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Abstract Ripening is crucial for the development of fleshy fruits that release their seeds following consumption by frugivores and are important contributors to human health and nutritional security. Many genetic ripening regulators have been identified, especially in the model system tomato, yet more remain to be discovered and integrated into comprehensive regulatory models. Most tomato ripening genes have been studied in pericarp tissue, though recent evidence indicates that locule tissue is a site of early ripening-gene activities. Here we identified and functionally characterized an Ethylene Response Factor gene,SlERF.D6, by investigating tomato transcriptome data throughout plant development, emphasizing genes elevated in the locule during fruit development and ripening.SlERF.D6loss-of-function mutants resulting from CRISPR/Cas9 gene editing delayed ripening initiation and carotenoid accumulation in both pericarp and locule tissues. Transcriptome analysis of lines altered inSlERF.D6expression revealed multiple classes of altered genes including ripening regulators, in addition to carotenoid, cell wall and ethylene pathway genes, suggesting comprehensive ripening control. Distinct regulatory patterns in pericarp versus locule tissues were observed indicating tissue-specific activity of this transcription factor. Analysis of SlERF.D6 interaction with target promoters revealed an AP2/ERF transcription factor(SlDEAR2) as a target of SlERF.D6. Furthermore, we show that a third transcription factor gene,SlTCP12, is a target of SlDEAR2, presenting a tri-component module of ripening control. 

Abstract Chromoplasts are plant organelles with a unique ability to sequester and store massive carotenoids. Chromoplasts have been hypothesized to enable high levels of carotenoid accumulation due to enhanced sequestration ability or sequestration substructure formation. However, the regulators that control the substructure component accumulation and substructure formation in chromoplasts remain unknown. In melon (Cucumis melo) fruit, β-carotene accumulation in chromoplasts is governed by ORANGE (OR), a key regulator for carotenoid accumulation in chromoplasts. By using comparative proteomic analysis of a high β-carotene melon variety and its isogenic line low-β mutant that is defective in CmOr with impaired chromoplast formation, we identified carotenoid sequestration protein FIBRILLIN1 (CmFBN1) as differentially expressed. CmFBN1 expresses highly in melon fruit tissue. Overexpression of CmFBN1 in transgenic Arabidopsis (Arabidopsis thaliana) containing ORHis that genetically mimics CmOr significantly enhances carotenoid accumulation, demonstrating its involvement in CmOR-induced carotenoid accumulation. Both in vitro and in vivo evidence showed that CmOR physically interacts with CmFBN1. Such an interaction occurs in plastoglobules and results in promoting CmFBN1 accumulation. CmOR greatly stabilizes CmFBN1, which stimulates plastoglobule proliferation and subsequently carotenoid accumulation in chromoplasts. Our findings show that CmOR directly regulates CmFBN1 protein levels and suggest a fundamental role of CmFBN1 in facilitating plastoglobule proliferation for carotenoid sequestration. This study also reveals an important genetic tool to further enhance OR-induced carotenoid accumulation in chromoplasts in crops. 

High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple 13 C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soil Pseudomonas species reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated ( Pseudomonas putida KT2440, Pseudomonas protegens Pf-5, and Pseudomonas putida S12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis–anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency. 

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening–specific LATERAL ORGAN BOUNDRIES ( LOB ) TF, SlLOB1 , up-regulates a suite of cell wall–associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 ( EXP1 ) are strongly suppressed in Sl LOB1 RNA interference lines, while EXP1 is induced in Sl LOB1 -overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, Sl LOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.