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First published online 31 January 2006
doi: 10.1242/jcs.02813

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The role of MAP65-1 in microtubule bundling during Zinnia tracheary element formation

Guojie Mao^*, Henrik Buschmann, John H. Doonan and Clive W. Lloyd

Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, UK

View larger version (81K): [in a new window]   Fig. 1. Microtubule bunching during Zinnia tracheary element differentiation. (A) Isolated mesophyll cell before addition of inductive hormones. (B) A tracheary element 72 hours post-induction, with cell wall thickenings visible under bright-field optics. (C) Bundles of cortical microtubules labelled by anti-tubulin immunofluorescence. (D) The same cell with coincident cellulose-rich secondary thickenings labelled with Calcofluor White. Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 2. Immunoblotting and immunofluorescence with an antibody against MAP65 peptide. Samples at equal protein loading immunoblotted with the antibody against the CEEESWLEDYNR peptide. (A) Immunoblot of Zinnia suspension cells at 0 hours (pre-induction); (B) differentiating cell culture at 48 hours. (C) Immunofluorescence of induced microtubule bundles at 48 hours. Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 3. Expression of ZeMAP65 isoforms during Zinnia cell differentiation. After a delay of 2 hours, ZeMAP65-1 was shown by RT-PCR to be expressed from 2-40 hours after induction, and decreased at 48 hours, similar to the pattern for the tracheary element differentiation marker TED2 (Demura and Fukuda, 1994).

View larger version (183K): [in a new window]   Fig. 4. Localisation of TED2 and ZeMAP65-1 transcripts in Zinnia. In situ hybridisations using cross-sections of 14-day-old Zinnia stems showed a similar pattern of expression in vascular bundles for ZeMAP65-1 (A) and TED2 (C), but this signal was not observed in the respective sense controls (B,D). Bar, 0.1 mm.

View larger version (11K): [in a new window]   Fig. 5. Phylogram of ZeMAP65-1 and known MAP65 family proteins. The following sequences were analysed: tobacco NtMAP65-1a (CAC17794), carrot DcMAP65-1 (CAD58680) and Arabidopsis AtMAP65-1 (At5g55230), AtMAP65-2 (At4g26760), AtMAP65-3 (At5g51600), AtMAP65-4 (At3g60840), AtMAP65-5 (At2g38720), AtMAP65-6 (At2g01910), AtMAP65-7 (At1g14690), AtMAP65-8 (At1g27920) and AtMAP65-9 (At5g62250). The Phylogram was created with the ClustalW software (Thompson, 1994).

View larger version (66K): [in a new window]   Fig. 6. GFP-ZeMAP65-1 induces microtubule bundles when expressed in Arabidopsis suspension cells. Massive bundling of microtubules still allows for the formation of ordered microtubule arrays, which may be helical (A) or transverse (B). Both pictures are z-projections of confocal sections. Bar, 8 µm.

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