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First published online 13 June 2007
doi: 10.1242/jcs.007393

Journal of Cell Science 120, 2241-2247 (2007)
Published by The Company of Biologists 2007
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AtMAP70-5, a divergent member of the MAP70 family of microtubule-associated proteins, is required for anisotropic cell growth in Arabidopsis

Andrey V. Korolev*, Henrik Buschmann, John H. Doonan and Clive W. Lloyd

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


Figure 1
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  		Fig. 1. Computational analysis of the AtMAP70-5 protein. (A) Prediction of coiled-coil domains by the COIL program (Lupas, 1996Go). Shown is an output based on parameters producing the highest stringency. The relative positions of the KLEEK motif and its imperfect repeats are indicated. The colour of the graphs indicates the window size used in the scan (red 28, blue 21 and green 14 amino acids window). (B) The KLEEK motif and neighbouring amino acids of AtMAP70-5 and the WAVE-DAMPENED 2 (WVD2) family. Identical amino acid positions are in red. The heptad pattern of the predicted coiled-coils (presented by amino acid positions a and d) is shown in the first line.

 

Figure 2
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  		Fig. 2. Recombinant AtMAP70-1 and AtMAP70-5 co-sediment with pre-assembled microtubules in vitro. Lanes 1 and 6: extracts of E. coli cells expressing 6xHis-AtMAP70-5 and 6xHis-AtMAP70-1, respectively; concentrations of the purified proteins were estimated as 50 µg/ml for 6xHis:AtMAP70-1 and 10 µg/ml for 6xHis:AtMAP70-5 using SDS-PAGE and BSA standards (not shown). Lane 7: purified tubulin used to form microtubules, but before assembly with Taxol. Lanes 2-5: co-sedimentation of 6xHis-AtMAP70-5 with pre-assembled microtubules. Lanes 8-11: co-sedimentation of 6xHis-AtMAP70-1 with pre-assembled microtubules; P, pellets (lanes 2, 4, 8 and 10); S, supernatants (lanes 3, 5, 9 and 11); + Mts, pre-assembled microtubules added to the reaction mixture (lanes 2, 3, 8 and 9); – Mts, no pre-assembled microtubules added (lanes 4, 5, 10 and 11).

 

Figure 3
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  		Fig. 3. The effect of added AtMAP70-5 on the length of pre-assembled microtubules in vitro. Tubulin was polymerized before either PME buffer (A) or AtMAP70-5 (B) was added. Addition of AtMAP70-5 shifts the size distribution towards longer microtubules.

 

Figure 4
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  		Fig. 4. Labelling patterns of cortical microtubules in tobacco BY-2 cells. (A-E) Untransformed cell (A) and cells stably transformed with GFP-TUA6 (B), GFP-AtMAP70-1 (C) and GFP-AtMAP70-5 (D,E). One in four cells expressing GFP-AtMAP70-5 had additional poles of growth (E). (F,G) Root epidermal cells of GFP-AtMAP70-5-overexpressing Arabidopsis plant show punctate labelling (F); when displayed as a time-lapse projection these punctae can be seen to resolve as linear cortical microtubules (G). Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Overexpression of GFP-AtMAP70-5 induces right-handed helical growth. (A,B) Three-day-old seedlings grown vertically on a Phytagel plate; A, wild type, B, GFP-AtMAP70-5 overexpressor showing right-twisting root. (C,D) Two-week-old plants; (C) wild type, (D) GFP-AtMAP70-5 overexpressor, illustrating right-twisting petioles. (E,F) Root epidermal cells from plants as in A and B, illustrating right-twisting epidermal files in the overexpressor (F). (G,H) Microtubules in root epidermal cells of the early elongation zone showing shallow left-handed microtubule helices in the overexpressor (H). (I) Semi-quantitative RT-PCR with AtMAP70-5 (70-5)- and APT1-specific primers shows that the twisted phenotype corresponds to high level of AtMAP70-5 expression. (J,K) Root growth under low levels of GFP-AtMAP70-5 expression (J) is straight but with higher expression levels the cell files twist (K). Bars, 1 mm (A-D); 30 µm (E-H); 100 µm (J,K).

 

Figure 6
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  		Fig. 6. Overexpression and RNAi knockdown of AtMAP70-5 generate stunted inflorescence stems. (A-C) Six-week-old wild-type (WT), overexpressor (OE) and RNAi-transformed Arabidopsis plants, respectively. (D) RT-PCR (25 cycles) based on total RNA from inflorescence stems (shown for two individuals each) using AtMAP70-5 (70-5) and APT1-specific primers.

 

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© The Company of Biologists Ltd 2007