3.27 4.88 CHARACTERIZATION OF TFTRES EXPRESSING PLANTS

Table 8: Observed characters on leaves after detachment lines TfTreS expressing plants Wild type plants Ctre Eact LWR TotWL DW Ctre Eact LWR TotWL DW 1 0.00 42.06 1.73 90.61 9.39 1.72 0.37 1.49 91.03 8.97 2 0.00 1.21 11.11 90.42 9.58 20.56 0.34 0.37 92.39 7.61 3 0.00 0.00 18.92 91.82 8.18 11.39 1.33 0.00 90.60 9.4 4 15.85 37.75 0.96 90.26 9.74 85.44 0.00 8.43 91.71 8.29 5 0.00 0.72 1.18 90.02 9.98 0.93 0.00 0.00 91.79 8.21 6 2.71 34.55 22.63 91.20 8.8 2.71 0.12 0.00 91.31 8.69 Average 3.09 9.42 19.38 90.72 9.28 20.46 0.36 1.72 91.47 8.53 STDEV 6.34 9.66 20.67 0.67 0.67 32.72 0.50 3.34 0.63 0.63 Note: CTre = Trehalose concentration, Eact = Enzyme activity, LWR = leaf water retention, TotWL = Total water loss and DW = Dry weight. Figure 26. Cluster Diagram of Detached Leaves Based on Characters shown Table 8. In this analysis 6 data of transgenic plants and 6 data of Wt plants were used Table 8, and the result is presented in Figure 26. Annotation with number 1-6 was TfTreS plants and 6-12 was the Wt plants. The Figure tells that the second most distance between the samples used about 30 similarity was achieved from sample 3 and 6 on one hand to samples no. 8 and 10 on other hand. While the rest of the samples seem to make a group but they did not mix indicating that there is not much different among them based on the given characters but there was a tendency to be different. Therefore, it suggested that among 6 TfTreS plants 2 plants might withstand drought. Using the third most distance based on the characters, other 2 plants 1 and 4 are considered as moderately withstand drought, while the rest 2 and 5 are not different from Wt in term of plant drought tolerance. Leaf Recovery after 16h incubation at room temperature RT Leaf recovery test might also indicate the capability of plant to withstand drought. Leaves that has been detached and dehydrated for 21h at RT were rehydrated saturatedly. The result showed that none of wild type leaves 5 leaves recovered from severe wilt; they remained very dull, dark in color, and were not rehydrated. One leaves only recovered and another one partly recovered among 15 TfTreS expressing leaves tested. The result suggests that 21h exposure to RT after detachment might be over exposure. For this reason, experiment was repeated, with the same number of samples of the same plants but the leaves were bared to RT for 16h, followed by rehydrating saturatedly. The result is presented in the Figure 27. It was observed that all the Wt plants were Figure 27. Leaf Recovery Test of dehydrated leaves. Fresh leaves A, leaves dehydrated for 16h and the WT is indicated B and leaves after 4h rehydration C, Wt leaves are also indicated. A B C WT TfTreS Wt Shrinkage, while some TfTreS expressing lines were less shrinkage than the Wt. Two and one leaves of TfTreS expressing lines recovered after 1h and 2h after rehydration respectively. There were six TfTreS expressing leaves showed the possibility to recover after rehydration, yet the recovering process was not ended with full rehydration. Three of TfTreS leaves recovered from drought possessed high trehalose content or enzyme activity Figure 28. Such mechanisms discussed at the end of this chapter might implicate to the phenomenon. Garg et al 2002 suggested that the increase of transgenic rice tolerance to drought, salt and cold that bear fusion trehalose synthesizing genes OtsA-OtsB from E. coli is due to the increased in soluble carbohydrate and photosynthetic activity, since the increase of trehalose level in the transgenic was too little to act as an osmo-protecting agent. They suggested further that the compound is functioning as sugar signaling and controlling carbohydrate allocation. Jang et al. 2003, suggested that trehalose is functioning as global protection against abiotic stresses. In agreement with these, Bray 2002 reported that there were 130 genes are up Figure 28. The trehalose content enzyme activity of recovered leaves from drought. Leaves were detached from the plant and kept at room temperature for 16h, followed by rehydration. Recovery occurred after 1-2h after rehydration. 101-m CT : 180 µmol EAct : 8 µmol h -1 CT : 434 µmol EAct : 276 µmol h -1 103-12 Wt CT average: 21 µmol EAct average : 2 µmol h -1 TfTreS CT average : 48 µmol EAct average : 28 µmol h -1 -1 CT : 9 µmol EAct : 10 µmol h -1 101-3 regulated in Arabidopsis thaliana in respond to water stress, those are involved in signaling evets, detoxification and celluler responses to specific water deficit. The capability of leaves to recover should relate with the integrity of the biological compounds within cells. In term of drought stress, membrane is the primary site of damage and membrane is intolerant to drought Crowe and Clegg, 1978; Leopold, 1986. Desiccation caused changes of structural damage such as removal of integral membrane protein, transition phase from fluid to gel phase or to hexagonal non-bilayers phase Crowe and Crowe, 1982; Anonym 2004 as illustrated in Figure 1. These are because; membrane bilayers are composed from many different lipids as well as proteins with different composition at any part of the membrane. Each compound of the membrane has their own transition phase at different temperature and different water status that would lead to membrane separation, membrane diffusion as well as membrane tiring off when dehydration occur at the same time with the same condition. Additionally, these also due to hydration force suction negative force that reduced membrane surface hence, changes of transition phase Bryant et al. 2001. The phospholipids chains become rigid which lead to the damage at rehydration and thawing state Crowe et al. 1990. Lypolitic genes that degrade membrane is also activated by drought, reducing lipid content especially the hydrophilic part in Arabidopsis thaliana Gigon et al. 2004. Taken together, drought lead to irreversible damage to membrane and cell wall Mundree et al. 2002, and protecting membrane component from drought is likely feature desiccation tolerance Crowe and Clegg, 1978; Leopold, 1986. Some theories were suggested e.g. by Crowe et al. 1987 in respond trehalose as stress protecting agent: trehalose as water replacement, water entrapment and vitrifying agent. The fact that some TfTreS expressing leaves performed better on drought physically as well as their water retention, hence, in this case trehalose may act as water entrapment. In agreement with the above mention, trehalose that tend to interact with water and concentrate it to surrounding biological molecules, there by maintaining salvation and native structures Sun et al 1996. Further more, Bordet et al. 2000 suggested that trehalose has tendency to interact with biological molecules at phosphate group laid between the head and the tail keeps the membrane fluidity Crowe et al. 1987; Rudolf et al. 1990, and adsorb water as well as reduce water molecule dynamic that is important for bio molecule stabilization. It has been reported that there is an interaction simulation between trehalose and membrane bilayers Pirera et al 2004. During the absence of water, trehalose is able to form energetically stable conformations bridging with a number of lipid molecules Luzardo et al. 2000, and the bridging reduce water molecule dynamic that is important for biological compound stabilization Bordet et al. 2004. Cottone et al. 2001 reported that a protein, carboxy myoglobin embedded in trehalose water matrix by which its molecular dynamic is reduced with even at elevated temperature. Water replacement, however, might as well occurred as revealed by Luzardo et al. 2000 suggested that trehalose also interact with lipid at carbonyl groups, and 11 of 14 water molecules could be substituted by 3 molecules of trehalose. While Bryant et al. 2001 suggested that solutes e.g. trehalose increase the osmotic pressure within cell, consequently reduced water removal from the cell. Potential cell is a sum of potential pressure, potential matrix, and potential osmotic. Potential pressure and potential matrix should be the same between control plants and TfTreS expressing plants. The presence of trehalose might increase the potential osmotic within the cell; hence, water removal is reduced. On wheat El-Bashiti 2003, the sensitive cultivar to stress has lower level of trehalose than the resistant cultivars either in normal condition or in drought and salt. Leaves that recovered from drought might happen through mechanism suggested above. Coincidently, 2 of 3 recovered from drought contained trehalose content and showed higher enzyme activity than that of control leaves. One, however, was only moderate in trehalose and enzyme activity level. In this situation, it might need to measure the trehalose and enzyme activity at interval of time during the time course of leaf incubation. More samples should be needed and it was not possible due to some constrain, such as number of sample limitation. While those that did not recovered might be due to unpreventable leakiness of the membrane, and when membrane is disrupted, major physiological processes are abolished, hence they were not able to recover after rehydration. The average of leaf dried weight of TfTreS expressing leaves was higher than that of Wt leaves. Although it was very light differences, however, it is statistically significant. This is paired with rice transgenic bearing OtsA-OtsB that produced more yield than the control plant due to the increase photosynthetic capacity as well as photosynthetic apparatus Garg et al. 2002; Jang et al. 2003. The capability of TfTreS expressing plants to slow down of water lost may indicate that the plant might withstand drought giving the possibility to obtain lines tolerant to drought by introducing TfTreS gene. Hence, parameters of drought tolerane observed on levaes suggested that it possible to obtain transgenic plants bering trehalose synthase gene from T. fusca that tolerant to grought might as well improved dry matter. TfTreS expressing lines in response to drought in planta TfTreS expressing lines were tested to see their capability to face drought. A week old seedlings resistant to 50 mgl kanamycin were transferred to ½ MS medium and kept for a week to eliminate the effect of kanamycin. The seedlings then were transferred to potting mix. When established growth was achieved, drought treatment was applied by withholding water. Observation was made at day 11 th after treatment and day 2 nd after rehydration. Result showed that growth at day 11 th all 3 wild type plants were severely wilted. Four and 9 plants over 38 transgenic plants of clone 816 and 918 respectively, were remained fresh. At day 12 th only one and 4 plants of clone 816 and clone 918 remain fresh respectively. None of clone 816 was able to withstand on day 13 th without irrigation, and only 2 plants of clone 918 remained fresh. At this point, water was given, and 2 and 9 plants of clone 816 and 918 respectively showed recovery from wilted after 24h rehydration. After 48 h rehydration, clone 816 did not increase the number of recovered plants remained 2 as before but some were started to recover, while clone 916 there were 12 plants had recovered from stress. Although this result is preliminary, since the wild type involved in the experiment were only three plants, however, it is interesting to know why clone 916 that has lower frequency of resistant plants on trehalose than that of clone 816, showed higher frequency of plants that withstand on drought. Both clones bear TfTreS expressing have 2 same site of point mutations at the 27 th base after the first base of start codon, from GCG to GCC, both codons encode the same amino acid, alanine; and at the site of 1406 from GCT to GCC, in which both codons also encode for alanine. Clone 918, however, beside possessed those two point mutations as clone 816, another point mutation occurred at 1257, from GGA to GGG, and both codons encode for glycine. Hence, these point mutations did not alter the amino acids; that might not change the properties of the protein. The codon usage comparison in Arabidopsis thaliana showed that the first point mutation increased slightly from 6-8.1, the second was drastically reduced from 31.8-8.5 and the last was increased from 8.1-20.2. Referring to assessing of the clones on trehalose and validamycin A, clone 918 showed more vigorous growth than clone 819. Hence, it seemed that 918 plants have stronger activity to combat stress than 816 plants. However, whether the different stringency of resistance was related with the mutation, thorough assessments are needed. Drought treatment produced some TfTreS expressing plants about 36 withstand drought and able to recover from severely wilted after rehydration. While all three of Wt plants did not survive on drought. This result needs further confirmation since the wild type plants that were involved were only three plants. It was done due to space limitation in growth chamber and it was assumed that biologically the wild type is less variable than the mutants are. Fresh and dry weight of recover plants need to be measured to clarify the presence of TfTreS on plant in response to drought. For this reason, second experiment was set up using T3 plants. Withholding water for a week was applied to 23 TfTreS expressing plants and 9 plants of Wild type. As a control, 17 TfTreS expressing plants and 5 Wt plants were watered normally non-stressesd as the control. Figure 29. TfTreS lines against drought or salt. Wild type was very wilted at day 13 A, some lines of 816 B and of 918 C were remained fresh. Two days after rehydration some of lines 918 D and 816 E showed more vigorous growth. A C B D E The number of recovered plants at the 4 th h and 24 th h after rehydration were recorded. Fresh weight were measured 2 weeks after rewatering to give a chance of those experienced to drought to establish their growth Figure 30. Among the Wt 9 plants that were subjected to drought, 2 plants were able to recover 4h after rehydration and 3 showed small green tips that seemed to be possible to recover 24h after rehydration. While all the rest died. Among 23 of transgenic plants, 8 plants were recover after 4h re-watering and 3 plants showed small green shoot tips after 24h Figure 30. Finally, however, those that showed small green tips were dead. Hence, only 2 22 Wt plant and 8 35 of TfTreS expressing plants develop their growth. It is interesting to note that the recovery of Wt plants occured at the meristematic tissue, all the developed leaves were dried and were unable to recover. While in the transgenic plants, process of recovery occurred not only in the meristematic tissue but also on developed leaves. After established growth was achieved, those experienced to drought showed a normal and healthy growth. Growth of the Wt plants experienced to drought were smaller then the TfTreS expressing lines. However, due to only 2 plants were survived, this observation needs further verification. Figure 30. Drought test of TfTreS expressing lines. Plant condition before test is shown A, plants after one week without irigation B and plants 4h after rehydration C. A C B B WT TfTreS Among TfTreS expressing plants recovered from drought, 50 contained trehalose at different level. The lowest was 17 µmol gr -1 FW and the highest was 434 µmol gr -1 FW 156 mg: out layer, it is not included in statistical analyses. Better situation shown by their enzyme activity, there were 75 of them showed the enzyme activity started from 2 µmol h -1 gr -1 FW the lowest and 275 µmol h -1 gr -1 FW of the highest. There was no report has been made about the presence of trehalose synthase encoded by TfTreS on plant. TSase, the genes involve in trehalose biosynthesis of the second pathway do not produce T6P from Grifola frondosa Figure 31. Plant recover from drought.. Watered wild type plants A, Watered Transgenic plants B, drought recovery plants of TfTreS Lines C and drought recovery plants of wild type D. has been introduced to tobacco Zhang et al 2005 and to sugarcane Zhang et al 2006 suggested that the presence of the gene improve transgenic tolerance to drought, salt and cold. Trehalose synthase from the first pathway of trehalose syntheses fused gene of OtsA-OtsB from E. coli that was expressed A B C D Figure 32. Fresh weight and dry weigh average of plants recovered from drought In Vivo of TfTreS expressing Lines. Drought means group of plant that were drought experienced. Watered means group of plants that were watered well. Scattered data of inflorescent FW A, the average of inflorescent FW B, and the average of inflorescent DW are presente d. in rice increased rice tolerance to drought Garg et al. 2002; Jang et al. 2003. From this experiment, there are some indications of the possibility to obtain transgenic plant with improved lines in responds to drought. The most dangerous of drought is the membrane disruption during desiccated and rehydration Scatered data of fresh weight draught tested plants 200 400 600 800 1000 1200 1400 5 10 15 Member of group Fresh weight mg Wt Dr TreS Dr Wt Ww TreS Ww 20 8 ± 68 25 7± 5 3 53 9 ± 13 6 64 4 ± 35 100 200 300 400 500 600 700 mgplant Draught Watered Fresh weight of draught tested plants WT TreS 14.8 ± 1.6 14.1 ± 1.5 12.41 ± 0.23 12.26 ± 1.4 5 10 15 ww dry Dry weight of inflorescent drought tested plantsts TreS Wt state that in turn to lead to membrane leaking, membrane fusion and membrane separation. T rehalose keeps native structure of biological compound that minimized dangerous effects of drought to membrane McDonald and Johari 2000; Pereira 2004; Hincha et al. 2004; Patist et al. 2005; Chen et al. 2001; Bryant et al. 2001. In this experiment, TfTreS expressing plants showed higher enzyme activity, and 35 of the tested plants instead of 22 of Wt plants, recovered from drought. Therefore, it might be concluded that there is an indication of the possibility to obtain transgenic plants withstand drought.

6.4 Conclusion

From the result and discussion, it is suggested that it might be possible 17 to obtain transgenic Arabidopsis bearing TfTreS gene with high level of trehalose content 62-180 µ M gr -1 FW. TRES activity of transgenic Arabidopsis was higher than the Wt, it magnified up to about 25 Fold of the average control. Excised leaf water loss of the transgenic plants was significantly different at the first 30 minutes and at longer than 21h up to constant dry weight. The total water loss of the transgenic was also smaller than the Wt excised leaves, while the Dry Weight was significantly higher than the Wt. Excised leaf recovery after drought was observed that 20 3 of 15 leaves were able to recover and none was observed on Wt. Taking together, cluster analysis based on those parameters subjected to excised leaves, suggests hat there is possibility 33 to obtained transgenic plants resistant to drought. Drought test in planta using both clone 918 and 816 indicated that the number of recovered plants 30 of total plants tested was higher than clone of 816 5 at P = 0.003 Appendix 9. The second experiment using clone 916 revealed the same result 30 recovery while the Wt 22. Hence from this study it is concluded that there is a possibility about 30 to obtain transgenic plant which withstand drought by introducing gene encoding trehalose synthase from Thermobifida fusca.

VII. GENERAL DISCUSSION

This study was conducted in the line of crop improvement. The demand of food supply is increasing along with the growth of human population, and where inversely, the quality of the environment is declining. Water, in particular, is becoming scarce, salt concentrations along with other pollutants are increasing at a time when crop production will be expanded to drier regions to produce biomass for food, energy or other purposes. Trehalose is well-known stress protecting sugar found to accumulate in organisms exposed to stressful conditions. Introducing genes encode trehalose metabolizing enzymes has been one of research interest. Plethoric phenotype is resulted from the accumulation of T6P when OtsA and or OtsB; TPS1 and or TPP are introduced to plant, e.g. on tobacco Goddijn et al. 1997: Pilon-Smith et al. 1998, and Welin et al. 2001. Introducing bifunctional fusion of E. coli OtsA-OtsB same as TPS1-TPP overcome the problem as reported by Garg et al. 2002 and Jang et al. 2003, since T6P produced by OtsA is directly converted to trehalose. This study assessed an alternative of gene encoding trehalose metabolizing enzyme for such purpose. This pathway involves a single gene only to produce trehalose from maltose without producing T6P, hoping that unexpected phenotypical defect might be prevented. TRES as trehalose synthesizing at the same time as trehalose degrading enzyme might be beneficial to the transgenic plant, by which metabolizable sugar is still available even if trehalose level reach the equilibrium. This proved with the capability of transgenic plants survive on high level trehalose eg 125 mM. As it shown in Chapter 6, that Arabidopsis bearing TreS grow normally and fertile. The plants were resistant to trehalose that might be as one of indication withstands stresses including in respone to trehalose-producing pathogens. Research has now shown that metabolism of trehalose in plants is critical to engineer drought stress resistant plants. It is clear that the presence of trehalose synthesizing gene improve plant responses toward stresses Garg et al 2002; Jang et al 2003; Zhang et al 2005; Zhang et al 2006. In this experiment using Arabidopsis in planta by with holding water showed that 30 of transgenic plants recover from drought stress and grow normal after rehydrated. Excised leaf tests with parameters of leaf water retention, leaf total water lost, leaf dry weight; and inflorescent trehalose content and inflorescent TRES activity