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The chloroplast-associated protein degradation pathway controls chromoplast development and fruit ripening in tomato

Abstract

The maturation of green fleshy fruit to become colourful and flavoursome is an important strategy for plant reproduction and dispersal. In tomato (Solanum lycopersicum) and many other species, fruit ripening is intimately linked to the biogenesis of chromoplasts, the plastids that are abundant in ripe fruit and specialized for the accumulation of carotenoid pigments. Chromoplasts develop from pre-existing chloroplasts in the fruit, but the mechanisms underlying this transition are poorly understood. Here, we reveal a role for the chloroplast-associated protein degradation (CHLORAD) proteolytic pathway in chromoplast differentiation. Knockdown of the plastid ubiquitin E3 ligase SP1, or its homologue SPL2, delays tomato fruit ripening, whereas overexpression of SP1 accelerates ripening, as judged by colour changes. We demonstrate that SP1 triggers broader effects on fruit ripening, including fruit softening, and gene expression and metabolism changes, by promoting the chloroplast-to-chromoplast transition. Moreover, we show that tomato SP1 and SPL2 regulate leaf senescence, revealing conserved functions of CHLORAD in plants. We conclude that SP1 homologues control plastid transitions during fruit ripening and leaf senescence by enabling reconfiguration of the plastid protein import machinery to effect proteome reorganization. The work highlights the critical role of chromoplasts in fruit ripening, and provides a theoretical basis for engineering crop improvements.

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Fig. 1: Sequence, localization and gene expression analysis of slSP1 and slSPL2.
Fig. 2: Analyses of the roles of slSP1 and slSPL2 in tomato leaf senescence.
Fig. 3: Examination of the effects of slSP1 and slSPL2 on tomato fruit ripening.
Fig. 4: Ultrastructural analysis of the effects of slSP1 on the chloroplast-to-chromoplast transition in ripening tomato fruit.
Fig. 5: Metabolic profile analyses of the effects of slSP1 on tomato fruit ripening.
Fig. 6: Analysis of the role of slSP1 in regulating the plastid proteome during leaf senescence and fruit ripening.

Data availability

All data generated or analysed during this study are included in this published article or its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank E. Johnson and R. Dhaliwal for transmission electron microscopy conducted in the Sir William Dunn School of Pathology EM Facility, D. Hauton and J. McCullagh for IC–MS conducted in the Mass Spectrometry Research Facility in the Department of Chemistry, P. Bota for GC–MS conducted in the Department of Plant Sciences, P. Bota and R. Ross for technical assistance, P. Pulido for assistance with the pigment analysis, and M. R. Rodriguez Goberna for HPLC conducted at CRAG, Spain. This work was supported by a Khazanah-Oxford Centre for Islamic Studies Merdeka Scholarship to N.M.S., by Strategic Priority Research Program (Type-B; project number: XDB27020107), Chinese Academy of Sciences to Q.L., by the Spanish Agencia Estatal de Investigación (grants BIO2017-84041-P and BIO2017-90877-REDT) to M.R.-C., and by the Biotechnology and Biological Sciences Research Council (BBSRC; grants BB/H008039/1, BB/K018442/1, BB/N006372/1, BB/R005591/1, BB/R009333/1 and BB/R016984/1) to R.P.J.

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Q.L. and N.M.S. designed and conducted the experiments, analysed the data and wrote the manuscript. Z.S., Y. Zhou, Y. Zeng and B.H. assisted with the fruit phenotypical analyses, qRT–PCR, immunoblotting, preparation of samples for the TEM and metabolomic experiments and data analysis. M.R.-C. performed the HPLC analysis of pigments and analysed the results. R.P.J. conceived the study, supervised the work, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to R. Paul Jarvis.

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The application of CHLORAD as a technology for crop improvement is covered by a patent application (no. WO2019/171091 A).

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Extended data

Extended Data Fig. 1 Expression profiles of the slSP1 and slSPL2 genes.

The expression profiles shown are based on Affymetrix GeneChip data and were generated using the Development (a) and Anatomy (b) functions of Genevestigator80. Data from ATH arrays are shown in scatter-plot diagrams. In a, the x-axis represents the following developmental stages, from left to right: young seedling, developed seedling, flower, green fruit, ripening fruit, and mature fruit. Values are means ± s.e.m., and for each data point the number of samples is indicated. Medium expression levels are defined as the interquartile range (IQR; light grey boxes); values below the IQR are defined as low expression (white boxes), and values above the IQR are defined as high expression (HIGH; dark grey boxes). The presented data provide a complement to the data in Fig. 1d, and confirm that slSPL2 is generally more weakly expressed than slSP1.

Extended Data Fig. 2 Assessment of the extent of knockdown or overexpression of the slSP1 and slSPL2 genes in the transgenic tomato plants.

Total leaf RNA was extracted from two-week-old tomato plants of the indicated genotypes; three independent T1 generation transformants (#1-3) were analysed for each construct. Quantitative RT–PCR analysis of slSP1 and slSPL2 expression was performed on the corresponding transgenic lines, in comparison with wild-type controls, as indicated. Relative gene expression levels were calculated by normalization using the reference gene, slACTIN. All values are expressed relative to the corresponding value for wild type, which in each case is set to 1. Values are means ± s.e.m. (n = 3 [WT-1, slSPL2-KD #2 and #3], 4 [slSP1-KD #3], or 5 [all other genotypes] technical replicates).

Extended Data Fig. 3 Determination of tomato fruit sizes.

Measurement of the maximal equatorial diameter of breaker-stage tomato fruit, from T2 generation transgenic plants, was performed using a calliper. The fruits were then detached from the plants and incubated at 25 °C in the dark for the ripening analysis presented in Fig. 3. Values are means ± s.e.m. (n = 26-27 fruits per genotype). The data demonstrate that fruit size in the slSP1-KD, slSP1-OX and slSPL2-KD transgenic lines at breaker stage was not significantly different from that in the wild type, as revealed by an unpaired two-tailed Student’s t-test (P = 0.4125 [slSP1-KD], 0.7132 [slSP1-OX], and 0.8001 [slSPL2-KD]). This rules out the possibility of nonspecific effects due to fruit size differences, which is important because ripening in detached tomato fruit is dependent on proper maturation up to the mature green stage17.

Extended Data Fig. 4 Determination of firmness of detached tomato fruits.

Fruit firmness was measured using a durometer at the breaker (Day 1) and red stages (a) (n = 20-28 [breaker stage] or 10-13 [red stage] fruits per genotype); or at specific days post breaker stage (b) (n = 20-28 [Day 1, Day 5, Day 9], 10-13 [Day 12], or 10-12 [Day 14] fruits per genotype). Note that slSP1-OX fruit at Day 14 were too soft to give a reading using the durometer. All values are means ± s.e.m. The fruit used in this analysis were randomly chosen from the fruit populations of T2 generation plants used in the ripening analysis in Fig. 3.

Extended Data Fig. 5 Analyses of the effects of slSP1 on ripening-related gene expression.

Total fruit RNA was extracted from wild-type (WT), slSP1-KD (KD), and slSP1-OX (OX) tomato plants at the Day 8 post-breaker stage (Day 8) stage and the red stage (the same fruit as those used in Fig. 6). Relative mRNA expression levels were analysed by qRT–PCR using primers specific for genes encoding ethylene synthesis (a), cell wall modification (b), carotenoid biosynthesis (c), and master, ripening-related transcription factors (d). It was reported previously that all of these ripening-related genes are upregulated during fruit ripening; typically, in wild-type fruit, their transcript levels will reach a peak at the pink stage, and then reduce at the red stage35,36,37. Correspondingly, in our analysis, wild-type fruit at the Day 8 post-breaker stage (pink-looking) show higher expression levels than fruit at the red stage. Although the slSP1-KD and slSP1-OX fruits both showed similar lower mRNA levels of ripening-related genes at the red stage, at Day 8 they showed striking differences in mRNA levels. In general, slSP1-KD fruit (green-looking) had markedly reduced mRNA levels, while slSP1-OX fruit (red-looking) had gene expression levels in between those of wild-type and slSP1-KD fruits. Overall, these results indicated that slSP1-KD fruit show delayed transcriptional changes of ripening-related genes relative to wild-type fruit, whereas slSP1-OX fruit displayed accelerated transcriptional changes relative to wild-type fruit. Expression data for the genes of interest were normalized using data for the reference gene, slACTIN. All values are expressed relative to the corresponding value for wild type, which in each case is set to 1. Values are means ± s.e.m. of three replicates. ACO1, 1-Aminocyclopropane-1-carboxylate oxidase 1; ACS2/4, 1-Aminocyclopropane carboxylic acid synthase 2/4; NR, Never ripe; PME, Pectin methylesterase; PG2a, Polygalacturonase 2a; PDS, Phytoene desaturase; PSY1, Phytoene synthase 1; RIN, Ripening inhibitor; TDR4, Agamous-like MADS-box protein AGL8 homolog.

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Ling, Q., Sadali, N.M., Soufi, Z. et al. The chloroplast-associated protein degradation pathway controls chromoplast development and fruit ripening in tomato. Nat. Plants 7, 655–666 (2021). https://doi.org/10.1038/s41477-021-00916-y

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