Identification of factors involved in Xylem Cell Differentiation Aarush Mohit Mittal 1, 2 1 Department of Biological Sciences and Bio-Engineering, Indian Institute of Technology, Kanpur, India 2 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan The vascular system of plants is composed of mainly two types of cells Xylem and Phloem. Xylem are the water carriers and the secondary cell wall of these cells also provides mechanical support to the plant body. This cell wall is primarily composed of cellulose which is deposited in several distinct patterns regulated by a two-dimensional network of microtubules. With the aim of finding which factors regulate this microtubule network, I tested 6 different inhibitors of cell differentiation and characterized each of them based on their effect on the differentiation rate and the pattern of cell wall formation. I then chose 2 inhibitors out of the 6 to further investigate their role in the process of microtubule regulation. As each inhibitor affected a different factor in the cell, these factors are possible candidates in the regulation of microtubule network and further studies need to be performed to fully elucidate their exact role in the process. 1 Introduction Xylem cells are the most important cells for water transport in plants and they form a large network of vessels throughout the plant body. Primary xylem vessels have two kinds of cells Protoxylem and Metaxylem. Protoxylem are the first cells to develop and they further differentiate into metaxylem as the plant matures. In the roots, the metaxylem are found close to the center and the protoxylem are arranged around the metaxylem cells. This arrangement is known as exarch (see Figure 1). The secondary cell wall of these cells is of special interest as it provides mechanical support to the plant organs. It is primarily compose of cellulose, hemicellulose and lignin. Xylem cells show different patterns of secondary cell wall depending on their stage of differentiation. Most common patterns are annular, spiral, reticulate and pitted. Protoxylem exhibit spiral patterns while mature Metaxylem is pitted (see Figure 1). The secondary cell wall pattern is determined by the deposition of cellulose on a two-dimensional network of microtubules. These microtubules are in turn regulated by several different factors in the cell which leads to such specific networks. It has been seen that certain inhibitors that affect cell differentiation also affect the pattern of secondary cell wall pattern. As each of these works in a different way, they would be useful in identifying the factors involved in the process. 2 Experimental Plan The first experiment (Experiment 1) was to check the optimum time at which most of the cultured cells are differentiated. This was done using an established cell culture protocol for Arabidopsis thaliana cells. The differentiation was induced in triplicate and the cells were characterized at 23hrs, 25hrs and 43hrs after induction. Page 1
Then I checked (Experiment 2) the effect of 5 inhibitors, namely Orizine (Orz), Taxol, Latrunculin B (LatB), RacI and Okadaic Acid (OA), on cell differentiation using cultured cells. All the inhibitors were at a concentration of 10µM except OA. OA was tested at 3 different concentrations (0.005µM, 0.05µM and 0.5µM). This was done the optimum concentration at which OA completely inhibits differentiation was not known. The cells were characterized at 24hrs after induction. The next step was to check the effect of these inhibitors on Arabidopsis plants grown in-vitro. For this purpose I chose 2 inhibitors, Taxol and OA as these two have different mechanisms by which they inhibit differentiation. Taxol stabilizes microtubules and thus prevents them from elongating. In contrast, OA inhibits protein phosphatases like PP1, PP2A etc. These phosphatases are responsible for a variety of functions in the cell including cell differentiation. Also time constraints prevented me from checking other inhibitors. The plants were grown (Experiment 3) on normal media for 10 days and then transferred to different plates containing different concentrations of Taxol (10µM and 20µM) and OA (0.05µM, 0.5µM and 2µM). The plant roots were observed under microscope on the 13 th day. To verify the importance of protein phosphatases in the process of cell differentiation, I tested (Experiment 4) another inhibitor, namely Calyculin A (CalA), as it has the same modus operandi as OA. CalA was tested at 7 different concentrations (0.05nM, 0.5nM, 0.005µM, 0.01 µm, 0.05µM, 0.1µM and 0.5µM). Cells were observed at 24hrs after induction. The last experiment (Experiment 5) was done to check the localization of the identified factors on the secondary cell wall. For this plasmids were made for different subunits of PP2B, tubulin and MAP70 (see Appendix). The cells were transformed using these plasmids and cultured. Differentiation was induced in these cells and the observations were recorded at 24hrs after induction. 3 Materials and Methods 3.1 Arabidopsis Cell Culture Arabidopsis thaliana suspension cells, strain Columbia-0, were cultured in 27mL of modified Murashige and Skoog (MS) medium, ph 5.8, containing 4.33g L 1 of MS inorganic salts, 4.1mM 2,4-D, 3.0% (w/v) sucrose, and vitamins, including 8mg L 1 nicotinic acid, 8mg L 1 pyridoxine-hcl, 80mg L 1 thiamine- HCl, and 800mg L 1 myoinositol. The cells were agitated on a rotary shaker at 124 rpm at 23 C in the dark. At weekly intervals, 12mL aliquots of the culture were transferred to 15mL of fresh medium in a 100mL culture flask. 3.2 Induction of Differentiation To induce differentiation, a 1mL aliquot of 7-dold cell culture was suspended in 9mL of 2,4-D free MS medium. After 3 min of settling, 5mL supernatant was removed to adjust cell density. The remaining cell suspension was supplied with 2µM estrogen and 2µM brassinolide, and cultured on a rotary shaker at 124 rpm at 23 C in the dark. 1µL of inhibitor was added in each of the wells. 3.3 In-vitro growth of Arabidopsis plants Arabidopsis ecotype Columbia was used as the wild-type plant. Seedlings were germinated on half-strength MS phytogel plates in a Percival incubator with 24hrs light for 7 to 10 d at 22 C. The plants were transferred to half strength MS phytogel plates containing different concentrations of inhibitor as mentioned above. Page 2
3.4 Cell Transformation Agrobacterium tumefaciens strain GV3101 (MP90) was first transformed using the 9 different plasmids (see Appendix) by electroporation. The bacteria carrying the expression constructs were grown in Luria- Bertani media with Spectinomycin for 3 days at 3 C. To establish a transformant strain, 3-d-old cells were co-cultured with the transformed bacterial cells in MS medium supplemented with 50mg L 1 acetosyringone for 2 d. Then, 0.5g L 1 claforan was added to the cell suspension and the cells were cultured for a further 5 d. Thereafter, the cell suspension was transferred into 15mL fresh medium and maintained as described above, and the surviving cells were used for experiments. 3.5 Characterization of cells To stain secondary cell walls, the cells were incubated with 1mg L 1 WGA AlexaFluor 594 (Invitrogen) for 15 min The plant roots were stained using 1% safranin in 30% EtOH. This was followed by washing in different concentrations of EtOH from 30% to 100% at 15 min intervals to remove the excess stain. The roots were then fixed in clear solution and the coverslip was sealed using manicure. Differential Interference Contrast (DIC) microscopy was used to visualize the cultured cells and the plant roots. Fluorescence microscopy was used to visualize the secondary cell walls at 561nm and GFP tagged proteins in the transformed cells at 488nm. Confocal Microscope was used to visualize the transformed cells and the cultured cells at high resolution. 4 Results 4.1 Experiment 1 The rate of differentiation started to increase at about 19hrs after induction and reached the saturation point at about 25hrs. After this time there was a slight reduction in the rate (Figure 2). Also under microscope, the control cells showed no secondary cell wall pattern while a spiral pattern was clearly visible in the secondary walls of the induced cells (Figure 3). 4.2 Experiment 2 The different inhibitors showed different characteristics regarding the rate of differentiation (Figure 4) and pattern of secondary cell wall (Figure 5). Orz completely disrupted the pattern but had no effect on differentiation rate. LatB highly affected the differentiation rate and also affected the pattern. RacI had a moderate effect on the differentiation rate but the pattern appeared almost normal. Taxol affected both differentiation rate and the pattern of secondary cell walls which appeared to be slightly lopsided as compared to the control. OA had no effect on both differentiation rate and pattern at low concentrations (0.005µM and 0.05µM). But at 0.5µM it severely affected the differentiation rate and completely disrupted the pattern. 4.3 Experiment 3 OA at concentration 2µM showed disruption of pattern in protoxylem. However the metaxylem seems to be unaffected. It inhibits root tip elongation and formation of root hair with increasing concentrations showing increased effect. Also at 0.05µM we can see that the gravity sensing of the roots is also affected. This is peculiar and needs further verification (Figure 6,7). Page 3
Taxol severely affected the protoxylem at the root tip showing the lopsided spiral pattern similar to that seen in the cultured cells before. Also the pattern of secondary cell wall in metaxylem seems to be very faint. It exhibits reduced rate of elongation of root tip as well (Figure 6,7). 4.4 Experiment 4 CalA shows an effect similar to that of OA, as predicted by previous studies. At concentrations below 5nM the differentiation rate is affected. However the pattern is similar to normal secondary cell wall pattern. At 5nM concentration the differentiation rate is reduced. The pattern is affected in most of the cells and some cells are dead. At 10nM concentration, there is very low differentiation rate. Only a few cells show pattern and many cells are dead. At concentrations above 10nM almost all the cells are dead (Figure 8). 4.5 Experiment 5 2052, 2129, 2131 and 2404 appear to be localized at the cytoplasm. 2051 and 2202 are localized at the cytoplasm but they show no differentiation. 2130 appears to localize along the secondary wall. Tub6 and MAP70 localize along the secondary cell wall pattern (see Appendix for complete nomenclature) (Figure 9). 5 Discussion The results from Experiment 1 suggested that the best time to observe differentiated cells is between 23-25hrs after induction. Thus for further experiments, the time point of 24hrs after induction was set to observe the cells. The different inhibitors tested in Experiment 2 show that different factors affect the differentiation pattern differently. Thus while some inhibitors affected both the differentiation rate as well as the type of pattern formed (Taxol, OA and LatB), others only affected the differentiation rate (RacI) and some only disrupted the pattern (Orz) and had no effect on the differentiation rate. Thus the factors inhibited by each of these become possible candidates for future studies to find out the exact mechanism of the pattern formation in secondary cell walls. Also, higher concentrations of OA eventually lead to cell death. This maybe because the protein phosphatases it inhibits may be responsible for other vital functions of the cell. The role of phosphatases was validated by the experiment using the inhibitor CalA. It showed the same inhibition characteristics as OA. The only difference was in the concentration required to inhibit completely the differentiation process. CalA was found to be 10 times more potent than OA as it completely inhibited at 0.05µM while OA showed the same at 0.5µM. Due to time constraints, I could only check for the effect of 2 inhibitors (Taxol and OA) on the WT plants grown in-vitro. As these have different mechanisms of action, they provide insight into the role of two different factors. As plants grown in OA showed decreasing rate of root tip elongation as the concentration increased from 0.05µM to 2µM, it is possible that due to reduced differentiation rate, the xylem vessels lose their ability to elongate. Similarly in the case of Taxol, the root tip elongation is disrupted. However due to the very different pattern of protoxylem in this case, the root tips show a very bulbous appearance. The root hairs are also reduced in the case of OA but they seem to be increased in the case of Taxol. The gravity sensing mechanism disruption in the case of OA is also a curious phenomenon. These need further studies to Page 4
verify and find out the exact reason behind these results. The experiment with transformed cells showed some interesting results. The transformed cells 2052, 2129, 2131 and 2404 don t seem to be associated with secondary cell wall patter n formation as they localized at the cytoplasm. 2051 and 2202 are potential candidates as inhibitor proteins because they showed no cell differentiation. 2130 is the only tested phosphatase that shows promise as being associated in the process of secondary wall pattern formation. Thus it needs to be investigated in future studies to figure out how exactly it controls the microtubule network. Tub6 and MAP70 are the two other proteins which have been shown to be associated in the pattern formation. Thus, in the above study I succeeded in testing a variety of inhibitors and found out 3 factors which may be directly involved in the process of pattern formation. These experimental results need to be verified and studied in future research. Also the other untested inhibitors provide ample material for future research. Knockout studies can be performed on the factors identified to elucidate their exact role in the cell. As we gain more information about how this whole process takes place, we can then modify different factors to invent novel patterns or structures that may have beneficial functions. 6 Acknowledgements I am extremely grateful to Professor Fukudasensei for offering me a place in his laboratory; Assistant Professor Oda-san, Katayama-san and Nakashima-san for their guidance and advice on the practical and analytical aspects of my project; Soeda-san for her support throughout the program; and finally all the members of the Fukuda Lab for making me feel so welcome. 7 References Yoshihisa Oda, Hiroo Fukuda, Secondary cell wall patterning during xylem differentiation, Current Opinion in Plant Biology, 2012. Kyoko Ohashi-Ito, Yoshihisa Oda, Hiroo Fukuda, Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that Govern programmed cell death and secondary wall formation during xylem differentiation, Plant Cell, 2010. Yoshihisa Oda, Seiichiro Hasezawa, Cytoskeletal organization during xylem cell differentiation, Journal of Plant Research, 2006. 8 Appendix The protein phosphatase PP2B is a protein composed of a number of subunits. Tubulin (Tub) is the main component of microtubules while MAP70 (Microtubule Associated Protein) has been shown to bind to microtubules. The plasmid constructs include the GFP (Green Fluorescent Protein) gene which can be used to visualize the translated protein. The plasmids used are: 2051PP2A-B -theta-gfp 2052PP2A-B -zeta-gfp 2129PP2A-B -eta-gfp 2130PP2A-B -gamma-gfp 2131PP2A-B -delta-gfp 2202PP2A-B -beta-gfp 2404PP2A-B -alpha-gfp 31GFP-MAP70-7 GFP-TUB6 Page 5
Figure 1: Exarch arrangement in Arabidopsis root. Note the spiral pattern in protoxylem and the pitted pattern in metaxylem. Figure 2: The percentage of differentiation as a function of time. The optimum time for observation of differentiated cells is between 23h to 25h. The Average is over the 3 experiments conducted simultaneously. Page 6
Figure 3: Extent of differentiation in cultured cells. Red colour shows the presence of secondary cell wall. 1.a,1.b,1.c: Control cells(uninduced) at 23h, 25h and 43h respectively. 2.a,2.b,2.c: Induced cells at 23h, 25h and 43h respectively. 3.a,3.b,3.c: Induced cells at 23h, 25h and 43h respectively. 4.a,4.b,4.c: Induced cells at 23h, 25h and 43h respectively. Page 7
Figure 4: The effect of different inhibitors on the differentiation rate. Values taken at 24h after induction. Figure 5: The effect of different inhibitors on the secondary cell wall pattern. Values taken at 24h after induction. The concentration of OA is in µm Page 8
Figure 6: The effect of Taxol and OA on the roots of the WT Arabidopsis plants grown in-vitro. Figure 7: The pattern of secondary cell walls in the roots of WT plants. a Control(MS only); b Protoxylem of plants grown in 2µM OA; c Metaxylem of plants grown in 2µM OA; d Protoxylem of plants grown in 10µM Taxol; e Metaxylem of plants grown in 10µM Taxol; Red colour shows the presence of secondary cell wall. Page 9
Figure 8: The effect of Calyculin A on the rate of differentiation at different concentrations. Figure 9: The localization of different proteins on the xylem cells. Green shows the presence of protein of interest. Red shows the presence of secondary cell wall. a 2051, b 2051, c 2129, d 2130, e 2131, f 2202, g 2404, h MAP70, i Tub6 Page 10