Fig 2: The second, chloroplast growth phase involves greening and is supported by protein accumulation profiles. a Chlorophyll content, quantified in each of three independent biological replicates per sample. Error bars represent standard error of the mean. See Additional file 5: Table S6 for calculations. b, c, d Expression (Z-scores) of pigment biosynthesis and thylakoid biogenesis (b), light reactions (c) and carbon fixation-associated genes (d). e Expression (Z-scores) of chloroplast development-associated transcripts reflecting two stages of plastid development, peaking in the early plastid phase (RCB, ARC5 and TIC40) and, second, chloroplast phase (PSBO2, LHCB1.4 and SBPAse). f Immunoblot analysis of the protein products of the genes displayed in e. In total, 20 µg of protein of samples 1–14 (for PSBO2, LHCB1.4 and SBPAse), 40 µg (for RCB, ARC5 and TIC40) or 10 µg (for Histone H3 as a constitutive control) was separated on denaturing SDS-PAGE gels, transferred to blots and probed with antibodies against the protein indicated. A Coomassie-stained total protein replica gel is also shown. Molecular weights (KDa) are indicated on the left. The results show one typical example from among three independent protein extraction and immunoblot experiments

Fig 3: The TIC-TOC translocon is not required for the internalization of NBR1 into photodamaged chloroplasts.(A) Confocal imaging of NBR1-GFP in wild-type Col-0 (WT) and toc132 cotyledon mesophyll cells from 1-week-old seedlings grown under LL or at 24 hr after HL exposure. Arrowheads indicate chloroplasts with internal NBR1-GFP signal. (B) Box and whisker plot displaying the percentages of chloroplasts associated with NBR1-GFP signal in WT and toc132 mutant mesophyll cells under LL or at 24 hr after HL exposure. Between 8 and 18 regions containing 20–30 chloroplasts from at least 3 cotyledons were used for quantification. (C) Protoplasts from 3-week-old wild-type Col-0 (WT) and tic40-4 expressing YFP-NBR1mPB1. Protoplasts were left in the dark or exposed to HL for 2 hr and imaged 12 hr later. Arrowheads indicate chloroplasts with internal YFP-mPB1-NBR1 signal. V, vacuole. (D) Box and whisker plot displaying the percentages of wild type and tic40-4 chloroplasts associated with YFP-mPB1-NBR1 signal in WT and tic40-4 mutant protoplasts kept in dark conditions or exposed to HL and left to recover for 12 hr. Between 9 and 10 protoplasts of each genotype and condition were used for quantification. In B and D, boxes show the variation in data through quartiles; the middle line indicates the median and whiskers show the upper and lower fences. Different letters denote significant differences from each other based on two-way ANOVA followed by Tukey’s test (p < 0.05). (E) Diagram summarizing a proposed mechanism for NBR1 association with photodamaged chloroplasts. HL exposure induces the breakdown of the chloroplast envelopes allowing the cytosolic ubiquitylation machinery to reach the stroma and thylakoids of photodamaged chloroplasts. As stromal and thylakoidal proteins become ubiquitylated, NBR1 diffuses into damaged chloroplasts and bind ubiquitylated proteins through its UBA2 domain. NBR1-decorated photodamaged chloroplasts are then delivered to the vacuole by microautophagy independently of ATG7. Scale bars: 5 µm in A and C.

Fig 4: Ultrastructure of chloroplasts in wild-type, atg7, and nbr1 cotyledons 24 hr after HL exposure.(A) Transmission electron micrograph of a high-pressure frozen/freeze-substituted atg7 cotyledon mesophyll cell from 8-day-old seedlings exposed to HL and left to recover for 24 hr. Three different types of chloroplasts based on their structural integrity are seen. Type-1 chloroplasts with electron dense stroma and tightly appressed thylakoids, Type-2 chloroplasts with lighter stroma and partially disorganized thylakoids, and Type-3 chloroplasts with ruptured envelopes, disorganized thylakoid membranes and a stroma region with similar electron density and appearance to the cytoplasm. (B, C) Representative Type-2 (B) and Type-3 (C) chloroplasts in an atg7 mesophyll cells. Note in (C) that the outer and inner envelopes (arrowheads) are disrupted in several sites (asterisks) exposing the interior of the chloroplast, including thylakoid membranes to the cytosol. (D) Box and whisker plots showing the percentage of Type-1,–2, and –3 chloroplasts per mesophyll cell section in wild-type Col-0 (WT), nbr1, and atg7 cotyledons. Different letters on the graph indicate significant difference (p<0.05) calculated by one-way ANOVA followed by Tukey’s test. Between 7 and 10 cells from two cotyledons of each genotype were used for this analysis. (E) Immunogold labeling with anti NBR1 antibodies on chloroplasts of WT, nbr1, and atg7 mutant mesophyll cells exposed to HL followed by 24 hr recovery. Red arrowheads indicate gold particles on chloroplasts. (F) Quantification of anti-NBR1 gold labeling on Type-1, –2, and –3 chloroplasts and cytoplasm from WT, atg7, and nbr1 mutant mesophyll cells exposed to HL. A t-test was used to compare values between mutant and WT samples; * and ** indicate p<0.05 and p<0.01, respectively. Between 5 and 11 chloroplasts or cytoplasmic regions from 2 cotyledons of each genotype were used for quantification. (G) Immunoblot detection of NBR1, TIC40 (chloroplast inner envelope protein), LHCIIb (thylakoid protein), and cFBP (cytosolic protein) in total protein extracts (T) and chloroplast protein fraction (C) from 4-week-old WT and atg7 plants grown under LL or exposed to HL and let recover for 24 hr. The numbers below indicate the ratios cFBP/TIC40 and NBR1/cFBPase based on the quantification of the western blots. The figure shows a representative set of western blots. The experiment was repeated twice. Box and whisker plots in D and F show the variation in data through quartiles; the middle line indicates the median and whiskers show the upper and lower fences. S, starch; St, stroma; Thy, thylakoids. Scale bars: 1 µm in A, B, C; 500 nm in E. Figure 2—source data 1.G.Original files of full raw unedited blots and figure with uncropped blots.