (Eurofins Genomics). The resulting cDNA samples were diluted 1:10 with nuclease-free water before quantitative PCR. Gene expression levels were measured using SYBR Green Master Mix (Thermo Fisher Scientific) and gene-specific primer pairs on a StepOnePlus Real-Time PCR System (Applied Biosystems). Each reaction was performed in technical duplicate. For quantification, serial dilutions (1:4) of a cDNA mass standard were included for each gene and run in parallel with the samples. EF1α (ATG07940) served as the internal reference gene for normalization of expression levels. All used primer pairs are listed in Supplementary Information (Supplementary Table 2).

Chlorophyll extraction and quantification
Fresh plant material (30–90 mg) was used for chlorophyll extraction in 95% ethanol (1 ml per 50 mg fresh weight). Samples were ground thoroughly and centrifuged for 15 min at 4°C at 10,000 × g to remove debris. Pigment concentrations were measured using a dual-wavelength spectrophotometer by recording absorbance at 664 nm, 649 nm, and 470 nm. Chlorophyll a, b, and total carotenoid contents were calculated according to established formulas as described previously61.

DCMU treatment
3-(3´,4´-dichlorophenyl)-1,1´-dimethylurea (DCMU) (Sigma-Aldrich) was applied after 7 days of seedling growth under red light. Seedlings were sprayed with 10 μM or 40 μM DCMU, diluted in water from a 10 mM stock solution prepared in 50% ethanol. Control plants were sprayed with an equivalent concentration of ethanol in water. Following treatment, all seedlings were transferred to blue light and grown for an additional 7 days prior to further analysis.

Chlorophyll fluorescence imaging
Photosynthetic capacity was assessed using the MAXI version of the Imaging-PAM fluorometer (Walz). Prior to measurement, seedlings were dark-adapted for 30 min to ensure accurate determination of photosystem II efficiency. Fluorescence parameters were recorded and analyzed using the ImagingWinGigE software (Walz).

BN-PAGE and western-immunoblot analysis
The assembly of the PEP complex was analyzed by Blue Native PAGE (BN-PAGE) followed by immunodetection according to previously described protocols62 with minor modifications63. Approximately 50 mg of Arabidopsis tissue, grown under the indicated conditions, was harvested and ground in liquid nitrogen. The resulting powder was extracted with three volumes of protein extraction buffer (100 mM Tris-HCl pH 7.2, 10 mM MgCl₂, 25% (v/v) glycerol, 1% (v/v) Triton X-100, 10 mM NaF, 5 mM β-mercaptoethanol, and 1× cOmplete™ EDTA-free protease inhibitor cocktail (Roche). Protein extracts were adjusted to contain 1% (w/v) β-D-dodecyl maltoside (DDM), 50 mM aminocaproic acid (ACA, pH 7.0), 25 mM Bis Tris (pH 7.0), and 1 μg/ml recombinant DNase I (Macherey-Nagel), and incubated for 1 h at room temperature. Samples were then supplemented with 0.25% (w/v) Coomassie Brilliant Blue G-250 (CBB-G250) and 20% (v/v) glycerol, followed by centrifugation at 16,000 × g for 10 min at 4°C. 25 μl of the resulting supernatant were loaded onto 4-16% acrylamide Bis Tris/ACA gradient gels (49% T, 3% C) with a 3% stacking gel. Electrophoresis was carried out using 50 mM Tricine/15 mM Bis Tris pH 7.0 with 0.01% (w/v) CBB-G250 as the cathode buffer and 50 mM Bis Tris-HCl pH 7.0 as the anode buffer, at 4°C. Gels were run initially at 40 V for 4 h, then continued overnight at 30 V after replacing the dark blue cathode buffer with light blue cathode buffer (same composition but 0.005% CBB-G250), and finally switched to clear cathode buffer (no dye) for a final run at 50 V until the dye front exited the gel. Molecular weight markers were included by loading 10 μl of 200-fold diluted horse spleen ferritin (Sigma-Aldrich, F4503), which was later stained using Coomassie Brilliant Blue R-250. For immunodetection, proteins were transferred to PVDF membranes (Roti®-PVDF, 0.45 μm, Carl Roth) over 3 h at 2 mA cm⁻² using transfer buffer (0.5% SDS, 25 mM Tris-HCl pH 7.6, 192 mM glycine, 10% ethanol). Membranes were destained in 100% methanol, blocked in 5% (w/v) skim milk powder in TTBS (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20), and incubated with either anti-RpoB antibody (PhytoAB, PHY1700, 1:10,000) or custom-made anti-PAP8 antibody (1:10,000) for 2.5 h. Membranes were washed 5 times in TTBS, incubated with HRP-conjugated Goat Anti-Mouse IgG (Agrisera, 1:25,000 in 15 ml) for 1.5 h, and washed again 5 times in TTBS. Chemiluminescence detection was performed using the SuperSignal™ West Atto Ultimate Sensitivity Substrate (Thermo Fisher), and images were acquired with a ChemoStar system (iNTAS) in sequence mode, recording 100 images of 5–10 s depending on signal intensity.

Statistics
Quantitative data given in the figures were subjected to statistical analyses wherever required for comparison. Sample number per experiment is indicated in the corresponding figure or figure legend. Plotting and statistical analysis were performed with Python using SciPy (v1.11.2) or Excel. Pairwise comparisons between relevant samples were initially evaluated using a two-tailed unpaired Student’s t-test. To control for multiple testing, p-values were subsequently adjusted using the Holm-Bonferroni method. Comparisons with adjusted p-values below 0.05 were considered statistically significant. Significance between groups is indicated in the figures with connecting bars and denoted as *p<0.05, **p<0.01, and ***p<0.001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overview\nUecker et al. present a method for controllable genetic complementation of Arabidopsis (PINC), which provides a means of\nanalysing the functional role of essential plant proteins. They demonstrate the use of PINC to control assembly of the\nchloroplast transcription complex PEP via induction of one of its critical subunits, PAP7. Two major conclusions are drawn:\n(1) that chloroplast biogenesis is coupled to the developmental state of the cell it is in, and (2) assembly of the PEP complex\ndoes not depend on light or photosynthetic activity. The manuscript thereby presents significant advances in multiple areas\nthrough a straightforward and elegant experimental design.\nThe findings presented are all supported by the displayed data, and I did not identify anything that prevents publication. The\nmethodology appears sound and reproducible, though this is not my area of expertise. However, there is substantial room to\nimprove clarity in both the main text and the way figures are presented as detailed below.\nMajor comments:\n• I can envisage PINC would be of broad utility to plant biologists as it does not depend on expensive technology. The\nmanuscript focuses on the use of PINC to study proteins essential to chloroplast biogenesis. However, PINC would\npresumably be useful to study proteins that are essential for any reason, not only chloroplast biogenesis. Can the authors\nclarify in the text if this is the case, or if it is uniquely suited to the study of chloroplast biogenesis? If the PINC system is\nindeed of general utility, I would suggest adapting the abstract and summary to emphasise this at the outset and attract more\nreaders than those interested in chloroplast transcription.\n• Dose-dependency is an important feature of the system. Photosynthetic parameters show dose-dependency that suggest\nPAP7 levels increase with more BLRE repetitions, but the data on PAP7 expression levels (1c) do not show the same trend\n– seemingly because only three measurements were taken and one is unusually low in line BVB12. Repetition of this\nexperiment to reduce uncertainty in BVB12 would give readers confidence that the system works in the way expected, and\nthat they could choose a number of BLREs to have some control over the extent of expression (closer to 70% of wt with 1x or\nto 100%? of wt with 3x).\n• The inability to completely complement to wt levels (evident in 1d-e) is an important limitation that could be more clearly\nquantified. The leaky expression in the 4xBLRE off condition led the authors to analyse plants with 3xBLREs. It would be\nhelpful for readers if (1) photosynthetic efficiency (fig 1d) was quantified, and (2) the main text stated the attributes of the line\nselected as useful (BVB09): PAP7 expression relative to wt as a %, photosynthetic efficiency as a % relative to wt, total\nchlorophyll levels as a % relative to wt. Each of these numbers will likely reassure readers that although the\ncomplementation level is not 100% it is still relatively high.\n• The conclusion there is a ‘time window’ (line 173) was not immediately obvious from the data. I strongly suggest the\nauthors explain the reasoning behind this conclusion. In particular, are they confident from this experiment alone there is\nboth a cell age that is too young and a cell age that is too old for PEP activation to be sufficient for greening? Or is it only the\nthreshold of too old that can be observed?\n• Regarding the time range (line 176): ‘crucial’ is a problematic choice of word as PEP is likely also crucial in other times. A\nbetter phrasing would be in terms of ‘necessary’ and ‘sufficient’: PEP is both necessary and sufficient in the first time period\nand is presumably still necessary but no longer sufficient in the second time period. Is that how the authors see it?\n• The ‘patchy’ complementation seen in the three-day shift may arise because the cells at the centre of the leaf are younger\nand were not exposed to blue light for enough time for sufficient PAP7 accumulation to occur, therefore remaining white. Is\nthis the authors interpretation too? Or do they propose that cells can be too young for their plastids to green? Could these\nalternatives be investigated by checking what the minimum duration of blue-light exposure needed for greening to occur is?\n• The authors note that DCMU increases the abundance of PEP specifically in BVB09 and not wt. This appears striking: the\ndifference appears to be 10-fold or more in the 40 uM treatment and exceeds PEP abundance in wt. Could the authors\ncomment in the Discussion what this might indicate? Can it be concluded that an increased expression of PAP7 relative to\nwt leads to increase in the production of all PEP subunits? PAP7 expression quantification on DCMU-treated plants may\nprovide the necessary insight.\n• In line 266 there is a risk of overstating how surprising this is. The data presented does not indicate that PEP does not have\na ‘predominant role’, only that another factor is also required and is lost with time. The major discovery appears less about\nthe role of PEP, and more about the identification there is another essential factor in chloroplast biogenesis (of which there\nare many) that is no longer available.\n• It is not introduced how this system differs from recent publications on plant optogenetics (refs 47, 48). This seems\nimportant context that could be provided in the introduction, rather than in the final paragraph. It is also not clear to a reader\nin what way the ‘experimental goal and working principle’ are different – additional detail would help emphasise the novelty\nof this work.\nMinor comments:\n• The authors seem to switch between PINC and BVB optoswitch to describe their technologies. Is it necessary to have\nboth? Could PINC be used throughout?\n• Line 32: ‘show the effect of temporally…’ would be clearer\n• Line 34: ‘allows reconstitution of PEP…’ (preferable) or ‘allows us to reconstitute PEP’\n• Line 37: ‘is effective only’ might be changed to ‘can only occur’\n• Line 39: ‘to grow’ should be ‘growth of’\n• Line 66: ‘photosynthesis’ should be ‘photosynthetic’\n• Line 68: ‘plastid-own genome’ should be ‘plastid genome’ (preferable) or ‘plastid’s own genome’.\n• Line 73: comma needed after ‘ancestor’\n• Line 77: ‘cyanobacterial crab-like structure’ should be ‘bacterial crab claw architecture’ or similar (the crab claw shape is\nnot unique to cyanobacteria).\n• Line 79: ‘put forward’ should be ‘advanced’ or similar\n• Line 80: ‘it does not provide direct clues’ – I would argue the structure does provide clues (or hypotheses more\nspecifically). What it does not provide is proof, which is what makes the described genetic analysis essential.\n• Line 82: should end with ‘in Arabidopsis’ for clarity?\n• Line 86: ‘hardly produce’ could be ‘produce few’ or ‘generally do not produce’ for clarity.\n• Line 92: ‘allow to grow’ should be ‘allow growth of’ (preferable) or ‘allow us to grow’.\n• Line 100: ‘As regulatory tools’ should be 'As the regulatory element' or similar.\n• Line 102: ‘Basic’ should be ‘The basic’\n• Line 112: ‘are’ should be ‘were’\n• Line 115: ‘a point-of-no-return’ is a somewhat unambiguous expression. Perhaps the authors would consider alternative\nwording, such as: 'that initiation of chloroplast biogenesis must occur before a specific timepoint during leaf development.'\n• Line 117: ‘potential photosynthetic influences’ is ambiguous. Perhaps the authors would consider the phrasing: 'to test\nwhether PEP formation depends on, or is influenced by, photosynthetic activity'\n• Line 119: The authors need to clarify whether they are referring to PEP assembly or activity – what has been measured?\n• Line 121: comma needed after ‘optoswitch’.\n• Line 128: ‘PEP complementation’ would be unclear to some readers. ‘for controlled complementation of PEP biogenesis’\nmay be clearer?\n• Line 133: could a citation be provided to the publication(s) pap7 phenotype was originally characterised?\n• Line 135: (BLRE)\n• Line 144: ‘effective’ could be ‘switchable/controllable’?\n• Line 144: A significant improvement would be to clarify what each line is in the main text – currently the reader must look at\nthe figure for this. For example: ‘representative lines with 2 (BVB04), 3 (BVB09) or 4 (BVB12) BLRE repetitions\ndeveloped…’\n• Line 145: ‘1c’ should be ‘1b’ and ‘2c’ should be ‘1c’\n• Figure 1c: ‘relative expression’ could be more specific on the figure: ‘Relative PAP7 expression’.\n• Line 145: comma needed before ‘with’\n• Line 145: ‘close to’ could be made more precise with a percentage: ‘approximately 70-90% of wild type levels’?\n• Line 149: ‘photosynthesis’ should be ‘photosynthetic’\n• Line 149-150: commas needed: ‘, especially in older plants, ‘\n• Line 157: could be clarified – this is presumably multiple individual plants, not one plant being shifted at different time\npoints? The word ‘it’ suggests an individual. ‘plants were grown in… before being shifted to..’ would be clearer. Figure 3a\nlegend would likewise benefit from clarifying these were different seedlings transferred to blue light at different time points,\nas it would be possible a reader would think this is the same seedling. Could the wording be ‘developmental timeline\nshowing representative images of albino ….’ Or similar?\n• Line 158: Could be stated more clearly that the conditional is simply enough time for follow up leaves to grow: perhaps\n‘and albino follow-up leaves if cultivated in Roff for long enough for these to develop’?\n• Line 165: comma needed before ‘where’. This statement would benefit from a citation.\n• Line 170: comma needed before ‘perfect’\n• Line 172: ‘i)’ should be before ‘demonstrate’ (as ‘suggest’ is after ii)\n• Line 173: ‘time window’ may not be the best way to describe as there is a limit to the latest point but not the earliest – see\ncomment above.\n• Line 177: ‘and performed’ could be ‘by performing’\n• Line 183: ‘biogenic’ is not clear to this reviewer. If this has a specific meaning, could the authors clarify in the text what is\nmeant? If not, this word could be removed.\n• Line 191: comma needed before ‘suggesting’\n• Line 194: Heading should be clearer, and would benefit from being in the form of a conclusion: ‘Production of PEP does\nnot depend on photosynthesis’ for example.\n• Line 196: comma needed before affecting\n• Line 197: ‘the perfect tool’ appears an overstatement: perhaps ‘provides a tool’?\n• Lines 219-220: commas needed ‘, and also that of … RNAP II, ‘\n• Line 229: ‘especially’ – do the authors mean ‘notably’, or that PRIN2 has particularly little responsiveness to DCMU?\n• Line 235: comma needed before ‘suggesting’\n• Line 236: Should be ‘In the case’\n• Line 239: ‘inhibitor’ should be ‘inhibitory’\n• Line 245: comma needed before ‘which’\n• Line 247: ‘tailor-made’ is not needed\n• Line 248: ‘alternatively’ should be ‘additionally’?\n• Line 249: comma needed before ‘allowing’\n• Line 251: ‘highly’ should be ‘greatly’ or ‘substantially’\n• Line 253: comma needed before ‘which’\n• Line 259: comma needed before ‘indicating’\n• Line 261: should be ‘complicated’\n• Line 265: comma needed before ‘indicating’\n• Line 266: a citation is needed for what the ‘current models’ are.\n• Line 271: ‘identical’ appears an overstatement: to what level of precision? Perhaps ‘consistent with the timescale of\nretrograde..’?\n• Line 272: should include ‘in wheat’ to clarify it is not necessarily directly comparable.\n• Line 272: should be ‘The physical nature’\n• Line 277: comma needed before ‘indicating’\n• Line 279: comma needed before ‘function’\n• Line 288: should be either ‘PAP6, PAP10…’ or ‘PAPs 6, 10,..’\n• Line 290: comma needed before ‘regulation’\n• Line 300: comma needed before ‘our’\n• Line 311: could this be ‘in standard growth cabinets equipped with…’ for simplicity?\n• Line 314: why must the phenotype be visible/trackable? Molecular techniques could be used too surely?\n• Figure 1e: there appear to be too few significance comparison on the chart. Could differences between groups be\npresented as letters using a suitable post-hoc test?\n• Figure 2 is labelled Figure 1.\n• Figure 1d and Figure 2d would benefit from quantification (bar charts).\n• Figure 6: the colouring and key are not clear. The key for ‘shift’ should at least include red/blue colours behind the lines.\nHowever, I strongly suggest having its own colour, rather than attempt to convey experimental approach inside a bar chart\nthat may be mistaken to be a divided bar chart where two measurements are shown within a column.", "reviewer_2": "This study from the Pfannschmidt laboratory generates an elegant, simple yet ingenious optogenetic system for use in\nplants. Several optogenetic systems have been developed and refined over the last two decades. Their aim is to control\ngene expression in an on/off manner, ideally fulfilling a number of requirements: (1) no need for exogenous chromophores,\n(2) minimal interference with endogenous signalling pathways, (3) compatibility with the need for light cycles for plant growth\nand (4) be widely applicable.\nThe system described by Uecker et al. beautifully fulfils requirements 1 and 3. 2 and 4 are achieved with some limitations.\nAs the data themselves show, monochromatic red light used for growth results in plants in which the lack of blue\nphotoreceptor action causes, among visible phenotypes, elongated petioles and leaf blades which are unable to face the\nlight sources. The applicability of the system will depend on the ability of the blue light-responsive element of the CHS gene,\nidentified in Arabidopsis thaliana, to be active in other species.\nWhile those limitations cannot be questioned, use of the system in Arabidopsis is sufficient to justify its utility. Furthermore\nthe authors develop it for one specific purpose: to generate genetic stocks of mutants whose homozygosity is either lethal or,\nat least, incompatible with seed production, and therefore with propagation. This then allows refined experiments with\nhomozygous material to identify the consequences of presence/absence of gene function, repeated multiple times: the\nrelevant, homozygous stock is generated under standard growth (white light) conditions, and the control achieved by turning\ncomplementation off by transfer to monochromatic red (i.e. blue-less) light. This is entirely, beautifully achieved, applying the\ntechnique to study the function of the plastid-encoded RNA polymerase (PEP), through its requirement of nucleus-encoded\nPAP7/pTAC14. The study demonstrates the usefulness of the technique, and reports three key findings: the requirement for\nPEP function during a critical time window in cells in which early stages of chloroplast development are taking place, but\nvery little later on, the fact that this requirement is (as expected) cell-autonomous, and lastly that PEP activity appears to be\nminimally dependent of photosynthetic functon(photosynthetic electron transport) during these biogenesis stages. The\nunexpected result of the activity of PEP under photosynthesis-inhibited conditions is precisely described by monitoring both\ntranscript levels and overall protein composition of the relevant, dissected tissues.\nOne issue may require further evidence to confirm the stated critical window and “point of no return” of PEP requirement. The\nevidence provided shows that seedlings grown for up to three days in red light (R), or in darkness, can have PAP7 activated\nby switch to blue (B) and the cotyledons green, but this is not the case after four days (of R or darkness). The authors\nconclude that PEP activation later cannot cause chloroplast greening. This interpretation would require demonstration that\nPEP activation by B did indeed take place after 4 days in R or darkness. The endogenous CHS activation system used\nmight, conceivably, also have a window of competence, and this would explain the same observation. One easy test would\nbe to monitor the transcript of the transgene, making use of the fused GFP, as done in Fig. 6.\nThe reviewer also found the cell-autonomy of the above response particularly intriguing. Thresholds of activity of essential,\ncell-autonomous processes in chloroplast biogenesis clearly exist, are probably operating in early proliferating cells in leaf\nprimordia, and explain the development of cell sectors of contrasting phenotype, as seen in variegated leaves. The evidence\nfor this cell-autonomous requirement is presented in Fig. 3 and shown by what seems a cotyledon (Fig. 3B, possibly derived\nfrom 3A). Cotyledons are, of course, embryonic organs. Was such a phenomenon ever observed in newly-developing leaves\nemerging from the meristem? A seedling in Fig. 3F may show this, but the image is insufficient to confirm. It would be of\nvalue if the authors could clarify.\nLastly, the authors display how transcript levels of a range of genes, nucleus- and plastid-encoded, are impacted by the\nabsence of PAP7 or its complementation. Among plastid-encoded genes, those transcribed by the nucleus-encoded\npolymerase are (with the exception of rpoB) all presented in Fig. S4. Do the authors have an explanation for the low\ntranscript levels of petD in the pap7 mutant and the R-grown BVB line?\nThe text is generally well written, and the figures beautifully constructed to easily interpret. Note, however, that Fig. 2 is also\nlabelled as a second Fig. 1. On several occasions Fig. 2 is referred to as Fig. 1, and viceversa.\nEditing comments:\nRepetitions > repeats.\nLine 79 put forward > advanced (to “put forward” is done only the first time)\nFig. 6 What is given a value of 1, y axis?\nFig. 1 legend Ron > Bon\nMethods should indicate the statistical tests used. Note that multiple testing increases errors, and there are ways to avoid\nthose.", "reviewer_3": "The manuscript by Uecker et al. addresses an important and timely question: the role of plastid-encoded RNA polymerase\n(PEP) in chloroplast biogenesis. Recent progress in structural understanding of PEP has only reinforced the importance of\nopen questions regarding the biological roles of this crucial enzyme. Advancement in this field has been hindered by a\nchallenging chicken-and-egg situation, where disruptions of PEP subunits impede chloroplast biogenesis, subsequently\ndisrupting various processes, potentially including transcription itself. This manuscript addresses this conundrum by\ndeveloping a novel inducible system, enabling precise control of PEP subunits (or any other proteins) through exposure to\ndifferent wavelengths of light. Utilizing this tool, the manuscript investigates the role of PEP and elucidates two important\naspects of its involvement in chloroplast biogenesis. (1) A limited time window for PEP-dependent chloroplast development,\nwhich is an interesting novel finding and (2) independence of chloroplast development of PEP transcription. Although these\ninsights do not exhaust the power of the new inducible system, the current scope of the manuscript is appropriate and fully\njustified by the impact of the novel optogenetic system.\nThis is a significant study that will establish a new standard for investigations of PEP subunits, other essential chloroplast\nproteins, and likely other essential plant proteins. The manuscript is well-written, technically solid and will be of interest to a\nbroad audience of scientists studying genetics, not only in plants.\nSpecific comments\n• There is no data showing behavior of the BVB optoswitch on the level of RNA or protein accumulation. It is important to\nknow the level of induction and the level of leaky expression, which is a common problem of many inducible systems.\nAdding new experimental data providing these insights would substantially strengthen the manuscript.\n• The mechanism responsible for the checkpoint that provides competence for chloroplast biogenesis should be discussed\nmore thoroughly. For instance, is it possible that this checkpoint is mediated by nutrient availability in seeds?\n• There is no justification of why the new optogenetic system is better than previously established inducible systems in\nplants. There is also no comparison to other optogenetic inducible systems.\n• There is no discussion of potential impacts of growing plants in narrow spectra (red or blue light only) compared to white\nlight, which is an important consideration for the interpretation of results obtained using the BVB optoswitch.\n• DCMU treatment has been used in the past and citing and discussing original literature describing its limited impacts on\nchloroplast biogenesis would strengthen the conclusions of this manuscript.\n• Impacts of DCMU on RNA accumulation are sometimes overinterpreted as impacts on gene expression. This part of the\nmanuscript has limited influence on its conclusions and could easily be shortened.\n• There are problems with labels of Fig 1F (referenced in the manuscript) and Fig 2 (mislabeled in figure legends)."}, "author_response": "Point-by-point response to reviewers´ comments\nWe went carefully through all comments and improved manuscript, figures and supplements\naccordingly either by adding experimental data and/or by revising the respective text. Changes in the\nmanuscript text are indicated by track changes function and are further detailed below. To facilitate\nthe overview of changes for the reviewers we highlighted the principle changes in green.\nReviewer #1 (Remarks to the Author):\nOverview\nUecker et al. present a method for controllable genetic complementation of Arabidopsis (PINC),\nwhich provides a means of analysing the functional role of essential plant proteins. They\ndemonstrate the use of PINC to control assembly of the chloroplast transcription complex PEP via\ninduction of one of its critical subunits, PAP7. Two major conclusions are drawn: (1) that chloroplast\nbiogenesis is coupled to the developmental state of the cell it is in, and (2) assembly of the PEP\ncomplex does not depend on light or photosynthetic activity. The manuscript thereby presents\nsignificant advances in multiple areas through a straightforward and elegant experimental design.\nThe findings presented are all supported by the displayed data, and I did not identify anything that\nprevents publication. The methodology appears sound and reproducible, though this is not my area\nof expertise. However, there is substantial room to improve clarity in both the main text and the way\nfigures are presented as detailed below.\nMajor comments:\n• I can envisage PINC would be of broad utility to plant biologists as it does not depend on expensive\ntechnology. The manuscript focuses on the use of PINC to study proteins essential to chloroplast\nbiogenesis. However, PINC would presumably be useful to study proteins that are essential for any\nreason, not only chloroplast biogenesis. Can the authors clarify in the text if this is the case, or if it is\nuniquely suited to the study of chloroplast biogenesis? If the PINC system is indeed of general utility,\nI would suggest adapting the abstract and summary to emphasise this at the outset and attract more\nreaders than those interested in chloroplast transcription.\nResponse: We totally agree with this comment. The basic principle of the PINC strategy is suitable for\nany kind of essential protein in plants and is NOT restricted to chloroplast transcription and\nbiogenesis although the phenotypic effects are here especially visible. We adapted the title and\nimproved the abstract accordingly, we further recommend the system for general utility at the end of\nintroduction and discussion. By choosing the right promoter elements the strategy might be also\nsuitable even for other model organisms such as bacteria, yeast or even animals (worms, flyes etc.).\nWithout experimental data this might be however to speculative for discussion.\n• Dose-dependency is an important feature of the system. Photosynthetic parameters show dose-\ndependency that suggest PAP7 levels increase with more BLRE repetitions, but the data on PAP7\nexpression levels (1c) do not show the same trend – seemingly because only three measurements\nwere taken and one is unusually low in line BVB12. Repetition of this experiment to reduce\nuncertainty in BVB12 would give readers confidence that the system works in the way expected, and\nthat they could choose a number of BLREs to have some control over the extent of expression (closer\nto 70% of wt with 1x or to 100%? of wt with 3x).\nResponse: The reviewer is absolutely right, dose-dependency is indeed an important feature of the\nBVB optoswitch. It becomes, however, more apparent with increasing plant age. While expression\nlevels of PAP7 in cotyledons of BVB04, BVB09 and BVB12 (Fig. 1B) do exhibit induction under B\non\nconditions with relatively similar overlapping values (Fig. 1c), the phenotypic consequences on\nphotosynthetic capacity and pigment content in later stages show a clear dose dependency (Fig.\n1e,g,h). We added additional data showing phenotypes of BVB04, BVB09 and BVB12 in later stages\n(with approx. 8-10 leaves) alongside with the respective PAP7 expression in these plants (new Fig.\n1d,e). Here, the dose dependency of the gene construct expression becomes clearly visible (with 50-\n60% of wt for 2xBLRE; 75% of wt for 3xBLRE and 85-95% of wt for 4xBLRE). This corresponds perfectly\nto the phenotypic observations at that developmental stage (which further corresponds to the data\nshown in former Fig. 1d,e now Fig. 1f,g,h). The figure was expanded accordingly and the text was\nadapted indicating the dose dependency in more detail.\n• The inability to completely complement to wt levels (evident in 1d-e) is an important limitation that\ncould be more clearly quantified. The leaky expression in the 4xBLRE off condition led the authors to\nanalyse plants with 3xBLREs. It would be helpful for readers if (1) photosynthetic efficiency (fig 1d)\nwas quantified, and (2) the main text stated the attributes of the line selected as useful (BVB09):\nPAP7 expression relative to wt as a %, photosynthetic efficiency as a % relative to wt, total\nchlorophyll levels as a % relative to wt. Each of these numbers will likely reassure readers that\nalthough the complementation level is not 100% it is still relatively high.\nResponse: The reviewer raises a highly important point that was very difficult for us to decide on.\nEither we could chose to go for a complete complementation but with some leakiness in the R\noff\ncondition or we could go for non-complete complementation but with full tightness in R . We chose\noff\nthe latter condition to avoid any ambiguities for further experiments since we were convinced that\nthis would provide more conclusive experimental data on the role of PEP in chloroplast biogenesis\nand its initiation. We now provide the requested quantification data and, in addition, explain why the\ntightness of the system is more important than a full complementation.\n• The conclusion there is a ‘time window’ (line 173) was not immediately obvious from the data. I\nstrongly suggest the authors explain the reasoning behind this conclusion. In particular, are they\nconfident from this experiment alone there is both a cell age that is too young and a cell age that is\ntoo old for PEP activation to be sufficient for greening? Or is it only the threshold of too old that can\nbe observed?\nResponse: In the last two years we made many different light switch experiments with varying time\nranges in R and B conditions. In the beginning of our studies we were just happy to get green\noff on\nleaves, but later we realized that fully developed albino leaves never started to green, although they\ngot the light induction. This led us to conclude that there is a sensitive phase in leaf cells in which B\non\ncondition are able to induce chloroplast biogenesis. Our further studies than demonstrated that cells\nthat exceed a certain age do not green anymore suggesting an age threshold that, if passed, does not\nallow chloroplast biogenesis anymore. In sum our data indicate that there is a threshold of too old.\nWe removed the term „time window“ and rephrased the text to make this more clear.\n• Regarding the time range (line 176): ‘crucial’ is a problematic choice of word as PEP is likely also\ncrucial in other times. A better phrasing would be in terms of ‘necessary’ and ‘sufficient’: PEP is both\nnecessary and sufficient in the first time period and is presumably still necessary but no longer\nsufficient in the second time period. Is that how the authors see it?\nResponse: The reviewer is right, PEP is crucial not only in early phases but also in later stages. We\nrephrased this sentence to avoid any misunderstandings.\n• The ‘patchy’ complementation seen in the three-day shift may arise because the cells at the centre\nof the leaf are younger and were not exposed to blue light for enough time for sufficient PAP7\naccumulation to occur, therefore remaining white. Is this the authors interpretation too? Or do they\npropose that cells can be too young for their plastids to green? Could these alternatives be\ninvestigated by checking what the minimum duration of blue-light exposure needed for greening to\noccur is?\nResponse: We think it works the other way around, the cells get enough blue light but the white cells\nare already too old. The figure is a bit tricky in this respect, but we had no idea for a figure outline\nthat is better than the present one. If the reviewer has any suggestion to improve it, we would be\nhappy to change the figure accordingly. In fact it works like this: Seeds are sown in parallel on several\nPetri dishes that obtain 1 or 2 or 3 or 4 or more days of red light light. Then they are shifted for 10\ndays to blue light. Thus they all obtain enough time in blue light to induce PAP7 even in cells in the\ncenter of the leaf blade. Seedlings that have obtained 4 days or more of red light before the shift to\nblue light did not generate green cotyledons anymore. Only subsequent leaves could green.\nSeedlings getting only 1 or 2 days red light after sowing, however, generated green cotyledons in the\nblue light and those getting 3 days in red light developed intermediate patterns with patchy leaves.\nWe think the white cells in these leaves are too old to induce chloroplast biogenesis, while the green\ncells are younger (due to later cell division because of the special leaf blade development programme\nin dicot plants) and still are susceptible to the blue light induction. Since the duration of blue light\nillumination is not limiting we conclude that the age of the cells is the critical parameter. This is what\nwe call the point-of-no-return. Once this point is passed by a cell it cannot generate chloroplasts\nanymore even if it gets enough blue light. In the case of the follow-up leaves in Fig. 2 the scenario is a\nbit different. Here tip and edges become green because these cells were developing in the leaf\nprimordia in Bon. Then the shift to Roff was done and all cells that developed subsequently could not\ninduce CP biogenesis. After a second switch to Bon these cells were however already too old for\ninduction. We think that this is due to a predetermined developmental cascade that couples cell age\nwith chloroplast development and that becomes desynchronized if PAP7 is not expressed in early\nstages of cell development. This is in full agreement with studies from Loudya et al who analysed\ndifferent cell types along the developmental gradient in monocots. We improved the text in results\nand discussion accordingly and explain this now in more detail.\n• The authors note that DCMU increases the abundance of PEP specifically in BVB09 and not wt. This\nappears striking: the difference appears to be 10-fold or more in the 40 uM treatment and exceeds\nPEP abundance in wt. Could the authors comment in the Discussion what this might indicate? Can it\nbe concluded that an increased expression of PAP7 relative to wt leads to increase in the production\nof all PEP subunits? PAP7 expression quantification on DCMU-treated plants may provide the\nnecessary insight.\nResponse: Yes, this observation is somewhat surprising. Apparently, DCMU treatment (actually done\nbefore the greening) promotes the accumulation of PEP complexes in BVB09, at least as detectable\nby the BN PAGE technique. We dont want to speculate too much on that since reviewer 3 is skeptical\nabout DCMU effects on gene expression. However, the increased transcript accumulation of rpoB\nand GFP under these conditions (see Fig. 6AB) suggest that the strong inhibition of all newly\nemerging photosystem II complexes by the DCMU treatment pretends a metabolic or developmental\nplastid stage that is similar to an initial step of chloroplast biogenesis when NEP promotes PEP\naccumulation. The effect might be induced indirectly, e.g. by lack of metabolites such as sugars or\nlipids. Further, it is possible that this effect is transient. The plastids in BVB09 are less well developed\nthan those in WT, because their development started with 7 days delay (after the shift to Bon).\nPossibly NEP is more active in BVB09 than in WT in that moment. We introduced these comments in\nthe discussion as requested.\nAdditional comment: The major determinant for PEP accumulation is likely the generation of RPO\nsubunits as these build the core to which the PAPs associate. We regard it as unlikely that enhanced\nexpression of a single nuclear-encoded PAP at transcription (as suggested by the reviewer) or\ntranslation levels governs the expression of RPOs. Currently, we assume that PAPs are produced in\nslight excess and attach to the RPO core in a stoichiometric number. We acknowledge the suggestion\nof the reviewer, but detection of PAP7 transcripts will not provide a sufficient explanation. Actually,\nthe expression balance of PAPs is very delicate. Many experiments in our lab indicate that both,\ntoo low as well as exceeding expression of PAPs lead to pale phenotypes, possibly because this disturbs\nthe number of PAPs that can be imported into plastids, for instance by cytosolic sequestration. We\ncannot explain this phenomenon right now since this requires precise quantification experiments\nwhich represent a project of its own. We aim to start a study on this, a project proposal is currently in\npreparation.\n• In line 266 there is a risk of overstating how surprising this is. The data presented does not indicate\nthat PEP does not have a ‘predominant role’, only that another factor is also required and is lost with\ntime. The major discovery appears less about the role of PEP, and more about the identification\nthere is another essential factor in chloroplast biogenesis (of which there are many) that is no longer\navailable.\nResponse: We agree, that this point is a question of interpretation and can be understood also in a\ndifferent way than we did. We rephrased this section in a way that is less strict leaving it more open\nand mentioning the possibility of a yet unidentified factor.\n• It is not introduced how this system differs from recent publications on plant optogenetics (refs 47,\n48). This seems important context that could be provided in the introduction, rather than in the final\nparagraph. It is also not clear to a reader in what way the ‘experimental goal and working principle’\nare different – additional detail would help emphasise the novelty of this work.\nResponse: We were reluctant in introducing too many technical descriptions in the introduction as\nthis would render our study into a more technological report which was not the aim of our project.\nHowever, we understand the concern of the reviewer that the progress of our PINC approach does\nnot become apparent without more detailed knowledge about the current state of other\napproaches. We, thus, introduced a paragraph in the introduction mentioning otptogenetics and\nchemical induction systems which should help to place our system into the current technological\ncontext.\nMinor comments:\n• The authors seem to switch between PINC and BVB optoswitch to describe their technologies. Is it\nnecessary to have both? Could PINC be used throughout?\nResponse: PINC is the general approach that can be technically modified in many ways (e.g. by\nchosing different promoters or light wavelengths). BVB is the tool (with BVB+number to indicate the\ndifferent genetic lines) that we have specifically developed for our study and scientific question. So\nwe think, both terms have their justification. We gave additional explanation in the introduction and\nresults sections to clarify this and adapted the terms where necessary.\nResponse to the improvements given below: We thank the reviewer for this really careful\nconsideration of our manuscript. We introduced all the changes suggested below or performed\nmodifications of the text that align with the reviewer comments (see tracked changes). Below, we\ngive only a few specific responses in cases were it seemed to us necessary for explanation.\n• Line 32: ‘show the effect of temporally…’ would be clearer\n• Line 34: ‘allows reconstitution of PEP…’ (preferable) or ‘allows us to reconstitute PEP’\n• Line 37: ‘is effective only’ might be changed to ‘can only occur’\n• Line 39: ‘to grow’ should be ‘growth of’\n• Line 66: ‘photosynthesis’ should be ‘photosynthetic’\n• Line 68: ‘plastid-own genome’ should be ‘plastid genome’ (preferable) or ‘plastid’s own genome’.\n• Line 73: comma needed after ‘ancestor’\n• Line 77: ‘cyanobacterial crab-like structure’ should be ‘bacterial crab claw architecture’ or similar\n(the crab claw shape is not unique to cyanobacteria).\n• Line 79: ‘put forward’ should be ‘advanced’ or similar\n• Line 80: ‘it does not provide direct clues’ – I would argue the structure does provide clues (or\nhypotheses more specifically). What it does not provide is proof, which is what makes the described\ngenetic analysis essential.\n• Line 82: should end with ‘in Arabidopsis’ for clarity?\n• Line 86: ‘hardly produce’ could be ‘produce few’ or ‘generally do not produce’ for clarity.\n• Line 92: ‘allow to grow’ should be ‘allow growth of’ (preferable) or ‘allow us to grow’.\n• Line 100: ‘As regulatory tools’ should be 'As the regulatory element' or similar.\n• Line 102: ‘Basic’ should be ‘The basic’\n• Line 112: ‘are’ should be ‘were’\n• Line 115: ‘a point-of-no-return’ is a somewhat unambiguous expression. Perhaps the authors would\nconsider alternative wording, such as: 'that initiation of chloroplast biogenesis must occur before a\nspecific timepoint during leaf development.'\n• Line 117: ‘potential photosynthetic influences’ is ambiguous. Perhaps the authors would consider\nthe phrasing: 'to test whether PEP formation depends on, or is influenced by, photosynthetic activity'\n• Line 119: The authors need to clarify whether they are referring to PEP assembly or activity – what\nhas been measured?\n• Line 121: comma needed after ‘optoswitch’.\n• Line 128: ‘PEP complementation’ would be unclear to some readers. ‘for controlled\ncomplementation of PEP biogenesis’ may be clearer?\n• Line 133: could a citation be provided to the publication(s) pap7 phenotype was originally\ncharacterised?\n• Line 135: (BLRE)\n• Line 144: ‘effective’ could be ‘switchable/controllable’?\nRephrased into: While transformation of constructs with a single BLRE cassette did not yield green\nplants (Supplementary Fig. 2b), experiments done with constructs containing two, three or four BLRE\nrepeats were successful.\n• Line 144: A significant improvement would be to clarify what each line is in the main text –\ncurrently the reader must look at the figure for this. For example: ‘representative lines with 2\n(BVB04), 3 (BVB09) or 4 (BVB12) BLRE repetitions developed…’\n• Line 145: ‘1c’ should be ‘1b’ and ‘2c’ should be ‘1c’\n• Figure 1c: ‘relative expression’ could be more specific on the figure: ‘Relative PAP7 expression’.\n• Line 145: comma needed before ‘with’\n• Line 145: ‘close to’ could be made more precise with a percentage: ‘approximately 70-90% of wild\ntype levels’?\n• Line 149: ‘photosynthesis’ should be ‘photosynthetic’\n• Line 149-150: commas needed: ‘, especially in older plants, ‘\n• Line 157: could be clarified – this is presumably multiple individual plants, not one plant being\nshifted at different time points? The word ‘it’ suggests an individual. ‘plants were grown in… before\nbeing shifted to..’ would be clearer.\nResponse: Yes, we did these experiments with many seeds/plants and show only one representive\nfor each condition. The plants depicted in the photographs are indeed different plants. In Fig2 f,g\nhowever we show one individual plant each to demonstrate the developmental timeline of a single\nplant in response to repeated light shifts. We improved text and figure legend accordingly to make\nthis clear.\nFigure 3a legend would likewise benefit from clarifying these were different seedlings transferred to\nblue light at different time points, as it would be possible a reader would think this is the same\nseedling. Could the wording be ‘developmental timeline showing representative images of albino ….’\nOr similar?\nResponse: Yes, each photograph depicts a different seedling since these are placed under blue light\nafter the indicated time in red light. We improved the figure legend accordingly.\n• Line 158: Could be stated more clearly that the conditional is simply enough time for follow up\nleaves to grow: perhaps ‘and albino follow-up leaves if cultivated in Roff for long enough for these to\ndevelop’?\n• Line 165: comma needed before ‘where’. This statement would benefit from a citation.\n• Line 170: comma needed before ‘perfect’\n• Line 172: ‘i)’ should be before ‘demonstrate’ (as ‘suggest’ is after ii)\n• Line 173: ‘time window’ may not be the best way to describe as there is a limit to the latest point\nbut not the earliest – see comment above.\nResponse: We rephrased this sentence.\n• Line 177: ‘and performed’ could be ‘by performing’\n• Line 183: ‘biogenic’ is not clear to this reviewer. If this has a specific meaning, could the authors\nclarify in the text what is meant? If not, this word could be removed.\n• Line 191: comma needed before ‘suggesting’\n• Line 194: Heading should be clearer, and would benefit from being in the form of a conclusion:\n‘Production of PEP does not depend on photosynthesis’ for example.\n• Line 196: comma needed before affecting\n• Line 197: ‘the perfect tool’ appears an overstatement: perhaps ‘provides a tool’?\nRephrased into: provides a suitable tool\n• Lines 219-220: commas needed ‘, and also that of … RNAP II, ‘\n• Line 229: ‘especially’ – do the authors mean ‘notably’, or that PRIN2 has particularly little\nresponsiveness to DCMU?\n• Line 235: comma needed before ‘suggesting’\n• Line 236: Should be ‘In the case’\n• Line 239: ‘inhibitor’ should be ‘inhibitory’\n• Line 245: comma needed before ‘which’\n• Line 247: ‘tailor-made’ is not needed\n• Line 248: ‘alternatively’ should be ‘additionally’?\n• Line 249: comma needed before ‘allowing’\n• Line 251: ‘highly’ should be ‘greatly’ or ‘substantially’\n• Line 253: comma needed before ‘which’\n• Line 259: comma needed before ‘indicating’\n• Line 261: should be ‘complicated’\n• Line 265: comma needed before ‘indicating’\n• Line 266: a citation is needed for what the ‘current models’ are.\n• Line 271: ‘identical’ appears an overstatement: to what level of precision? Perhaps ‘consistent with\nthe timescale of retrograde..’?\nRephrased into: corresponds\n• Line 272: should include ‘in wheat’ to clarify it is not necessarily directly comparable.\n• Line 272: should be ‘The physical nature’\n• Line 277: comma needed before ‘indicating’\n• Line 279: comma needed before ‘function’\n• Line 288: should be either ‘PAP6, PAP10…’ or ‘PAPs 6, 10,..’\n• Line 290: comma needed before ‘regulation’\n• Line 300: comma needed before ‘our’\n• Line 311: could this be ‘in standard growth cabinets equipped with…’ for simplicity?\n• Line 314: why must the phenotype be visible/trackable? Molecular techniques could be used too\nsurely?\n• Figure 1e: there appear to be too few significance comparison on the chart. Could differences\nbetween groups be presented as letters using a suitable post-hoc test?\nResponse: For clarity reasons we gave only those significance comparisons with immediate relevance\nto the parameter in question. In the Roff condition only the comparison between BVB12 and pap7-1\nis important (because it indicates the potential leakiness) while comparisons between BVB04,\nBVB09 and pap7-1 were not significant. For a comparison to the wt control a statistical test was not\nrequired because of the eminent data differences. In the Bon condition only the differences between\nBVB04 with wt were significant, while those of BVB09 and BVB12 were not (indicating the dose\ndependency). For a comparison to the pap7-1 control a statistical test was not required again\nbecause of the eminent data differences. We now added additional information about the non-\nsignificance in the differences and a comment in the figure legend where we indicate this including\nthe statistical test used.\n• Figure 2 is labelled Figure 1.\nCorrected.\n• Figure 1d and Figure 2d would benefit from quantification (bar charts).\nIntroduced.\n• Figure 6: the colouring and key are not clear. The key for ‘shift’ should at least include red/blue\ncolours behind the lines. However, I strongly suggest having its own colour, rather than attempt to\nconvey experimental approach inside a bar chart that may be mistaken to be a divided bar chart\nwhere two measurements are shown within a column.\nResponse: We agree, the colouring of the bars and the key for the shifted samples were neither\nconsistent nor intuitive. Using a different colour would, however, destroy to some extent the style of\nour figures in our manuscript in which we always tried to support the understanding of the data by\nthe colouring. We therefore decided to label the bars by a perpendicular red/blue colouring that\nsuggests the red/blue switch during the growth period of the seedlings, but avoids the potential\nmisunderstanding of a divided bar chart with two additive measurements. The key was adapted\naccordingly.\nReviewer #2 (Remarks to the Author):\nThis study from the Pfannschmidt laboratory generates an elegant, simple yet ingenious optogenetic\nsystem for use in plants. Several optogenetic systems have been developed and refined over the last\ntwo decades. Their aim is to control gene expression in an on/off manner, ideally fulfilling a number\nof requirements: (1) no need for exogenous chromophores, (2) minimal interference with\nendogenous signalling pathways, (3) compatibility with the need for light cycles for plant growth and\n(4) be widely applicable.\nThe system described by Uecker et al. beautifully fulfils requirements 1 and 3. 2 and 4 are achieved\nwith some limitations. As the data themselves show, monochromatic red light used for growth\nresults in plants in which the lack of blue photoreceptor action causes, among visible phenotypes,\nelongated petioles and leaf blades which are unable to face the light sources. The applicability of the\nsystem will depend on the ability of the blue light-responsive element of the CHS gene, identified in\nArabidopsis thaliana, to be active in other species.\nResponse: Yes, the red light illumination causes an elongation of petioles and a downward turning of\nthe leaves. This is a well-known phytochrome response that we cannot avoid in our system. It,\nhowever, has no critical influence on our approach since the induction is done in blue light and hits\neither cotyledons or the apical meristem, both face the light sources perfectly. Further the tissues are\nalbino and the light penetration can be expected to be efficient even in unfavourable leaf positions.\nFor a transfer into another plant species we would first compare the Arabidopsis BLREs to those of\nthe target species. If sequence differences can be identified we would isolate the corresponding\nsequences of the target species and implement them in our system.\nWhile those limitations cannot be questioned, use of the system in Arabidopsis is sufficient to justify\nits utility. Furthermore the authors develop it for one specific purpose: to generate genetic stocks of\nmutants whose homozygosity is either lethal or, at least, incompatible with seed production, and\ntherefore with propagation. This then allows refined experiments with homozygous material to\nidentify the consequences of presence/absence of gene function, repeated multiple times: the\nrelevant, homozygous stock is generated under standard growth (white light) conditions, and the\ncontrol achieved by turning complementation off by transfer to monochromatic red (i.e. blue-less)\nlight. This is entirely, beautifully achieved, applying the technique to study the function of the plastid-\nencoded RNA polymerase (PEP), through its requirement of nucleus-encoded PAP7/pTAC14. The\nstudy demonstrates the usefulness of the technique, and reports three key findings: the requirement\nfor PEP function during a critical time window in cells in which early stages of chloroplast\ndevelopment are taking place, but very little later on, the fact that this requirement is (as expected)\ncell-autonomous, and lastly that PEP activity appears to be minimally dependent of photosynthetic\nfuncton(photosynthetic electron transport) during these biogenesis stages. The unexpected result of\nthe activity of PEP under photosynthesis-inhibited conditions is precisely described by monitoring\nboth transcript levels and overall protein composition of the relevant, dissected tissues.\nOne issue may require further evidence to confirm the stated critical window and “point of no return” of PEP requirement. The\nevidence provided shows that seedlings grown for up to three days in red light (R), or in darkness, can have PAP7 activated\nby switch to blue (B) and the cotyledons green, but this is not the case after four days (of R or darkness). The authors\nconclude that PEP activation later cannot cause chloroplast greening. This interpretation would require demonstration that\nPEP activation by B did indeed take place after 4 days in R or darkness. The endogenous CHS activation system used\nmight, conceivably, also have a window of competence, and this would explain the same observation. One easy test would\nbe to monitor the transcript of the transgene, making use of the fused GFP, as done in Fig. 6.\nResponse: We thank the reviewer for this insightful suggestion which is a highly valuable\nexperimental addition to our study. Actually, we have not thought about the possibility that the blue\nlight induced CHS promoter system could have also a time window for competence. We thus\nfollowed the proposal of this reviewer and grew BVB09 seeds 2, 3, 4 or 5 days in either dark or R\noff\nfollowed by a switch to B , respectively. We then measured the expression of the PAP7 construct in\non\nthe cotyledons by qRT-PCR after 2 days of blue light illumination (a time point far before new leaves\ncould emerge and compromise the experiment). We found a strong expression of the construct after\nthe shift to Bon in all samples regardless of the tested pre-cultivation time. This indicates that the\nlack of chloroplast formation after 4 or more days of non-inducing pre-cultivation is not caused by a\nlack of responsiveness of the system. This observation is in favor of our hypothesis that intrinsic\ndevelopmental constraints are responsible for the missing chloroplast biogenesis. The data were\nadded as Supplementary Fig. 3g and the corresponding text in the results section was adapted\naccordingly.\nThe reviewer also found the cell-autonomy of the above response particularly intriguing. Thresholds\nof activity of essential, cell-autonomous processes in chloroplast biogenesis clearly exist, are\nprobably operating in early proliferating cells in leaf primordia, and explain the development of cell\nsectors of contrasting phenotype, as seen in variegated leaves. The evidence for this cell-\nautonomous requirement is presented in Fig. 3 and shown by what seems a cotyledon (Fig. 3B,\npossibly derived from 3A).\nResponse: This is correct, Fig. 3B is a magnification of Fig. 3A. We mention that in the figure legends,\nbut to make it more clear we connected now both images by lines and included a frame in Fig. 3A. In\nSupplementary Fig. 3 we added additional examples from other plants to demonstrate that the\nappearance of patchy cotyledons can be robustly reproduced.\nCotyledons are, of course, embryonic organs. Was such a phenomenon ever observed in newly-\ndeveloping leaves emerging from the meristem? A seedling in Fig. 3F may show this, but the image is\ninsufficient to confirm. It would be of value if the authors could clarify.\nResponse: The reviewer is absolutely right, cotyledons are embryonic tissues that may exhibit\nproperties that deviate from follow-up true leaves. We, however, observed very similar\nphenomenons in true leaves when we performed our light shift experiments presented in Fig. 2. For\ninstance look to leaves 3a and 3b in Fig. 2e. These leaves emerged from the apical meristem at the\nend of the B condition and the leaf blade expanded later in R conditions. As a result you can see\non off\nat the end of the photo time line (all photos show the same plant even if the orientation rotates) that\nthe leaves 3a and 3b have green tips and edge while the leaf blades remain white with some light\ngreen patches. Such kind of mosaic patterns could be observed often in the shift experiments since\nthe BVB09 line grows faster in Bon than in Roff preventing a perfect match between induction times\nand emergence of leaf primordia. Actually we aimed to obtain perfect alternating green and white\nleaf pairs but realized that this would require extensive testing of variations in induction times to\nobtain perfect synchronicity (a project for the future). We pointed towards leaves 3a and 3b in the\nresults section and show additional follow-up leaves in Supplementary Fig. 3f. We further rephrased\nthe discussion about the cell-autonomy and explain the phenomenon in more detail.\nLastly, the authors display how transcript levels of a range of genes, nucleus- and plastid-encoded,\nare impacted by the absence of PAP7 or its complementation. Among plastid-encoded genes, those\ntranscribed by the nucleus-encoded polymerase are (with the exception of rpoB) all presented in Fig.\nS4. Do the authors have an explanation for the low transcript levels of petD in the pap7 mutant and\nthe R-grown BVB line?\nResponse: Because of the space constraints we selected only a few representative genes with\ngeneral relevance for the study to appear in Fig. 6. This does not mean that the other genes included\nin the supplement are less important or less relevant. In fact, also these data include important\nresults which are of great interest, however, more for a specialist rather than for a broad audience.\nTherefore, we show and discuss only the most important data.\nThe plastid petD gene belongs to the group of class II genes, thus it is transcribed by NEP and PEP.\nHowever, PEP is the dominant polymerase for its transcription and consequently petD transcript\naccumulation is very low in the homozygous pap7-1 control as well as in the BVB09 line grown in\nRoff. These expression data correspond well with those we reported earlier for petD (compare\nGrübler et al 2017 Plant Phys).\nThe text is generally well written, and the figures beautifully constructed to easily interpret. Note,\nhowever, that Fig. 2 is also labelled as a second Fig. 1. On several occasions Fig. 2 is referred to as Fig.\n1, and viceversa.\nResponse: Numbering has been corrected.\nEditing comments:\nRepetitions > repeats. done\nLine 79 put forward > advanced (to “put forward” is done only the first time) done\nResponse: Fig 1f was corrected to Fig 1e. Remark on Fig 2f likely refers to Fig. 1e. Leakiness in Chl\nproduction is poor but detectable and statistically significant when compared to pap7-1 as indicated\nby asterisks. It is however not well visible in the bar chart because of the high wt values. The precise\nvalues are provided with the Source Data sets now. Indeed, the leakiness is not reflected at the level\nof PAP7 expression, but to avoid any potential ambiguity in further experiments we chose the 3-\nrepeat BVB line that is tight in both parameters. This is discussed now in more detail as also\nrequested by another reviewer.\nFig. 6 What is given a value of 1, y axis?\nResponse: The expression data are given as „relative expression“ where the detected value refers to\nEF1alpa expression as internal control. This is indicated in the figure legend now.\nFig. 1 legend Ron > Bon done\nMethods should indicate the statistical tests used. Note that multiple testing increases errors, and\nthere are ways to avoid those.\nResponse: The statistical tests have been mentioned now in the methods section and appropriate\nplaces in the manuscript.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Uecker et al. addresses an important and timely question: the role of plastid-\nencoded RNA polymerase (PEP) in chloroplast biogenesis. Recent progress in structural\nunderstanding of PEP has only reinforced the importance of open questions regarding the biological\nroles of this crucial enzyme. Advancement in this field has been hindered by a challenging chicken-\nand-egg situation, where disruptions of PEP subunits impede chloroplast biogenesis, subsequently\ndisrupting various processes, potentially including transcription itself. This manuscript addresses this\nconundrum by developing a novel inducible system, enabling precise control of PEP subunits (or any\nother proteins) through exposure to different wavelengths of light. Utilizing this tool, the manuscript\ninvestigates the role of PEP and elucidates two important aspects of its involvement in chloroplast\nbiogenesis. (1) A limited time window for PEP-dependent chloroplast development, which is an\ninteresting novel finding and (2) independence of chloroplast development of PEP transcription.\nAlthough these insights do not exhaust the power of the new inducible system, the current scope of\nthe manuscript is appropriate and fully justified by the impact of the novel optogenetic system.\nThis is a significant study that will establish a new standard for investigations of PEP subunits, other\nessential chloroplast proteins, and likely other essential plant proteins. The manuscript is well-\nwritten, technically solid and will be of interest to a broad audience of scientists studying genetics,\nnot only in plants.\nSpecific comments\n• There is no data showing behavior of the BVB optoswitch on the level of RNA or protein\naccumulation. It is important to know the level of induction and the level of leaky expression, which\nis a common problem of many inducible systems. Adding new experimental data providing these\ninsights would substantially strengthen the manuscript.\nResponse: We gave expression data of the BVB gene construct in Fig. 1C and Fig. 3D as well as\nprotein data in Fig. 6. These data are however endpoint data. We assume that with the term\n„behaviour“ the reviewer is asking for the properties of the system covering speed of responsiveness,\nmaximum level of expression and the ability to shut off. These are indeed important properties to\nknow especially in comparison to other inducible systems using chemical inductors. To this end we\nperformed a detailed kinetic experiment. BVB09 and wt seeds were grown for 7 days in Roff\nconditions and PAP7 expression was determined by qRT-PCR. Subsequently, the seedlings were\nexposed to Bon for 24h to observe the induction followed by another shift to Roff to determine the\nshut-off properties. The induction of the BVB optoswitch reached wt levels within 30 min and\nmaximum level after 4h with more than 2 times the expression level of wt. The shut-off was rapid\n(reaching less than 10% of max level within 1h and almost zero after 4h). With respect to\nresponsiveness and tightness this system is definitely of high value. We generated a new figure (Fig.\n2h) and introduced these data into the manuscript right after the shift experiment data described in\nFig. 2 including a description of the expression behaviour of the BVB optoswitch.\n• The mechanism responsible for the checkpoint that provides competence for chloroplast\nbiogenesis should be discussed more thoroughly. For instance, is it possible that this checkpoint is\nmediated by nutrient availability in seeds?\nResponse: We expanded the discussion of this point as also requested by reviewer 1. Nutrient\navailability could be one possibility, but also other influences such as defined developmental check\npoints could be imagined. This question certainly will be a major focus of our future research.\n• There is no justification of why the new optogenetic system is better than previously established\ninducible systems in plants. There is also no comparison to other optogenetic inducible systems.\nResponse: The reviewer is absolutely right, we did not describe and discuss the advantages of the\noptoswitch in comparison to other well established systems. The new figure describing the temporal\nand quantitative properties of the system, however, now provides a sound data base for such kind of\ndiscussion. Correspondingly, we included some remarks about speed, degree of responsiveness and\nleakiness as well as some more general consideration such as simplicity and suitability of the system\nin the discussion. Please see the revised manuscript for details.\n• There is no discussion of potential impacts of growing plants in narrow spectra (red or blue light\nonly) compared to white light, which is an important consideration for the interpretation of results\nobtained using the BVB optoswitch.\nResponse: Here, the reviewer raises another important point for the evaluation of the system. One\ncould assume that the different light qualities generate side effects, and indeed in red light we see a\nclear petiole elongation, a well-known phytochrome response under continuous red light. However,\nwe observed no major impacts of the narrow bandwith lights on growth behaviour, seed production\nand photosynthetic capacity when compared to white light. This is likely caused by the adjusted light\nintensities (see Supplemental Fig 1). For the reaction center of PSII an equal number of red or blue\nphotons will result in the same number of charge separations since the higher energy content of the\nblue photons is released as heat and only the energy content to excite P680 is used. From the\nviewpoint of photosynthesis the different light qualities are thus negligible. With respect to\nphotomorphogenic responses, the different light qualities definitely have an impact which however is\nnot relevant for the scientific questions we follow in our present study. We discuss this now in more\ndetail and conclude that the BVB optoswitch is well suited for many different questions except if the\nstudied process is affected by the light qualities (for example through the action of specific\nphotoreceptors).\n• DCMU treatment has been used in the past and citing and discussing original literature describing\nits limited impacts on chloroplast biogenesis would strengthen the conclusions of this manuscript.\nResponse: DCMU as photosynthesis inhibiting herbicide has been used in a plethora of studies, but\nmostly in analyses that study photosynthetic functions. DCMU effects targeting the process of\nchloroplast biogenesis were studied in only very few cases, and to the best of our knowledge the\nherbicide has been never used to elucidate a potential impact of photosynthesis on PEP formation.\nDoing an extensive literature search we identified some publications that deal with related aspects,\nbut no one used DCMU in a way that corresponds to our specific experimental question. We,\nhowever, added two references describing DCMU effects on chloroplast biogenesis in the discussion\nthat appear to us appropriate to meet the reviewers concerns.\n• Impacts of DCMU on RNA accumulation are sometimes overinterpreted as impacts on gene\nexpression. This part of the manuscript has limited influence on its conclusions and could easily be\nshortened.\nResponse: Actually the DCMU treatment aimed to block photosynthetic signals that were proposed\nto affect or regulate the build-up of the chloroplast RNA polymerase in early chloroplast biogenesis\n(see Diaz et al 2017 Nat. Commun.). The oberved greening alone (Fig. 4) was not sufficient to prove\nthis question. Therefore we did a micro-dissection of the greening tissues and checked the presence\n(by BN-PAGE, see Fig. 5) and the activity of PEP by determining the accumulation of plastid\ntranscripts (Fig. 6). We detected only minor effects of the DCMU treatment on transcript\naccumulation indicating that photosynthetic electron flow has only very limited effects at this level,\nat least during the early stages of chloroplast biogenesis. While this appears as confirmatory\nevidence on first sight, it is however of high importance for the conclusions of our study as it\ndemonstrates that the assembled PEP complex identified in the subsequent green leaves by BN-\nPAGE is indeed functional. Further, this figure provides additional information about efficiency and\ntightness of the BVB tool. It directly compares transcript accumulation in Roff conditions for BVB09\nand pap7-1 indicating the tightness of the BVB tools (since we do not observe any increase in gene\nexpression in BVB09 vs. Pap7-1). The figure further demonstrates the increase in expression in Bon\nconditions (including the increase after a shift) and finally provides also a comparison to WT under all\nthese conditions. We therefore would like to keep this figure as it is (in addition also to meet\nrequests from reviewers 1 and 2), but we now include more cross-references in the manuscript to\nunderline its importance and rephrased parts of the paragraph to make this more clear.\n• There are problems with labels of Fig 1F (referenced in the manuscript) and Fig 2 (mislabeled in\nfigure legends).\nResponse: This has been corrected.\nPoint-by-point response\nReviewer 2\nMinor corrections:\nLine 405: Please replace “pretends” with “mimics”\nResponse: done as requested\nLine 443 (and possibly elsewhere): Replace “a well known phytochrome response” with “a\nresult to absence of cryptochrome and phototropin photoreceptors signalling”, or an equivalent\nexpression. The current statement is incorrect. Phytochrome mutants, some in particular\nexhibit elongated petioles, therefore phytochrome signalling would shorten the petioles, and\nshade-light, therefore REDUCED phytochrome signalling, elongates them. The elongated\npetioles are a result of low cryptochrome action, and the down-turned or twisted laminae a\nconsequence of absence of phototropin signalling.\nResponse: The reviewer is absolutely right, our statement was too cryptic and therefore\nmisleading. We replaced it by a more detailed statement: „a result of absent cryptochrome and\nphototropin photoreceptors signalling due to the lack of blue light“.\nBoth changes are marked in yellow in the manuscript text.\nWe further added all other details (mostly statistical p-values) requested from your in-house\neditors as stated in the Author Check List. Please see further details in our responses added\nin that document.\nIn Figure 2 and Figure 5 we added the requested scale bars and molecular weight markers,\nrespectively."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All comments were appropriately addressed, and I see no barrier to publication in its current form. Congratulations to the\nauthors on this important contribution.", "reviewer_2": "This manuscript, already of high quality in its first version, has been significantly strengthened by the additional data,\nspecially on PEP protein accumulation and function and on functionality of the blue-dependent promoter element, the\nimproved figures, and the substantially revised text. The efficiency of the optoswitch in particular is robustly confirmed. The\nauthors refer to their responses (as is customary) as a rebuttal letter, but by-and-large they have taken the majority of the\nreviewers’ comments on board. The study does, without question, contribute new understanding of chloroplast development\nand provide a useful, promising research and biotechnological tool.\nMinor corrections:\nLine 405: Please replace “pretends” with “mimics”\nLine 443 (and possibly elsewhere): Replace “a well known phytochrome response” with “a result to absence of\ncryptochrome and phototropin photoreceptors signalling”, or an equivalent expression. The current statement is incorrect.\nPhytochrome mutants, some in particular exhibit elongated petioles, therefore phytochrome signalling would shorten the\npetioles, and shade-light, therefore REDUCED phytochrome signalling, elongates them. The elongated petioles are a result\nof low cryptochrome action, and the down-turned or twisted laminae a consequence of absence of phototropin signalling.", "reviewer_3": "I am satisfied with the revisions and support publication of the revised manuscript."}, "author_response": ""}]

Decision letter