The rin, nor and Cnr spontaneous mutations inhibit tomato fruit ripening in additive and epistatic manners
Wang, Rufang ; Lammers, Michiel ; Tikunov, Yury ; Bovy, Arnaud G. ; Angenent, Gerco C. ; Maagd, Ruud A. de - \ 2020
Plant Science 294 (2020). - ISSN 0168-9452
Colorless non-ripening - Fruit ripening - non-ripening - ripening inhibitor - Spontaneous mutation - Tomato
Tomato fruit ripening is regulated by transcription factors (TFs), their downstream effector genes, and the ethylene biosynthesis and signalling pathway. Spontaneous non-ripening mutants ripening inhibitor (rin), non-ripening (nor) and Colorless non-ripening (Cnr) correspond with mutations in or near the TF-encoding genes MADS-RIN, NAC-NOR and SPL-CNR, respectively. Here, we produced heterozygous single and double mutants of rin, nor and Cnr and evaluated their functions and genetic interactions in the same genetic background. We showed how these mutations interact at the level of phenotype, individual effector gene expression, and sensory and quality aspects, in a dose-dependent manner. Rin and nor have broadly similar quantitative effects on all aspects, demonstrating their additivity in fruit ripening regulation. We also found that the Cnr allele is epistatic to rin and nor and that its pleiotropic effects on fruit size and volatile production, in contrast to the well-known dominant effect on ripening, are incompletely dominant, or recessive.
Genetic and epigenetic regulation of tomato fruit ripening
Wang, Rufang - \ 2020
Wageningen University. Promotor(en): G.C. Angenent, co-promotor(en): R.A. de Maagd. - Wageningen : Wageningen University - ISBN 9789463952354 - 175
Tomato is one of the top consumed vegetables and an excellent model for studies on fleshy fruit ripening. In tomato, ripening is a complex process regulated by genetic and epigenetic mechanisms together with plant hormones. So far mostly one-on-one regulatory relationships between regulators and effectors were explored, but the true function of some major regulators is still not clear, as are the interactions of their spontaneous mutant alleles in ripening regulation.
This thesis studies tomato fruit ripening regulation at the molecular level by focusing on the evaluation of the true function of major transcription factors (TFs), on the interaction of their spontaneous mutant alleles and on the effects of non-coding elements. To achieve that, CRISPR/Cas9-mutagenesis as well as crosses were used to obtain real knock-out mutants and genotypes with different and combined doses of spontaneous mutant alleles, followed by phenotyping and gene expression analysis to evaluate their functions in ripening. Moreover, some suggestions for future CRISPR mutagenesis experiments are proposed based on our observations.
In conclusion, this thesis illustrates the function of some major TFs and interactions of their spontaneous mutant alleles in tomato ripening regulation, which enhances our knowledge, and points the way to future work on ripening regulation.
Revisiting the Role of Master Regulators in Tomato Ripening
Wang, Rufang ; Angenent, Gerco C. ; Seymour, Graham ; Maagd, Ruud A. de - \ 2020
Trends in Plant Science 25 (2020)3. - ISSN 1360-1385 - p. 291 - 301.
CRISPR- mutagenesis - gain-of-function - mutants - ripening - tomato - transcription factors
The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are ‘master regulators’. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.
Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis
Wang, Rufang ; Rocha Tavano, Eveline Carla da; Lammers, Michiel ; Martinelli, Adriana Pinheiro ; Angenent, Gerco C. ; Maagd, Ruud A. de - \ 2019
Scientific Reports 9 (2019)1. - ISSN 2045-2322
Tomato (Solanum lycopersicum) is a model for climacteric fleshy fruit ripening studies. Tomato ripening is regulated by multiple transcription factors together with the plant hormone ethylene and their downstream effector genes. Transcription Factors APETALA2a (AP2a), NON-RIPENING (NOR) and FRUITFULL (FUL1/TDR4 and FUL2/MBP7) were reported as master regulators controlling tomato fruit ripening. Their proposed functions were derived from studies of the phenotype of spontaneous mutants or RNAi knock-down lines rather than, as it appears now, actual null mutants. To study TF function in tomato fruit ripening in more detail, we used CRISPR/Cas9-mediated mutagenesis to knock out the encoding genes, and phenotypes of these mutants are reported for the first time. While the earlier ripening, orange-ripe phenotype of ap2a mutants was confirmed, the nor null mutant exhibited a much milder phenotype than the spontaneous nor mutant. Additional analyses revealed that the severe phenotype in the spontaneous mutant is caused by a dominant-negative allele. Our approach also provides new insight into the independent and overlapping functions of FUL1 and FUL2. Single and combined null alleles of FUL1 and FUL2 illustrate that these two genes have partially redundant functions in fruit ripening, but also unveil an additional role for FUL2 in early fruit development.