Targeted gene disruption coupled with metabolic screen approach to uncover the LEAFY COTYLEDON1-LIKE4 (L1L4) function in tomato fruit metabolism

Abstract

Functional analysis of tomato L1L4 master transcription factor resulted in important metabolic changes affecting tomato fruit quality. Tomato fruits from mutant lines bearing targeted disruption of the heterotrimeric nuclear transcription factor Y (NF-Y) transcription factor (TF) gene LEAFY-COTYLEDON1-LIKE4 (L1L4, NF-YB6), a master regulator of biosynthesis for seed storage proteins and fatty acids, were evaluated for metabolites content and morphology. Metabolic screens using LC-MS/MS-based analysis and physico-chemical methods in different L1L4 mutants of the fourth generation allowed a comparative assessment of the effects of the TF disruption. Mutagenesis resulted in fruits phenotypically similar to wild-type with subtle shape differences in the distal end protrusion and symmetry. Conversely, mutant fruits from independent lines had significant variation in moisture content, titratable acidity and overall metabolite profiles including oxalic and citric acid, fructose, β-carotene, total polyphenols and antioxidants. Lines 6, 7 and 9 were the richest in β-carotene and antioxidant activity, line 4 in ascorbic acid and lines 4 and 8 in succinic acid. The reduced content of the anti-nutrient oxalic acid in several mutant fruits suggests that L1L4 gene may regulate the accumulation of this compound during fruit development. Detailed LC-MS/MS analysis of mutant seeds showed substantial differences in bioactive compounds compared to wild-type seeds. Taken together, the results suggest that the L1L4 TF is a significant regulator of metabolites both in tomato fruit and seeds providing a molecular target for crop improvement. Elucidation of the candidate genes encoding key enzymes in the affected metabolic pathways aimed to facilitate the L1L4 gene network exploration and eventually lead to systems biology approaches in tomato fruit quality.

Qualitative evaluation of fruits from different Opuntia ficus‐indica ecotypes/cultivars harvested in South Portugal

“…Half of the peel and pulp (including seeds) of each of the 10 fruits from each replicate was pulped with an UltraTurrax T 18 (IKA, Germany) for 2 min, then centrifuge for 5 min at 5,000 rpm (Gago et al, ). The resulting juice was used for the assessment of pH, titratable acidity (TA) and total soluble solids content (TSS).…”

The Interplay among Polyamines and Nitrogen in Plant Stress Responses

“…So far, a large number of functional and regulatory genes implicated in abiotic and biotic stress responses have been identified in a diversity of plants. In particular, we characterized some functional or regulatory genes controlling metabolism of N, PAs, ethylene, and abscisic acid that are involved in various biological processes [22,33,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72].…”

NF‐Y plays essential roles in flavonoid biosynthesis by modulating histone modifications in tomato

“…Indeed, silencing particular NF‐YB genes led to pink‐coloured fruits with colourless peels (Figs 2a, 3a). Previous studies have reported that L1L4 (NF‐YB6) affects tomato fruit quality (Gago et al ., 2017), high‐level expression of a tomato HAP5a gene can reduce the flowering time of Arabidopsis (Ben‐Naim et al ., 2006) and SlNF‐YA‐L1 is implicated in the regulation of salt and osmotic stress tolerance (Filichkin et al ., 2018). Combined with our observation on colour change in transgenic fruits (Figs 2a, 3a, S3a), these results indicated that NF‐Y complexes composed of different subunits may regulate different biological processes.…”