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.
Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis
Es, S.W. van; Silveira, S.R. ; Rocha, D.I. ; Bimbo, A. ; Martinelli, A.P. ; Dornelas, M.C. ; Angenent, G.C. ; Immink, G.H. - \ 2018
The Plant Journal 94 (2018)5. - ISSN 0960-7412 - p. 867 - 879.
The flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5-like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have
wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5-like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1-amino-cyclopropane-1-carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation–quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5-like TFs in the regulation of ethylenemediated petal development and growth.
Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition
Urbanus, S.L. ; Martinelli, A.P. ; Dinh, Q.D. ; Aizza, L.C.B. ; Dornelas, M. ; Angenent, G.C. ; Immink, G.H. - \ 2010
The Plant Journal 63 (2010)3. - ISSN 0960-7412 - p. 60 - 72.
homeotic gene apetala3 - stem-cell maintenance - organ identity - arabidopsis-thaliana - flower development - homeobox genes - ectopic expression - meristem identity - ovule development - pound-foolish
During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins