GENERAL DISCUSSION Cloning Trehalose Synthase Gene And Its Expression In Arabidopsis thaliana
followed by a cluster analysis based on the characters suggests that 33 of the transgenic plants resistant to drought. From leaf water recovery test, it was
observed 20 of leaves recover after 16h keeping on RT. Hence it might be suggested that there is possibility about 30 to obtain transgenic plant with
improved drought response by introducing trehalose synthase gene from Thermobifida fusca
. It was suggested that the improved response toward stress of
transgenic plant bearing trehalose metabolizing genes was due to the increase of soluble carbohydrate as result of the increase of photosynthetic
capacity Garg et al 2002. While Jang et al 2003 suggested, trehalose give global protection against abiotic stresses, yet, they did not suggest the
mechanism. While Avonce et al. 2004 suggested that AtTPS1 is regulator of glucose, abcisic acid, and stress signaling. In this experiment, the capability of
some TfTreS expressing lines withstand drought might also has been implicated with the increase of T6P. Arabidopsis expressing AtTPS1 is sugar and ABA
insensitive, and T6P regulate the expression of ABI4. ABI4 is down regulated by T6P when glucose is presence and is up regulated in the absence of
metabolizable sugar. T6P affect growth and development Schluepmann et al 2004, mutant of AtTPS1 is embryo lethal. Hence, trehalose metabolism is
indispensable for growth Easmond et la. 2002; Schluepmann et al. 2004. This effect on growth was also shown on M. smegmatis Woodruff et al 2007. The
presence of T6P, however, leads to induction of APL3 hence starch is accumulated in chloroplast and growth is arrested. Feeding trehalose to
seedlings increased T6P level Schluepmann et al. 2004. Expressing TfTreS that increase trehalose level, might increase increase T6P level on plant in some
extend, as starch accumulation was proved occurred to some lines Figure 33. The increase, of T6P however, did not retard growth that might be due to the
reverse TfTreS activity that still provides sugar for energy source. Furthermore, the presence of the sugar down regulates ABI4, hence ABA production is
reduced, and growth retardation is rendered Avonce et al. 2004. In more general about the linking of trehalose and ABA is suggested by
Brodmann 2006. He revealed that application of 25 mM trehalosevalidamycin incresease or decrease the expression of 2277 genes, and more than a third of
them are regulated by ABA, linking trehalose to ABA or ABA signaling. Interestigly, on ABA deficient mutant abi-1 the application of trehalose and validamycin affect
ABA inducible genes, indicating that the induction of Trehalosevalisdamycin A is independent from ABA. However, some the induced genes and ABA induced
genes were sppressed in mutant abi2-1, suggesting that signaling cascade triggered by trehalosevalidamycin affect the ABA sigbaling pathway downstream
of the ABA binding site but upstream of ABI2 activity. He suggested further that those genes that are regulated by trehalosevalidamycin but not by ABA are those
involve in disease resistant and secondary metabolites, hence trehalose act also as an elicitor in plant.
Figure 33. Starch staining of seedlings grown on trehalose or ½ MS. TfTreS expressing
leave grown on 12 MS A and its magnification C or on trehalose B and its magnification D. Leave of sensitive as Wt grown on 12MS E and its
magnification G or on trehalose F, and its agnification H. Some TfTreS expressing plants accumulated starch I.
It is very recently reported new role of trehalose that controls the dynamic mRNA of
heat shock protein Hsf1 by activating transcriptional activity. This acceleration and the presence of trehalose enhance phosphorylation of the protein Collin and
C
A B
D
E D
C
F G
H C
D
I J
I G
H
Nelson 2007. This report support previous suggestions that trehalose prevent protein denaturation and agregation of denaturated protein as heat shock protein
involves in molecular chaperon that refold protein back to natural structure. TfTreS
plants resistance to trehalose open a question about the possibility of the used of TfTreS as selectable marker on trehalose. As earlier discussed
there are several non toxic selectable marker has been developed, to meet the public concern about bio safety of the use antibioticherbicide resistance genes.
Method to engineer marker free transgenic plants is also developing, however, analog to Darbani et al. 2007 suggesting that one of the methods to produce
free marker transgenic plant is by using non toxic selectable marker. Selection TfTreS
on trehalose as well as on a combination of trehalose and validamycin A, may obtain marker free transgenic plants. The combination is stronger selection
system, since validamycin A is a trehalase inhibitor that keeps trehalose remain un-degraded and increases trehalose level within tissue Godijn et al. 1997;
Muller et al. 2001. As the selection system gave transgenic lines, hence, both selection system can be used with considerable amount of transgenic plants
obtained. The combination, however, gave fewer lines than when trehalose alone is used as the selection agent. None of the wild type plants survived either on
trehalose alone 100 mM or on the combination 10 µ
M validamycin A and 50 mM or more trehalose. These lead to the conclusion that TreS can be used as a
selectable marker. The use of the gene as an alternative selectable marker might be favorable to the transgenic obtained for some reasons. First, about 30 of
transgenic plants might withstand stresses, 2 the product of the gene, trehalose, is biologically friendly and has been used widely for many aspects of human life
and 3 the selection agent is harmless sugar to the environment. The validamycin A as the waste after selection is biodegradable with half life less than 5h in soil
Sticher et al. 1997.