Ethylene response factor ERF.D7 activates auxin response factor 2 paralogs to regulate tomato fruit ripening

Abstract

Despite the obligatory role of ethylene in climacteric fruit ripening and the identification of 77 ethylene response factors (ERFs) in the tomato (Solanum lycopersicum) genome, the role of few ERFs has been validated in the ripening process. Here, using a comprehensive morpho-physiological, molecular and biochemical approach, we demonstrate the regulatory role of Ethylene Response Factor D7 (SlERF.D7) in tomato fruit ripening. SlERF.D7 expression positively responded to exogenous ethylene and auxin treatments, most likely in a Ripening Inhibitor (RIN)-independent manner. SlERF.D7 overexpression promoted ripening, and its silencing had the opposite effect. Alterations in its expression modulated ethylene production, pigment accumulation, and fruit firmness. Consistently, genes involved in ethylene biosynthesis and signalling, lycopene biosynthesis, and cell wall loosening were upregulated in the overexpression lines and downregulated in RNAi lines. These transgenic lines also accumulated altered levels of IAA at late-breaker stages. A positive association between Auxin Response Factor 2 (ARF2) paralog’s transcripts and SlERF.D7 mRNA levels and that SlARF2A and SlARF2B are direct targets of SlERF.D7 underpinned the perturbed auxin-ethylene crosstalk for the altered ripening program observed in the transgenic fruits. Overall, this study uncovers that SlERF.D7 positively regulates SlARF2A/B abundance to amalgamate auxin and ethylene signalling pathways for controlling tomato fruit ripening.

Methylglyoxal controls tomato fruit ripening by regulating ethylene biosynthesis

“…Exogenous application of ethylene and 1-MCP was done as previously described (Gambhir et al ., 2022). Briefly, for ethylene and ACC treatment, wild type tomato fruits (Pusa Ruby) were harvested at the mature green stage and injected with a buffer solution containing 10 mM MES, pH 5.6, sorbitol (3% w/v), and 100 μM of Ethrel (2-Chloroethylphosphonic Acid, 40% Solution, Sisco Research Laboratories Pvt.…”

“…The assessment of fruit firmness was carried out using TA.XT Plus Texture Analyser (Stable Micro Systems, England) as previously described (Gambhir et al ., 2022). Briefly, fruits of wild type and SlGLYI4-silenced genotypes during different stages of ripening stage were subjected to a puncture test with a 2 mm needle probe, and the force and distance measurements were recorded.…”

“…Ethylene measurement was done as previously described (Gambhir et al ., 2022). Briefly, fruits at different ripening stages were harvested and stored in open 250-ml jars for 2 h to minimize the effect of wound-induced ethylene caused by the harvesting of the fruits.…”

“…For the effector vector, the full open reading frame (ORF) of RIN was cloned in the binary vector pBI121 driven by the CaMV35S promoter. Both reporter and effector constructs were co-transformed into 4-week-old N. benthamiana leaves, as described previously (Gambhir et al ., 2022). GFP fluorescence measurement assay using was done after 48 h of infiltrations.…”

“…Ethylene in the headspace gas was measured thrice for each sample, with at least five biological replicates for each ripening stage. Lycopene and β-carotene extractions for HPLC experiments were performed as described previously (Gambhir et al, 2022). Briefly, 150 mg of ground lyophilized tomato fruit powder were extracted with chloroform and methanol (2:1 v/v); Then, 1 volume of 50 mM Tris buffer (pH 7.5, containing 1M NaCl) was added followed by the incubation of samples on ice for 20 min.…”