Ketelsen, Bernd

Cytokinins are phytohormones involved in regulating plant growth and plant development. They regulate cell division, metabolism, chloroplast development, shoot and root development and senescence (Haberer and Kieber (2002)).
Cytokinin signalling occurs mediated by a two-component system permutated to a multistep phosphorelay (To and Kieber (2008)), which eventually activates type B ARRs (Arabidopsis response regulators) via AHPs (Arabidopsis histidine-containing phosphotransfer proteins). Type B ARRs induce transcription of type A ARRs which regulate negatively the cytokinin signal transduction pathway as well as other downstream responses (Argyros et al. (2008)). In parallel, AHPs also activate members of another protein family: The Cytokinin Responsive Factors (CRFs) (Rashotte et al. (2006)). These 6 transcription factors belong to the group of AP2/EREBP transcription factors. Many of which are playing important roles in plant growth and development and they are especially known to respond to environmental stresses and hormones (Feng et al. (2005)).
Half of the target genes of type B ARRs are also regulated by CRFs and CRFs seem to play an important role during seed development (Rashotte et al. (2006), Day et al. (2008)) (Figure 1).

Figure 1
Model of cytokinin signaling. Both AHPs and CRFs move into the nucleus in response to cytokinin. AHPs activate type B ARRs which, together with CRFs, mediate cytokinin-regulated gene expression. (Rashotte et al. (2006))
 

2 LOKALIZATION OF CRFs

Recently it was shown that CRFs migrate from cytoplasm into the nucleus after cells were treated with cytokinins (Rashotte et al. (2006)). CRF5 and CRF6, however, contain a putative import sequence for their import into mitochondria (Schwacke et al. (2007)). Nonetheless, fusing the pre-sequence of CRF5 to GFP did not enable GFP to migrate into mitochondria.
Transient transformations of Arabidopsis protoplasts and onion epidermal cells with crf5-gfp by PEG led to an interesting GFP-pattern within the nucleus. It accumulates there as nuclear bodies, which are spread allover the nucleus excluding the nucleolus and might be, for example, spliceosome-dependent speckles or cajal bodies (Figure 2).

Figure 2
Intracellular location of the CRF5-GFP-fusion protein after transient transformation of Arabidopsis thaliana protoplasts (upper part, left) and onion epidermal cells (lower part). The picture top right shows the GFP location if fused just to the predicted pre-sequence of CRF5. Green: GFP fluorescence, red: chlorophyll fluorescence.  

3 CRF SPECIFIC DOMAINS AND CRF HOMOLGUES IN OTHER PLANT SPECIES

Peptide sequence alignments of all six Cytokinin Responsive Factors in Arabidopsis revealed four N-terminal located conserved regions (CRF specific domains I-IV) as well as one laying in the middle of the peptide sequence (CRF specific domain V) behind the AP2/EREBP transcription factor characterizing DNA binding domain AP2 (Figure 3, upper part).
Blasting the resulting consensus sequence of the previous alignment against all on the NCBI database available peptide sequences verified all five conserved regions and additionally led to homologue peptide sequences in the castor oil plant (Ricinus communis), in tomato (Solanum lycopersicum), in common grape wine (Vitis vinifera) and in the California poplar (Populus trichocarpa) (Figure 3, lower part).

Figure 3
Upper figure: Multiple alignment of all Arabidopsis thaliana Cytokinin Responsive Factors (CRF1 - CRF6) and one independent AP2/EREBP transcription factor (At2g44940). Lower figure: Schematic presentation of a peptide blast (blastp) of the consensus sequence resulting from the CRF peptide sequence alignment (upper figure) against all peptide sequences in the NCBI database.
Red annotations: CRF-specific elements. Blue annotation: AP2 DNA binding domain. (Geneious v4.6, Drummond et al., (2009))

We expect at least the first four CRF specific domains (I-IV) being strong interaction domains with the transcription complex. Auto-activation assays within a yeast-2-hybrid system showed a extremely strong self-induction of promoter genes when linking CRF5 and CRF6 to the promoter binding domain (data not shown).

4 METHODS OF TRANSIENT TRANSFORMATION OF PLANT CELLS

The transient transformation of plant cells is a established method to analyze the intra-cellular localization of proteins.
GFP tagged versions of CRF5 have already been used in plant cells mediated by several systems for transient transformation. We are currently using Agrobacterium tumefaciens leaf infiltration as well as PEG mediated Arabidopsis protoplast transformation (Fig. 5) and particle bombardment (Fig. 4).
We have established a modification of the particle-gun-mediated transient transformation technique using leaf cells of the moss Funaria hygrometrica (Fig. 4). These leafs consist of only one layer of large cells, like the commonly used onion epidermal cells, but they do in contrast to the onion cells contain chloroplasts instead of leukoplasts. Another advantage is the large size of chloroplasts in Funaria cells which makes it possible to easily detect GFP-fluorescence in these organells and to obtain informations about the intra-organellar localization of the proteins.

Figure 4
Transient transformation of Funaria hygrometrica leaf cells or onion epidermis cells mediated by particle bombardment.

Figure 5
PEG mediated transient transformation of protoplasts derived from an Arabidopsis thaliana cell suspension culture.

5 GFP PATTERNS OF TRANSIENTLY TRANSFORMED PLANT CELLS

Figure 6 shows GFP-constructs used as marker (for cytoplasm and nucleus (35S-GFP), chloroplasts(At-Why1-GFP) and mitochondria (CoxIV-presequence-GFP) in different transformation systems. Especially the results of the lately established transformation system based on Funaria hygrometrica show the accuracy of this system. All GFP-constructs were detected at the same inner-cellular location as they were in higher plant cells.