Abstract
The mechanisms associated with the regulation of iron (Fe) homeostasis have been extensively examined, however, epigenetic regulation of these processes remains largely unknown. Here, we report that a naturally occurring epigenetic mutant, Colorless non-ripening (Cnr), displayed increased Fe-deficiency responses compared to its wild-type Ailsa Craig (AC). RNA-sequencing revealed that a total of 947 and 1,432 genes were up-regulated by Fe deficiency in AC and Cnr roots, respectively, while 923 and 1,432 genes were, respectively, down-regulated. Gene ontology analysis of differentially expressed genes showed that genes encoding enzymes, transporters, and transcription factors were preferentially affected by Fe deficiency. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed differential metabolic responses to Fe deficiency between AC and Cnr. Based on comparative transcriptomic analyses, 24 genes were identified as potential targets of Cnr epimutation, and many of them were found to be implicated in Fe homeostasis. By developing CRISPR/Cas9 genome editing SlSPL-CNR knockout (KO) lines, we found that some Cnr-mediated Fe-deficiency responsive genes showed similar expression patterns between SlSPL-CNR KO plants and the Cnr epimutant. Moreover, both two KO lines displayed Fe-deficiency-induced chlorosis more severe than AC plants. Additionally, the Cnr mutant displayed hypermethylation in the 286-bp epi-mutated region on the SlSPL-CNR promoter, which contributes to repressed expression of SlSPL-CNR when compared with AC plants. However, Fe-deficiency induced no change in DNA methylation both at the 286-bp epi-allele region and the entire region of SlSPL-CNR gene. Taken together, using RNA-sequencing and genetic approaches, we identified Fe-deficiency responsive genes in tomato roots, and demonstrated that SlSPL-CNR is a novel regulator of Fe-deficiency responses in tomato, thereby, paving the way for further functional characterization and regulatory network dissection.
Highlights
Iron (Fe) is an essential microelement for higher plants, involved in many physiological and metabolic processes such as photosynthesis, substance metabolism, and respiration
Since the induction of root ferric chelate reductase (FCR) activity has been proved to be a rate-limiting step for Fe uptake in Strategy I plants (Connolly et al, 2003), root FCR activity was examined in both Ailsa Craig (AC) and Colorless non-ripening (Cnr) plants under Fe deficiency
A significant enhancement of FCR activity was found both in AC and Cnr roots under Fe deficiency, and this Fe-deficiency-induced FCR activity was much higher in Cnr than that in AC (Figures 1C,D), indicating that Cnr is more sensitive to Fe deficiency than AC
Summary
Iron (Fe) is an essential microelement for higher plants, involved in many physiological and metabolic processes such as photosynthesis, substance metabolism, and respiration. When suffering from Fe deficiency, two strategies have been developed for higher plants to acquire Fe from rhizosphere. In Strategy I plants, such as Arabidopsis and tomato, Fe3+ is first reduced by a plasma membrane-localized Ferric Reduction Oxidase 2 (FRO2) (Robinson et al, 1999), and transported across the membrane into cells by Ironregulated Transporter 1 (IRT1) (Vert et al, 2002). Strategy II plants such as maize, rice, and barley secrete phytosiderophores to chelate Fe3+, which is transported across the membrane by oligopeptide transporter Yellow-stripe 1 (YS1) in maize and its functional homolog OsYSL15 in rice (Curie et al, 2001; Inoue et al, 2009). Fe3+ solubilization is mediated by both H+-ATPase-dependent rhizosphere acidification and secretion of Fe3+-mobilizing coumarins (Santi and Schmidt, 2009; Tsai and Schmidt, 2017)
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