Blots were then given five washes, for 5 min each, with washing solution. cotton fibers, we hypothesized that microtubule-based motors likely participated in quick reorganization of microtubules and transport of various materials and organelles. In this study, we statement the identification of the cotton KCBP homolog GhKCBP from a cotton fiber-specific cDNA library. Furthermore, we present results showing that in this differentiated cell type, GhKCBP localizes to the cortical microtubules. RESULTS Isolation of GhKCBP cDNA To identify cotton kinesin cDNA sequences, available herb kinesin sequences were used to compare with cotton fiber expressed sequencing tag (EST) sequences. One sequence from your Clemson University cotton EST database (http://www.genome.clemson.edu/projects/cotton/est/) was found to match the KCBP sequence significantly. Probes based on this sequence were used to screen a 21-DPA cotton fiber-specific library (Pear et al., 1996). Two clones were isolated that overlapped and together covered the full-length coding region of spp. endosperm cells, GhKCBP is also present in mitotic microtubule arrays in cotton meristematic cells. Open in a separate window Physique 4 Immunolocalization of GhKCBP in cotton cells undergoing mitotic cell division. Cotton root cells were stained by immunoflurescence techniques for GhKCBP (A, D, G, J, and M) or for -tubulin (B, E, H, K, and N) or were stained with DAPI to detect DNA (C, I, and L). Pseudocolored images (F and O) show anti-GhKCBP in green and anti–tubulin in reddish. GhKCBP could be detected Tofogliflozin (hydrate) in the microtubule preprophase band (arrow, A, B, and DCF). Abundant GhKCBP transmission was present in metaphase cells (GCI). GhKCBP clearly was present among phragmoplast microtubules (J, K, M, to O). Level bar = 10 m. Conversation The kinesin-related protein KCBP is usually conserved at least in algae and flowering plants (Abdel-Ghany et al., 2000; Abdel-Ghany and Reddy, 2000). A related protein was also recognized in sea urchin (Rogers et al., 1999). Our results indicated that GhKCBP clearly associated with cortical microtubules in cotton fibers, which supported the notion that KCBP plays a role in interphase cells, as suggested by genetic studies of trichome morphogenesis (Oppenheimer et al., 1997). Our work and previous studies (Seagull, 1992) with fixed cells at different stages of fiber development clearly show that reorientation of cortical microtubules take place, although we have not directly observed microtubule reorganization in living cotton fibers. It has been acknowledged that cortical microtubule reorganization requires energy (Wymer et al., 1996). Such an energy requirement could be due to the functions of motor proteins. On the basis of our localization data, we suggest that GhKCBP plays a role in reorganization of cortical microtubules during cotton fiber development. Because KCBP has a nu-cleotide-independent microtubule-binding site outside PVRL2 the motor domain name (Narasimhulu and Reddy, 1998), it is affordable to presume that microtubules could be a cargo of this motor. KCBP could contribute to organizing cortical microtubules as they move against each other by Tofogliflozin (hydrate) such a minus end-directed motor would converge their minus ends. Newly converged microtubules could presume new orientation depending on how microtubules were stabilized at a certain direction. Alternatively, GhKCBP may contribute to microtubule stability directly. One possibility Tofogliflozin (hydrate) is usually that KCBP acts as a microtubule stabilizer. KCBP could participate in stabilizing cortical microtubules in a certain orientation so that a cell could undergo proper elongation and morphogenesis. This hypothesis is usually supported by the fact that this phenotype of reduced branches of the mutation can be suppressed by the microtubule stabilization agent taxol (Mathur and Chua, 2000). Conversely, KCBP may act as a microtubule destabilizer. A C terminus motor in yeast, Kar3p, has been shown to be able to destabilize microtubules at their minus ends (Endow et al., 1994). Loss of the Kar3p protein in yeast increases the quantity of cytoplasmic microtubules (Huyett et al., 1998). If GhKCBP has a comparable function in microtubule stabilization, we could expect that a lower level of GhKCBP would be detected at Tofogliflozin (hydrate) later stages of fiber development. In our immunoblotting experiments, there was a significant reduction of GhKCBP protein level at 21 DPA when secondary wall synthesis was about to initiate, whereas -tubulin content increased at 21 DPA compared.