Predicting the Impact of Alternative Splicing on Plant MADS Domain Protein Function
Severing, E.I. ; Dijk, A.D.J. van; Morabito, G. ; Busscher-Lange, J. ; Immink, G.H. ; Ham, R.C.H.J. van - \ 2012
PLoS ONE 7 (2012)1. - ISSN 1932-6203 - 13 p.
flowering time - box genes - transcription factors - arabidopsis-thaliana - messenger-rna - evolutionary analysis - exon duplication - organ identity - sequence - identification
Several genome-wide studies demonstrated that alternative splicing (AS) significantly increases the transcriptome complexity in plants. However, the impact of AS on the functional diversity of proteins is difficult to assess using genome-wide approaches. The availability of detailed sequence annotations for specific genes and gene families allows for a more detailed assessment of the potential effect of AS on their function. One example is the plant MADS-domain transcription factor family, members of which interact to form protein complexes that function in transcription regulation. Here, we perform an in silico analysis of the potential impact of AS on the protein-protein interaction capabilities of MIKC-type MADS-domain proteins. We first confirmed the expression of transcript isoforms resulting from predicted AS events. Expressed transcript isoforms were considered functional if they were likely to be translated and if their corresponding AS events either had an effect on predicted dimerisation motifs or occurred in regions known to be involved in multimeric complex formation, or otherwise, if their effect was conserved in different species. Nine out of twelve MIKC MADS-box genes predicted to produce multiple protein isoforms harbored putative functional AS events according to those criteria. AS events with conserved effects were only found at the borders of or within the K-box domain. We illustrate how AS can contribute to the evolution of interaction networks through an example of selective inclusion of a recently evolved interaction motif in the MADS AFFECTING FLOWERING1-3 (MAF1–3) subclade. Furthermore, we demonstrate the potential effect of an AS event in SHORT VEGETATIVE PHASE (SVP), resulting in the deletion of a short sequence stretch including a predicted interaction motif, by overexpression of the fully spliced and the alternatively spliced SVP transcripts. For most of the AS events we were able to formulate hypotheses about the potential impact on the interaction capabilities of the encoded MIKC proteins
Transcriptome and Metabolite Profiling Show That APETALA2a Is a Major Regulator of Tomato Fruit Ripening
Karlova, R.B. ; Rosin, F.M.A. ; Busscher-Lange, J. ; Parapunova, V.A. ; Do, P.T. ; Fernie, A.R. ; Fraser, P.D. ; Baxter, C. ; Angenent, G.C. ; Maagd, R.A. de - \ 2011
The Plant Cell 23 (2011)3. - ISSN 1040-4651 - p. 923 - 941.
homeotic gene apetala2 - ethylene biosynthesis - flower development - 1-aminocyclopropane-1-carboxylate synthase - chromoplast differentiation - lycopersicon-esculentum - expression analysis - arabidopsis flower - seed development - organ identity
Fruit ripening in tomato (Solanum lycopersicum) requires the coordination of both developmental cues as well as the plant hormone ethylene. Although the role of ethylene in mediating climacteric ripening has been established, knowledge regarding the developmental regulators that modulate the involvement of ethylene in tomato fruit ripening is still lacking. Here, we show that the tomato APETALA2a (AP2a) transcription factor regulates fruit ripening via regulation of ethylene biosynthesis and signaling. RNA interference (RNAi)-mediated repression of AP2a resulted in alterations in fruit shape, orange ripe fruits, and altered carotenoid accumulation. Microarray expression analyses of the ripe AP2 RNAi fruits showed altered expression of genes involved in various metabolic pathways, such as the phenylpropanoid and carotenoid pathways, as well as in hormone synthesis and perception. Genes involved in chromoplast differentiation and other ripening-associated processes were also differentially expressed, but softening and ethylene biosynthesis occurred in the transgenic plants. Ripening regulators RIPENING-INHIBITOR, NON-RIPENING, and COLORLESS NON-RIPENING (CNR) function upstream of AP2a and positively regulate its expression. In the pericarp of AP2 RNAi fruits, mRNA levels of CNR were elevated, indicating that AP2a and CNR are part of a negative feedback loop in the regulation of ripening. Moreover, we demonstrated that CNR binds to the promoter of AP2a in vitro
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
In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana
Urbanus, S.L. ; Folter, S. de; Shchennikova, A. ; Kaufmann, K. ; Immink, G.H. ; Angenent, G.C. - \ 2009
BMC Plant Biology 9 (2009). - ISSN 1471-2229
green-fluorescent protein - box transcription factors - homeotic gene apetala1 - flower development - ovule development - meristem identity - cell-differentiation - negative regulation - fruit-development - organ identity
Background: MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results: We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion: This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses.
Tagging of MADS domain proteins for chromatin immunoprecipitation
Folter, S. de; Urbanus, S.L. ; Zuijlen, L. ; Kaufmann, K. ; Angenent, G.C. - \ 2007
BMC Plant Biology (2007). - ISSN 1471-2229 - p. 7 - 47.
floral homeotic gene - arabidopsis fruit-development - box gene - recombinant proteins - regulatory elements - negative regulation - ectopic expression - flower development - organ identity - purification
Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow in vivo identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.
Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis
Gomez-Mena, C. ; Folter, S. de; Costa, M.M.R. ; Angenent, G.C. ; Sablowski, R. - \ 2005
Development 132 (2005)3. - ISSN 0950-1991 - p. 429 - 438.
mads-box genes - arabidopsis-thaliana - flower development - organ identity - meristem identity - gibberellin 3-beta-hydroxylase - molecular characterization - expression analysis - stem elongation - target genes
Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. The AG target genes included most genes for which mutant screens revealed a function downstream of AG. Novel targets were validated by in situ hybridisation and binding to AG in vitro and in vivo. Transcription factors formed a large fraction of AG targets, suggesting that during early organogenesis, much of the genetic program is concerned with elaborating gene expression patterns. The results also suggest that AG and other homeotic proteins with which it interacts (SEPALLATA3, APETALA3, PISTILLATA) are coordinately regulated in a positive-feedback loop to maintain their own expression, and that AG activates biosynthesis of gibberellin, which has been proposed to promote the shift from meristem identity to differentiation.
Ectopic expression of LLAG1, an AGAMOUS homologue from lily (Lilium longiflorum Thunb.) causes floral homeotic modifications in Arabidopsis.
Benedito, V.A. ; Visser, P.B. ; Tuyl, J.M. van; Angenent, G.C. ; Vries, S.C. de; Krens, F.A. - \ 2004
Journal of Experimental Botany 55 (2004)401. - ISSN 0022-0957 - p. 1391 - 1399.
mads-box genes - controlling flower development - protein-protein interactions - transcription factors - organ identity - gerbera-hybrida - plant biology - wild-type - petunia - transformation
The ABC model for floral development was proposed more than 10 years ago and since then many studies have been performed on model species, such as Arabidopsis thaliana, Antirrhinum majus, and many other species in order to confirm this hypothesis. This led to additional information on flower development and to more complex molecular models. AGAMOUS (AG) is the only C type gene in Arabidopsis and it is responsible for stamen and carpel development as well as floral determinacy. LLAG1, an AG homologue from lily (Lilium longiflorum Thunb.) was isolated by screening a cDNA library derived from developing floral buds. The deduced amino acid sequence revealed the MIKC structure and a high homology in the MADS-box among AG and other orthologues. Phylogenetic analysis indicated a close relationship between LLAG1 and AG orthologues from monocot species. Spatial expression data showed LLAG1 transcripts exclusively in stamens and carpels, constituting the C domain of the ABC model. Functional analysis was carried out in Arabidopsis by overexpression of LLAG1 driven by the CaMV35S promoter. Transformed plants showed homeotic changes in the two outer floral whorls with some plants presenting the second whorl completely converted into stamens. Altogether, these data strongly indicated the functional homology between LLAG1 and AG.