Project description:In recent years our group has been constructing an activation tag library based on the En-I transposon system which resulted in the identification of novel Arabidopsis mutants (Marsch Martinez et al 2002; manuscript accepted for publication in Plant Phys). Among them the bountiful (bou) mutant showed dominant alterations in leaf size and morphology, delayed flowering, vertically oriented siliques and higher yield. Sequence analysis of the genomic region flanking the transposon insertion in combination with expression analysis indicated that the mutant phenotype observed was presumably caused by over-expression of a gene encoding a yet uncharacterised DNA binding protein. In particular over-expression in the activation tagged mutant seems to cause ectopic expression of the BOU gene in all vegetative and reproductive tissues while the endogenous pattern of expression appeared to be restricted only to root tissue. Over-expression lines in which the BOU ORF was driven by the 35S promoter displayed the bountiful phenotype confirming that the activation tagged phenotype was indeed caused by activation of the BOU gene. Protein sequence analysis indicates a putative role for BOU as a chromatin remodelling factor which in association with the expression pattern suggests a possible involvement of BOU in high-hierarchy order of regulation of gene expression. Hence microarrays could be very useful for the identification of downstream interacting factors or target geneswhich will help us to gain insights towards unravelling the biological role of the BOU gene. The specific interaction to flowering time genes will be studied independently using RT-PCR to reveal the relationship to other genes in the regulatory pathway. As experimental setup we intend to compare gene expression between bountiful mutant and wild-type arabidopsis plants using rosette leaf tissue as source for RNA. Though the BOU gene is endogenously expressed in a root-specific mannerits ectopic expression in the bountiful mutant phenotipically affects the whole plant including leaves. Experimenter name: Raffaella Greco; Experimenter department: Pereira Lab; Experimenter institute: Plant Research International; Experimenter address: Business Unit Genomics; Plant Research International; Postbus 16; Wageningen; Experimenter zip/postal_code: 6700 AA; Experimenter country: The Netherlands Experiment Overall Design: 2 samples were used in this experiment

Project description:In Antirrhinum, the equivalent mutant to the Arabidopsis cuc1 cuc2 double is called cup. We have cloned CUP and shown that it encodes a NAC-domain transcription factor homologous to CUC1 and CUC2. Yeast two-hybrid analysis shows that CUP interacts with TIC, an Antirrhinum TCP transcription factor. Moving back to Arabidopsis, the closest homologues to TIC encode TCP factors TCP13 and TCP14 which, we have now shown, also interact in two-hybrid experiments with CUC1 and CUC2. We have identified insertions in both TCP13 and TCP14. CUP, CUC1 and CUC2 play a role in the establishment of boundaries between lateral organs. As evolutionarily conserved interactors, we expect TCP13 and TCP14 to act in the same process. Homozygous tcp13 mutant flowers show mixed cell identity (mci) with the boundaries of organ identity out of register with those of physical organ development. tcp 14 mutants show a very weak phenotype, but the double heterozygote is identical to the tcp13 homozygote. The double homozygote is underway at the moment and this application takes into account the time required to generate these plants.The aim of this microarray experiment is to identify targets of TCP13 and TCP14. We propose to compare expression levels in WT, tcp13, tcp14, tcp13/+ tcp14/+, and tcp13 tcp14 plants. Although we initially propose only duplicate experiments for each mutant, requiring a total of 10 chips, the experimental material will serve as internal replicates, since we expect to see different degrees of inactivation/activation of at least a core of conserved genes. The fact that all of the mutant combinations observed so far produce flowers with the appropriate cell types present, but in a different position, suggests that, unlike in homeotic mutants, very similar sets of floral genes will be present in each sample. This gives us hope that we might find a smaller number of genes with altered expression. Our tissue samples will be wild type and mutant inflorescences and, as such, will contain a variety of flowers at different stages of development. By taking a large number of flowers and buds we hope to overcome any sampling errors. Although we have requested 10 chips - for duplicate analyses of WT, both single mutants, the heterozygote and the double mutant - it remains a formal possibility that the double homozygote is inviable, or produces no inflorescence. In this unlikely eventuality we would only submit 8 of the 10 samples.

Project description:In Antirrhinum, the equivalent mutant to the Arabidopsis cuc1 cuc2 double is called cup. We have cloned CUP and shown that it encodes a NAC-domain transcription factor homologous to CUC1 and CUC2. Yeast two-hybrid analysis shows that CUP interacts with TIC, an Antirrhinum TCP transcription factor. Moving back to Arabidopsis, the closest homologues to TIC encode TCP factors TCP13 and TCP14 which, we have now shown, also interact in two-hybrid experiments with CUC1 and CUC2. We have identified insertions in both TCP13 and TCP14. CUP, CUC1 and CUC2 play a role in the establishment of boundaries between lateral organs. As evolutionarily conserved interactors, we expect TCP13 and TCP14 to act in the same process. Homozygous tcp13 mutant flowers show mixed cell identity (mci) with the boundaries of organ identity out of register with those of physical organ development. tcp 14 mutants show a very weak phenotype, but the double heterozygote is identical to the tcp13 homozygote. The double homozygote is underway at the moment and this application takes into account the time required to generate these plants.The aim of this microarray experiment is to identify targets of TCP13 and TCP14. We propose to compare expression levels in WT, tcp13, tcp14, tcp13/+ tcp14/+, and tcp13 tcp14 plants. Although we initially propose only duplicate experiments for each mutant, requiring a total of 10 chips, the experimental material will serve as internal replicates, since we expect to see different degrees of inactivation/activation of at least a core of conserved genes. The fact that all of the mutant combinations observed so far produce flowers with the appropriate cell types present, but in a different position, suggests that, unlike in homeotic mutants, very similar sets of floral genes will be present in each sample. This gives us hope that we might find a smaller number of genes with altered expression. Our tissue samples will be wild type and mutant inflorescences and, as such, will contain a variety of flowers at different stages of development. By taking a large number of flowers and buds we hope to overcome any sampling errors. Although we have requested 10 chips - for duplicate analyses of WT, both single mutants, the heterozygote and the double mutant - it remains a formal possibility that the double homozygote is inviable, or produces no inflorescence. In this unlikely eventuality we would only submit 8 of the 10 samples. Experiment Overall Design: Number of plants pooled:10

Project description:Our analysis of the sfr6 freezing-sensitive mutant (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.) and cls8 (unpublished) chilling-sensitive mutant of Arabidopsis, has revealed that the expression of certain cold-regulated genes is aberrant in both these mutants. In order to understand the molecular basis of chilling and freezing stress in Arabidopsis and also to determine commonalities and differences between these 2 different physiological stress-tolerance processes, we request transcriptome analysis for both of these mutants compared to wild type in one experiment, upon cold treatment and at ambient conditions. The sfr6 mutant shows the most severe phenotype with respect to cold gene expression, but is tolerant to chilling (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999). Plant Cell 11, 875-886.). However, it is unable to cold acclimate and hence is sensitive to freezing. The cls8 mutant, on the other hand, has a relatively mild phenotype relative to the cold-regulated genes we have examined, but is very sensitive to chilling temperatures (15 to 10 degree centigrade). It is thus likely that in cls8 we have not yet identified the genes which are most affected, and which account for the physiological phenotype. Both sfr6 and cls8 have been fine-mapped and are close to being cloned. The cls8 mutant has an altered calcium signature in response to cold which means it is likely to be affected in early signalling, e.g. cold perception itself.We will compare the expression profiles of genes in sfr6, cls8 and Columbia (parental line for both mutants), both at ambient, and after treatment with cold (5 degrees) for 3 hours. This timepoint is designed to ÃÂcaptureÃÂ both rapidly responding genes e.g. CBF/DREB1 transcription factors, and also more slow genes e.g. COR genes (KIN1/2 and LTI78). Pilot northerns confirm that this time point is suitable.This analysis will provide new insight into 2 novel genes required for tolerance to low temperature in Arabidopsis, and additionally will determine the nature of overlap between the separate processes of chilling and freezing tolerance.

Project description:Plant growth and development are strongly affected by light signals perceived by phytochromes (phy) and cryptochromes (cry). The physical interaction between photoreceptors and the cross-talk among downstream signalling steps creates a network of interactions in which the action of one photoreceptor depends on the status of the others. The processes modulated by phy and or cry include seed germination, seedling de-etiolation, plant body formation and flowering. Compared to the wild type the leaves of the phyB mutant are characterised by their extended petioles and pale colour. These features resemble the phenotype of normal plants grown under the low red to far-red ratios typical of dense plant canopies thus providing support to the idea that phyB plays a key role in the perception of high red to far-red ratios. The triple mutant lacking phyA cry1 and cry2 shows little differences in leaf size and shape with the wild typebut the quadruple phyA phyB cry1 cry2 mutant shows severely impaired leaf expansion. In addition to the specific effects on leaf sizeshape and pigmentation the phyB mutation accelerates flowering in the wild type background. This effect can be observed as a reduced number of days between sowing and visible flower buds (time scale) and as a reduced final number of leaves (developmental scale). However in the phyA cry1 cry2 triple mutant background the phyB mutation accelerates flowering on a developmental scale but severely delays flowering on a time scale. The long photoperiods inducing flowering are perceived by the leaves. Flowering signals that could include hormones and sugars migrate from the leaves to the apex in response to the light stimulus. We propose to compare the following mRNA samples of expanding leaves: 1) wild type 2) phyB mutant 3) phyA cry1 cry2 triple mutant 4) phyA phyB cry1 cry2 quadruple mutant. The comparison between the wild type and the phyB mutant will help to uncover the changes in leaf mRNA patterns underlying enhanced petiole growthreduced pigmentation and early flowering in response to the absence of phyB activity. The comparison between the phyA cry1 cry2 triple mutant and the phyA phyB cry1 cry2 quadruple mutant should reveal the mRNA differences between photosynthetic leaves with dramatically different abilities to expand. The added value of the simultaneous analysis of the two comparisons is that effect of phyB is different in both genetic backgrounds and this should provide information on the mechanisms and significance of photoreceptor interactions.

Dataset Information

Transcription profiling of Arabidopsis of gain of function (BOU) bountiful mutant bs wild type arabidopsis plants using rosette leaf tissue

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