Protocol for evaluating seedling greening capacity during dark-to-light transition

Summary

We present a protocol to evaluate greening capacity in etiolated Arabidopsis seedlings during the critical dark-to-light transition. We describe steps for sample preparation and sowing and then detail procedures for quantifying protochlorophyllide accumulation in darkness, the greening rate upon illumination, and reactive oxygen species levels as an indicator of photo-oxidative stress. This protocol can be used for screening and phenotypic quantification across genetic backgrounds.

For complete details of this protocol, please refer to Zhong et al.¹ and Zhong et al.²

Graphical abstract

Highlights

• •An integrated framework for assessing greening capacity in Arabidopsis seedlings
• •Fluorometric quantification of protochlorophyllide in etiolated seedlings
• •Quantitative scoring of seedling greening rates during dark-to-light transition
• •Live imaging of reactive oxygen species via H₂DCFDA staining

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

We present a protocol to evaluate greening capacity in etiolated Arabidopsis seedlings during the critical dark-to-light transition. We describe steps for sample preparation and sowing and then detail procedures for quantifying protochlorophyllide accumulation in darkness, the greening rate upon illumination, and reactive oxygen species levels as an indicator of photo-oxidative stress. This protocol can be used for screening and phenotypic quantification across genetic backgrounds.

Before you begin

The transition from skotomorphogenesis to photoautotrophic growth is a critical and vulnerable stage in seedling establishment, making the assessment of greening capacity fundamental for understanding plant adaptive strategies. During skotomorphogenesis, seedlings accumulate the chlorophyll precursor protochlorophyllide (Pchlide).³ Upon illumination, Pchlide is converted to chlorophyll by enzymes such as protochlorophyllide oxidoreductases (PORs).⁴ An excess of free Pchlide, however, leads to photo-oxidative damage and cotyledon bleaching upon light exposure.^5,6,7,8,9 To prevent this, seedling greening is tightly coordinated by multiple internal and external signaling pathways, which synchronize to inhibit premature greening and avoid photo-oxidative damage.^{1,2,10,11,12,13,14,15,16,17} This assay provides a quantitative measure of seedling greening capacity by assessing three key parameters: Pchlide levels in darkness, greening rates following illumination, and reactive oxygen species (ROS) accumulation in cotyledons. Our method integrates multiple physiological and biochemical dimensions to deliver a versatile, quantitative framework for assessing seedling greening capacity. The protocol can be designed for forward genetic screening (e.g., identifying greening-defective or greening-enhanced mutants), functional characterization of genetic perturbations, and investigating greening responses under environmental stresses.

Innovation

This protocol presents a significant methodological innovation by moving beyond single-parameter assays to establish a integrated, quantitative framework for holistically evaluating seedling greening capacity during the critical dark-to-light transition. This protocol systematically combines three key physiological and biochemical dimensions into a unified, streamlined workflow. The protocol is meticulously optimized for rigor and reproducibility. Designed for simplicity and scalability, this comprehensive framework is uniquely suited for genetic screening, detailed phenotypic analysis across mutants, and evaluating environmental stress responses, offering a versatile tool that advances the study of de-etiolation from a descriptive to a systems-level analysis.

After-ripening of sample seeds

Timing: ≥2 months

• 1.Store the harvested seeds at 25°C in airtight containers for at least two months to ensure complete after-ripening.

CRITICAL: Maintain the relative humidity inside the containers at 30%–40% throughout the storage period.

Note: The after-ripening conditions and storage period of differnet species may differs significantly. When applying this protocol to other species, if unsatisfactory germination is observed in subsequent steps, the after-ripening duration and storage conditions should be adjusted accordingly.

Germination viability test

Timing: 5 days

• 2.
  Seed plating and stratification.

  • a.
    Surface-sterilize a minimum of 100 seeds for each genotype (Table 1).
  • b.
    Plate the seeds equidistantly (spacing >5 mm) on 1/2 MS solid medium (Table 2).

    Note: Include at least three independent biological replicates per genotype.

  • c.
    Wrap the plates in aluminum foil and stratify them at 4°C in darkness for 2 days.
• 3.
  Germination assay and viability determination.

  • a.
    Transfer the stratified plates to a growth chamber set at 22°C under continuous white light (50 μmol m⁻² s⁻¹) for 3 days.

    CRITICAL: Do not unwrap the aluminum foil until the plates have been placed inside the light incubator.

  • b.
    Calculate the germination rate: Germination rate (%) = (Number of germinated seeds/Total seeds plated) × 100.

    CRITICAL: Proceed with subsequent experiments only if all tested genotypes exhibit a germination rate >95%.

Table 1.

Seed surface-sterilization solution

Reagent	Final concentration	Amount
Triton X-100	0.5% (v/v)	500 μL
Ethanol absolute	75% (v/v)	750 mL
ddH2O	–	Up to 1 L
Total	–	1000 mL

Table 2.

1/2 MS solid medium

Reagent	Final concentration	Amount
Murashige and Skoog (MS) Basal Salt Mixture	1/2 strength	2.2 g
MES	0.1% (w/v)	1 g
Sucrose	1% (w/v)	10 g
Agar	1.5% (w/v)	15 g
ddH2O	–	Up to 1 L
Total	–	1000 mL

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
EDTA	Sigma-Aldrich	CAS# 60-00-4
Agar	Difco	Ref. 214010
Murashige and skoog (MS) basal medium	Phytotech	Cat# M524
Tris Base	Sigma-Aldrich	CAS# 77-86-1
Acetone	Sigma-Aldrich	CAS# 67-64-1
Ammonium hydroxide, 35% solution in water	Thermo scientific	CAS# 1336-21-6
Triton X-100	Sigma-Aldrich	CAS# 9002-93-1
Ethanol	Sigma-Aldrich	CAS# 64-17-5
Sucrose	Sinopharm chemical reagent	CAS# 57-50-1
MES	Sigma-Aldrich	CAS# 4432-31-9
H2DCFDA	Sigma-Aldrich	CAS# 4091-99-0
Hydrochloric acid	Sigma-Aldrich	CAS# 7647-01-0
Experimental models: Organisms/strains
Arabidopsis: Col-0	Widely distributed	N/A
Arabidopsis: EIN3ox	Zhong et al.1	N/A
Arabidopsis: ein3eil1	Zhong et al.1	N/A
Software and algorithms
ZEISS ZEN microscopy software	ZEISS	https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html
Gen5	BioTek	https://www.agilent.com.cn/en/support/biotek-software-releases
Prism7	GraphPad	www.graphpad.com
Other
96-well plates	Corning	Cat# 3590
6-well plates	Corning	Cat# 3335
Plant growth chamber	Swift Automation	Cat# PT-G1060
Rotary mixer	Benchmark	Cat# R5010
Zeiss LSM 800 confocal microscope	ZEISS	N/A
Zeiss axio zoom.v16 stereoscopic microscope	ZEISS	N/A
BioTek cytation 5 spectrofluorometer	BioTek	N/A
Glass petri dish	Zhong et al.1	N/A
Dim green safe light	Zhong et al.1	N/A
NK System LA-105 light analyzer	NK System	N/A
3M™ Micropore™ Tape	3M	Cat# 1530-1

Materials and equipment

CRITICAL: Triton X-100 is severe eye irritant and may cause skin sensitization. Prevent direct contact by wearing nitrile gloves and safety goggles. Ethanol is highly flammable and its vapors are explosive; ensure no ignition sources are present and use in a well-ventilated area. Solution can be stored at 22°C–28°C for up to 1 month.

Note: Adjust pH to 5.8 with 0.5 M KOH, then autoclave at 121°C for 15 min. The medium can be stored at 22°C–28°C for up to 1 month.

CRITICAL: Prepare freshly on the day of use. Acetone is highly flammable and volatile; ammonia is corrosive and releases toxic vapors. Prepare and use this solution exclusively in a fume hood while wearing nitrile gloves and safety goggles.

CRITICAL: Prepare freshly on the day of use, protect from light. H₂DCFDA is cytotoxic. Handle using nitrile gloves in a fume hood. Avoid skin contact and eyes. Decontaminate spills immediately with appropriate absorbent material.

Step-by-step method details

Sample preparation and sowing

Timing: 8 days

• 1.
  Preparation of glass culture dishes.

  • a.
    Select glass culture dishes with a height of at least 25 mm.

    CRITICAL: Do not use plastic dishes. The difference in refractive index between plastic and glass can interfere with accurate greening assessment. Dishes with insufficient height will cause etiolated seedlings to contact the lid, invalidating greening rate measurements.

  • b.
    Sterilize the dishes by autoclaving.
  • c.
    Pour approximately 20 mL of molten ½ MS medium into each dish to achieve a uniform depth of about 5 mm.

    Note: Maintain this medium depth. Insufficient depth can lead to aberrant seedling growth, while excessive depth may allow seedlings to contact the lid during the etiolation phase, potentially biasing results.

• 2.
  Seed surface sterilization and plating.

  • a.
    Aliquot more than 100 seeds per genotype into separate 1.5 mL microcentrifuge tubes.
  • b.
    Add 1 mL of seed surface-sterilization solution to each tube.
  • c.
    Cap the tubes and place them on a rotary mixer for 10–15 min.
  • d.
    In a laminar flow hood, carefully remove and discard the sterilization solution.
  • e.
    Wash the seeds thoroughly with sterile distilled water at least three times to remove all traces of sterilant.
  • f.
    Plate 100 seeds per sample onto the surface of the prepared 1/2 MS medium in the glass dish (Figure 1).

    CRITICAL: Space seeds more than 5 mm apart to prevent overlapping growth of seedlings.

  • g.
    Seal each plate with micropore tape (e.g., 3M™ Micropore™).
• 3.
  Stratification and germination initiation.

  • a.
    Wrap the sealed plates with aluminum foil to ensure complete darkness.
  • b.
    Incubate the plates at 4°C for 2 days for stratification.

    Note: If reduced germination is observed due to inadequate stratification, the stratification period can be extended to 3 days.

  • c.
    Unwrap the plates and transfer them to a growth chamber under continuous high-intensity white light (∼100 μmol photons m⁻² s⁻¹).

    Note: Verify light intensity at plate level using a calibrated light meter.

  • d.
    Expose the plates to light for 8 hours to trigger uniform seed germination.
• 4.
  Dark incubation for etiolation.

  • a.
    After the 8-hour light pulse, re-wrap the plates thoroughly in aluminum foil to exclude all light.
  • b.
    Transfer the foil-wrapped plates to a dark incubator set at 22°C.

    Note: Dark incubators are recommended to be placed in a dark room to prevent light-leak.

  • c.
    Incubate for 5 days to obtain uniformly etiolated seedling growth.

    Note: Etiolated seedlings should ideally be processed immediately for subsequent assays. If necessary, they can be stored in the dark at 4°C for up to 24 h; prolonged storage may affect physiological responsiveness.

    Note: To adapt this protocol for use with other plant species, optimization of the etiolation period is critical. We recommend first performing a pilot experiment using a range of dark-growth durations (e.g., 1 to 7 days) for the new species. The optimal period for subsequent experiments is the one yielding a greening rate of approximately 60%–80% in wild-type seedlings. Additional parameters, such as light intensity and the duration of growth after light exposure, may also require empirical adjustment.

Figure 1.

Representative image of seed planting on 1/2 MS medium

Seeds are plated on 1/2 MS solid medium in glass petri dishes. Each genotype includes ≥ 100 seeds to ensure statistical robustness for greening rate quantification. Seeds are spaced to prevent mutual shading during growth. Ruler divisions = 1 mm.

Protochlorophyllide content measurement

Timing: 2 days

This section details the quantification of Pchlide, whose accumulation in darkness is indicative of the seedling’s photomorphogenic potential upon light exposure.

• 5.Prepare reagents. Prepare the Pchlide extraction buffer fresh on the day of sample collection (Table 3).
• 6.
  Harvest seedlings and extract Pchlide.

  • a.
    After 5 days of dark incubation, harvest etiolated seedlings under dim green light (Figure 2A). For each sample, collect 50 dark-grown seedlings into a 2 mL microcentrifuge tube.
  • b.
    Immediately add 1.5 mL of ice-cold Pchlide extraction buffer to each tube.
  • c.
    Incubate the tubes on a rotary mixer at 4°C in complete darkness for 24 h to extract Pchlide.
• 7.
  Quantification of Pchlide by fluorometry.

  • a.
    Centrifuge the samples at 12,000 × g for 10 min at 4°C.
  • b.
    Transfer the supernatant to a new 1.5 mL microcentrifuge tube.
  • c.
    Piper 200 μL of the clarified supernatant into individual wells of a 96-well microplate. Include at least three technical replicates per sample.
  • d.
    Place the microplate in a spectrofluorometer.

    CRITICAL: All steps up to this point must be performed under dim green light in a darkroom. Exposure to other wavelengths will trigger the photoconversion of Pchlide and invalidate the measurement.

  • e.
    Set the excitation wavelength to 440 nm and record the fluorescence emission spectrum from 600 nm to 700 nm (1 nm bandwidth) (Figure 2B).
  • f.
    Plot the full emission spectrum for each sample (Figure 3A), and quantify Pchlide content by measuring the peak fluorescence intensity at 632 nm (Figure 3B). To minimize measurement error, each sample should be assayed with at least 3 technical replicates.

    Note: Perform this assay with a minimum of three independent biological replicates.

    Note: If the Pchlide fluorescence signal is too low to yeild a distinct peak, refer to troubleshooting, problem 1.

Table 3.

Pchlide extracting solution

Reagent	Final concentration	Amount
Acetone	90% (v/v)	90 mL
Ammonia	5% (v/v)	0.5 mL
dd H2O	-	9.5 mL
Total	-	100 mL

Figure 2.

Pchlide quantification in etiolated seedlings

(A) Harvest of dark-grown seedlings under dim green safelight in a darkroom to prevent photoconversion of Pchlide.

(B) Spectrofluorometric analysis of Pchlide using a pre-chilled spectrofluorometer (BioTek Cytation 5). Samples are loaded in a 96-well plate for emission scanning.

Figure 3.

Pchlide levels in Col-0, ein3 eil1 and EIN3ox etiolated seedlings

(A) Emission spectra (λex = 440 nm, λem = 600–700 nm, 1-nm step) showing Pchlide-specific peaks from Col-0, ein3eil1 and EIN3ox etiolated seedlings. Data are mean ± SD of three biological replicates.

(B) Relative Pchlide fluorescence intensity at 632 nm. Data are mean ± SD (n = 3 biological replicates, ≥20 seedlings each). ∗p < 0.05 by one-way ANOVA with Tukey’s HSD post-hoc test.

Greening rate measurement

Timing: 2–3 days

This section describes the light-triggered greening process and its quantitative assessment, which directly reflects the seedling’s ability to execute de-etiolation.

• 8.
  Light-induced greening of etiolated seedlings.

  • a.
    Transfer the foil-wrapped plates containing 5-day-old etiolated seedlings to a growth chamber providing continuous high-intensity white light (>100 μmol photons m⁻² s⁻¹).
  • b.
    Only then unwrap the plates to expose seedlings to light.

    CRITICAL: Do not open the foil until plates are inside the high-light environment. Exposure to lower light during transfer can trigger abnormal photomorphogenic responses and compromise greening uniformity.

  • c.
    Allow seedlings to grow under continuous high-intensity white light for 48 h to complete the greening transition (Figure 4A).

    CRITICAL: Maintain light intensity at approximately 100 μmol m⁻² s⁻¹ and ensure uniform illumination across all plates. Deviations from this intensity will systematically bias greening rates (too high or too low).

• 9.
  Phenotype scoring and greening rate determination.

  • a.
    After 48 h of light, score each seedling based on cotyledon appearance (Figures 4B–4D). Normal greening seedlings exhibiting expanded and dark-green cotyledons (Figure 4B), while impaired greening seedlings exhibiting white, yellowish, or light-green cotyledons (Figures 4C and 4D).
  • b.
    Calculate the greening rate for each sample as:

Figure 4.

Phenotypic classification of greening competence

(A) Representative five-day-old etiolated seedlings grown at 22°C in complete darkness, showing closed, yellow cotyledons and apical hooks.

(B–D) Phenotypes after 48 h of light exposure: normally greened seedlings with expanded, dark-green cotyledons (B); intermediate greening with partial chlorophyll accumulation and light-green cotyledons (C); and greening-defective seedlings exhibiting closed, white cotyledons as a result of photo-oxidative damage (D). Scale bar = 1 mm.

Greening rate (%) = (Number of normally greened seedlings/Total viable seedlings) × 100 where Total viable seedlings = total plated seedlings – germination-defective seeds (Figure 5).

Note: Include at least three independent biological replicates per genotype.

Note: If the wild-type greening rate is abnormally high or low, refer to troubleshooting, problems 2 and 3. If germination failure is observed in test lines, refer to troubleshooting, problem 4.

Figure 5.

Greening rates of Col-0, ein3 eil1 and EIN3ox seedlings

(A–C) Representative images of Col-0 (A), ein3 eil1 (B) and EIN3ox (C) seedlings after 48 h of continuous white light (100 μmol m⁻² s⁻¹). Expanded, green cotyledons indicate successful greening; closed, yellow cotyledons indicate photobleaching.

(D) Quantification of greening rates of Col-0, ein3 eil1 and EIN3ox. Five-day-old etiolated seedlings were exposed to white light for 48 h. Data are mean ± SD (n = 3 biological replicates, ≥100 seedlings each). ∗p < 0.05 by one-way ANOVA with Tukey’s HSD test. Scale bar = 1 mm.

H₂DCFDA staining for ROS detection

Timing: ∼2 h

This section describes the detection of ROS in cotyledons using the cell-permeable fluorescent probe H₂DCFDA. The probe is hydrolyzed intracellularly to H₂DCF, which is then oxidized by ROS to yield highly fluorescent DCF, thereby providing a quantitative indicator of photo-oxidative stress during de-etiolation.

• 10.Prepare staining solution (Table 4).

Table 4.

H₂DCFDA staining solution

Reagent	Final concentration	Amount
1M Tris-HCl (pH = 7.5)	2 mM	100 μL
200 mM H2DCFDA	2 mM	500 μL
ddH2O	–	49.3 mL
Total	–	50 mL

Prepare H₂DCFDA staining solution fresh on the day of use.

Note: All subsequent steps must be performed under dim green safelight.

• 11.
  Seedling staining.

  • a.
    Collect at least 20 seedlings (grown under the same conditions as for greening assays) and transfer them to a well of a 6-well plate.
  • b.
    Add 3 mL of freshly prepared H₂DCFDA staining solution to cover the seedlings completely.
  • c.
    Incubate the plate in complete darkness at 22°C–28°C for 30 min.
• 12.
  Wash to remove unbound dye.

  • a.
    After the 30-min incubation, carefully aspirate and discard the staining solution using a pipette.
  • b.
    Wash the seedlings by adding 3 mL of 10 mM Tris-HCl buffer (pH 7.5) to the well.
  • c.
    Gently agitate the plate on an orbital shaker at low speed (∼50 rpm) for 1 min in the dark, then remove the buffer.
  • d.
    Repeat this wash step for a total of five times.

CRITICAL: Thorough washing is essential to reduce background fluorescence. Incomplete removal of unbound H₂DCFDA will result in elevated nonspecific signals.

• 13.
  Confocal imaging.

  • a.
    Mount the washed seedlings onto a microscope slide with a minimal amount of 10 mM Tris-HCl buffer (pH 7.5).
  • b.
    Image using a laser-scanning confocal microscope (e.g., Zeiss LSM 800).

    • i.
      Set the excitation wavelength to 488 nm.
    • ii.
      Collect emission between 500–530 nm for H₂DCFDA fluorescence.
    • iii.
      Acquire chlorophyll autofluorescence in a separate channel (ex: 640 nm, em: 660–750 nm) for tissue reference (Figure 6).

CRITICAL: Use identical laser power, gain, and pinhole settings for all samples to ensure comparability.

Note: If high background fluorescence is observed, refer to troubleshooting, problem 5.

Note: H₂DCFDA is commonly employed to detect intracellular reactive oxygen species (ROS), primarily hydrogen peroxide and hydroxyl radicals, offering a sensitive and convenient method for assessing ROS levels in seedlings. For the detection of other specific ROS types, alternative histochemical stains are recommended: nitroblue tetrazolium (NBT) staining for superoxide and 3,3′-diaminobenzidine (DAB) staining for hydrogen peroxide.

Note: Microscope settings may vary depending on the specific instrument model and sample preparation. The following parameters from our setup are provided as a reference: Objective lens: Zeiss Plan-Apochromat 10×/0.45 M27 (NA = 0.45, WD = 2.6 mm) Two tracks were configured: Track 1 (for H₂DCFDA): Excitation laser = 488 nm; Detector gain = 638 V; Digital gain = 2.0. Track 2 (for chlorophyll autofluorescence): Excitation laser = 640 nm; Detector gain = 500 V; Digital gain = 1.0.

Figure 6.

ROS detection and chlorophyll autofluorescence in de-etiolating cotyledons

Seedlings were grown in darkness for 5 days, then exposed to continuous white light for 2 days. ROS were visualized by H₂DCFDA fluorescence (green); chlorophyll autofluorescence (red) indicates chloroplast development. Scale bar = 100 μm.

Expected outcomes

This protocol provides a comprehensive framework for assessing seedling survival during the critical dark-to-light transition. Three key parameters are quantified: (1) protochlorophyllide (Pchlide) content as a biochemical predictor of photoconversion capacity; (2) greening rate as the core phenotypic metric; and (3) reactive oxygen species (ROS) accumulation, visualized by H₂DCFDA staining, as an indicator of photo-oxidative stress.

Pchlide accumulation in darkness serves as a predictive marker for subsequent chlorophyll synthesis. Hypogreening mutants, such as ein3 eil1, typically accumulate higher Pchlide levels than the wild-type (Col-0), whereas hypergreening lines (e.g., EIN3ox) show reduced Pchlide (Figure 3). Discrepancies between greening rate and Pchlide content may indicate alterations in POR activation kinetics or ROS-scavenging capacity.¹⁸

For greening-rate quantification, successfully de-etiolated seedlings display expanded, dark-green cotyledons, while impaired seedlings show yellowish-green, yellow, or white cotyledons (Figures 4B–4D). Under standardized conditions, wild-type Arabidopsis (Col-0) seedlings typically exhibit a greening rate of 60%–80% (Figure 5A). Key controls include EIN3ox, which show near-complete greening (≥95%; Figure 5B), and the ein3 eil1 double mutant, which displays severe photo-oxidation with very low greening (<10%; Figure 5C). Assay validity is confirmed when control genotypes differ significantly from the wild-type baseline in statistical analysis.

Upon illumination, Pchlide that is not promptly converted to chlorophyll can generate high-energy electrons, leading to ROS production. Excessive ROS accumulation compromises seedling survival and greening ability. Fully greened seedlings exhibit low H₂DCFDA fluorescence (low ROS) and strong chlorophyll autofluorescence. In contrast, photobleached seedlings show intense H₂DCFDA signal and minimal chlorophyll fluorescence (Figure 6). Lines with impaired ROS-scavenging capacity display elevated H₂DCFDA fluorescence, correlating with their greening deficiency.

Quantification and statistical analysis

One-way analysis of variance (ANOVA) was used to compare greening rates, Pchlide content, and ROS levels across genotypes or treatment groups, followed by Tukey’s honestly significant difference (HSD) post hoc test for all pairwise comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05. All analyses were performed in Prism 9 (GraphPad).

Limitations

The reliability of this protocol is highly dependent on strict control of light conditions. Unintended light exposure during dark incubation may prematurely activate protochlorophyllide oxidoreductase (POR), leading to artificially elevated greening rates. In addition, results are influenced by seed after-ripening quality and storage history; seeds from plants grown under suboptimal conditions often show reduced germination synchrony, which can confound phenotypic scoring.

This protocol has been validated primarily in Arabidopsis thaliana. When applied to other plant species—particularly those with thicker cotyledons or inherently slower de-etiolation kinetics—the prescribed dark-incubation and greening periods may not adequately capture the full phenotypic readout. In such cases, re-optimization of dark/light duration, light intensity, and sampling time points may be required for cross-species application.

Troubleshooting

Problem 1

Weak or undetectable Pchlide fluorescence signal during quantification (Step 7).

Potential solution

Conduct all harvesting and extraction steps under dim green safelight in a darkroom. Keep extraction time to a minimum to avoid spontaneous degradation of Pchlide. During fluorometer measurement, cover the 96-well plate with aluminum foil before scanning to reduce photobleaching and solvent evaporation.

Problem 2

Excessively high greening rate in wild-type seedlings (Step 9).

Potential solution

Excessively high greening rate may be caused by light contamination during dark incubation. Ensure that no light leaks occur during the 5-day dark incubation. Use multiple layers of aluminum foil and check for pinholes. When transferring plates to light, unwrap the foil only after the plates are inside the growth chamber set at 100 μmol m⁻² s⁻¹. Exposure to intermediate light during transfer can alter de-etiolation process. Maintain a minimum inter-seed spacing of 5 mm to avoid mutual shading, which creates micro-environmental variability. Avoid placing seeds near the dish periphery which reflects light unevenly. Regularly calibrate the light meter to ensure accurate and uniform illumination intensity.

Problem 3

Abnormally low greening rate in wild-type seedlings (Step 9).

Potential solution

Use seeds harvested from healthy, unstressed plants and ensure they have been after-ripened adequately (≥2 months). Confirm that the light intensity during the 48-h greening period is maintained at 100 μmol m⁻² s⁻¹. Excessive light can induce photo-oxidative stress and reduce greening efficiency. Check that the growth-chamber temperature is stable at 22°C.

Problem 4

Poor seed germination rate (Step 9).

Potential solution

Ensure that the 8-h light pulse used to trigger germination is provided. Insufficient light exposure can lead to uneven or failed germination. Limit the surface-sterilization time to 10–15 min; prolonged exposure to sterilant can reduce seed viability. After sterilization, rinse seeds thoroughly with sterile water to remove any residual chemicals that may inhibit germination.

Problem 5

High fluorescente background in ROS detection (Step 13).

Potential solutions

High background fluorescence signal may be caused by incomplete dye removal during elution. Ensure thorough washing after H₂DCFDA incubation. Perform five complete washes with 10 mM Tris-HCl (pH 7.5), with gentle agitation for 1 min per wash. Prepare the H₂DCFDA staining solution immediately before use and protect it from light to minimize pre-loading oxidation.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shangwei Zhong (shangwei.zhong@pku.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Yixuan Fan (yixuan_fan@stu.pku.edu.cn).

Materials availability

This study did not generate new unique reagents. All the non-commercial materials described in this study are available upon request.

Data and code availability

This study did not generate or analyze datasets.

Acknowledgments

We thank Huan Li, Mohan Lyu, and Sheng Xu for technical assistance and experimental guidance. We are grateful to Hui Shi and Dingcheng Yi for their helpful comments during manuscript preparation. We acknowledge the National Center for Protein Sciences at Peking University (Beijing, China) for providing access to confocal microscopy facilities. Figures were created in part using BioRender.com. This work is supported by the National Natural Science Foundation of China (32325007 to S.Z.).

Author contributions

S.Z. designed the experiments; Y.F. performed the experiments, analyzed the data, and wrote the manuscript; and Z.S. reviewed and edited the manuscript. All the authors have read and approved the manuscript.

Declaration of interests

The authors declare no competing interests.