The Roles Of Kinesins In Exponential Cell Production And Enlargement During Early Fruit Development

Cucumber, as high production and consumption is widely cultivated in open field and protected cultivation in the world and account for a pivotal position of vegetable. In recent years, the yield and quality of cucumber become the study focus of the breeders. Unlike fruits that are eaten at a mature stage, cucumbers, which are consumed as both a fresh product and a processed food, are typically harvested at an early phase of fruit development. Therefore, the rapid cell division and expansion in early fruit development are important phases for cucumber fruit production. A number of studies have focused on fruit ripening and post-harvest physiology; however, the mechanism of early fruit growth, especially the mechanism of rapid cell division and expansion in cucumber, is not well understood.Recent studies have revealed that tubulin is highly expressed during the exponential growth stage in cucumber fruit. Tubulin is the major building block of microtubules. Kinesins constitute a superfamily of MT-dependent motor proteins and play critical roles in mitosis, meiosis, cell morphology, and the transport of vesicles and organelles. Some studies have revealed that cyclin and cyclin-dependent kinases are important facilitators of cell production during fruit development; phosphorylate a mitotic kinesin protein for the appropriate onset and/or progression of cytokinesis. However, it is still not known whether specific kinesin proteins are required for regulating exponential cell production and expansion during early fruit development. Therefore, the function of kinesin proteins in this process is expected to have unique characteristics. In this study, we investigated the Kinesin family factors in cucumber and the expression patterns of the Kinesin genes in early phase of fruit development. We found at least 8 candidate kinesins that are involved in these processes and we focus on the three cucumber Kinesin Cs KF1、Cs KF2 and Cs KF3.We determined the intracellular localization pattern in fruit cells via immunofluorescence labeling. The results are as follows:(1) We analyzed physiological changes, cell production, and cell expansion during early cucumber fruit development in 9930. The fruit length and fruit diameter showed similar growth curves, increasing almost exponentially between 3 DAA and 5 DAA. The fruit firmness was relatively constant before 3 DAA and then decreased rapidly. Rapid increases in cell number were observed between 0 DAA and 3 DAA in both the cross-section and the longitudinal-section, with an approximate 200% increase. The average cell area increased by <2-fold from 0 DAA to 5 DAA. However, the majority of the exponential increase in cell size(almost 30-fold) occurred between 5 DAA and 16 DAA, after the cells exited from mitotic cell production. These results indicated that the period from 3 DAA to 5 DAA was an obvious boundary for fast growth of the fruit of ‘9930’.(2) 47 genes were identified in the cucumber genome by using the conserved Kinesin motor domains of human, mouse and Arabidopsis as BLAST queries in the cucumber genome database.The expression of 47 cucumber kinesin genes during vegetative and reproductive development, measured by using RT-PCR, indicated that all of these predicted kinesins were expressed genes and were differentially expressed in various tissues. 47 kinesin proteins genes were measured using quantitative RT-PCR in early cucumber fruit development(-2BF to 16DAA). Further analysis indicated that 10 kinesin genes showed peak expression levels in the rapid cell division phase(before 3 DAA), while 2 kinesin genes showed peak expression levels during the rapid cell expansion(after 5 DAA). Eight of these kinesin genes were selected by hierarchical clustering for further study.(3) The expression of the 8 predicted kinesins was further analyzed in these four characterized varieties during early fruit development. 5 kinesin genes were upregulated before 3 DAA, and the expression levels of these genes correlated with the difference of cell numbers in these four varieties. Additionally, 2 kinesin genes were upregulated after 5 DAA, and the expression levels of these genes correlated with the order of cell area in these four varieties. Cs KF2、Cs KF3、Cs KF4、Cs KF5 and Cs KF6 play roles in rapid cell production, while Cs KF1 and Cs KF7 function in exponential cell expansion.(4) Cs KF1, Cs KF2 and Cs KF3 were cloned from cucumber and the secondary structure were predicted by bioinformatics tools. A histidine-tagged Cs KF1 motor domain and a histidine-tagged Cs KF2 motor domain were solubly expressed in E. coli and purified from the cell extract by Ni-NTA agarose affinity chromatography. MT-stimulated ATPase activity assay and MT co-sedimentation experiments results indicate that His-Cs KF1-motor has an MT-stimulated ATPase activity and binds to MTs in an ATP dependent manner, which is consistent with the traditional characterization of a kinesin protein. However, the ATPase activity of the His-Cs KF2-motor is independent of MT concentration, and this protein binds to MTs in a nucleotide independent manner.(5) Polyclonal anti-KF1, anti-KF2, and anti-KF3 antibodies were raised in rabbits using the purified His-KF1-N, His-KF2-C, and His-KF3-C protein as the antigens. The anti-KF1 antibody recognized an approximately 80 k Da band, which is similar to the predicted size of Cs KF1, in cucumber root, leaf, shoot, and fruit(5 DAA). The anti-KF2 antibody recognized an approximately 125 k Da band in cucumber leaf, shoot, and fruit(0 DAA). The anti-KF3 antibody recognized an approximately 110 k Da band in cucumber root, stem, male flower, female flower and fruit(2 DAA).(6) The specific antibodies were used to localize Cs KF1, Cs KF2, and Cs KF3 in fruit cells via immunofluorescence. Cs KF1 was detected at the plasma membrane in fruit cells at 5 DAA. It was also found that the Cs KF2 fluorescence signals could predominantly be detected in fruit that was harvested at –2DAA to 2 DAA during rapid cell division. To understand the detailed localization of Cs KF2 in chloroplasts, its detailed localization was studied in cucumber fruit cells by combining the chloroplast fraction with immunoblotting detection. Cs KF2 proteins exist predominantly in chloroplasts, in which proteins from both membrane and stoma showed Cs KF2 immunoblotting signals. A localization assay showed that Cs KF3 localizes mainly at the midzone of the phragmoplast in fruit telophase cells.(7) The over-expression vectors Cs KF1-OE 、 Cs KF2-OE and Cs KF3-OE, and the RNAi transformation vectors Cs KF1-RNAi、Cs KF2-RNAi and Cs KF3-RNAi for biolistic or Agrobacterium- mediated transformations were constructed using Gateway technology.