about 68 kDa, however in the active form it has 390 kDa. Hence it is an hexamere of 6 identical monomers. This enzyme catalyzes maltose to trehalose
and capable of interconversion with equilibrium state at 42-45 of each that can be reached at incubation for 6h with 0.5 mM maltose as the substrate. Higher
concentration reduced the conversion rate. When trehalose was used as substrate, 30 maltose was obtained after et least 12h incubation with 8-10 of
glucose was obtained. The optimum pH was 7 and experiment was conducted at 37
o
C. When maltose is used as substrate it has K
m
was 10 mM but he K
m
for trehalose was 90 mM.
The character of the enzyme TRES from Thermobifida fusca was also reported Wei et al. 2004 reported. It has a molecular weight of 66 kDa with
optimum condition of pH at 6.5 and temperature at 25
o
C. This enzyme converts matose into trehalose, and has capability to reverse back trehalose into maltose
at equilibrium state, about 60 trehalose concentration. At higher temperature 37
o
C, or when the enzyme concentration is high, the enzyme also diverts matose into glucose up to 15 of the substrate. Reaction mixture with15
maltose, it converts substrate about 55-60 at optimal condition. Heavy metal Cu
2-
, Mn
2-
and Zn
2-
at 5 mM did not inhibite its activity. The catalytic sites of this enzyme laid at 223-257 amino acids and 400-439 amino acids.
TreS is also found in Mycobacterium tuberculosis Murphy et al. 2005. In
this bacteria, there 3 pathways are present, and TreS is not the used major pathway compared to the two others. While Cardoso and Castro 2007 reported
2 pathways OtsA-OtsB and TreS found in Propionibacterium freudenreichii. This bacteria accumulated high level of trehalose especially in response to stresses
osmotic, oxidative and acid stresses. The first pathway is suggested to function in trehalose synthesis while the second is functioning as trehalose degradation.
2.7 Foreign genes encoding for Trehalose Synthase on plants
Trehalose producing organism tolerated desiccation and other stresses rises expectation that plants expressing trehalose synthase might increase plant
to stresses. Tobacco expressing yeast trehalose 6 phosphate synthase Tps improve drought tolerance, although multiple alteration of phenotype was shown
such as small, dark and lancet leaves Romero et al. 1997. This abnormality was in agreement with tobacco expressing OtsA E. coli reported by Goddijn et al
1997. While Smith et al. 1998 found that tobacco expressing bacterial
Trehalose 6 phosphate synthase OtsA and trehalose 6-phosphate phosphatase OtsB showed larger leaves and better growth on drought. Phenotypic alteration
of tobacco expressing trehalose synthase was corrected by expressing both genes of yeast Tps and Tpp and tolerant to drought was conserved Welin et al.
2001. One-step further was done by Garg et al. 2002 and Jang et al. 2003, they introduced fused of both genes OtsA-OtsB into rice, and the transgenic rice
was tolerant to drought, cold and salt with higher yield. They suggested further that the increase of trehalose migt not be enough to act as osmolytes, instead as
regulator of sugar sensing and expression of genes involve in carbon metabolism.
Unlike the previous report by Paul et al. 2001 suggested that the increase of photosynthetic capacity was modulated by T6P, while Garg et al. 2002
suggested that it was due to trehalose, since introducing fusion OtsA-OtsB low
T6P resulted in high photosynthetic capasity. Using the third pathway of trehalose synthesis genes proved to overcome high trehalose induced
morphological defect as well as increased the resistance of transgenic plant to drought and salt, such as tobacco and sugarcane bearing TSase from Grifola
frondosa Zhang et al. 2005; Zhang et al. 2006.
2.8 Trehalose induces starch biosynthesis in Arabidopsis thaliana
Starch is the main storage carbon in plant. In chloroplast starch is synthesized during the day as a temporary store, and it is degraded during the
night and exported to non-photosynthetic tissue. For long-term storage such as in tuberous plant, e.g. potato tuber, starch is resulted from sucrose conversion.
ADP-glucose pyrophosphorylase AGPase is the first enzyme in starch formation. Glucose-P and ATP is catalyzed to produce ADP-glucose and PPi.
ADP-glucose then is used to form starch by elongating glucan chain of starch granule.
ADP glucose phyrophosphorylase AGPase activation can be achieved via several steps in the pathway. First, it is regulated via alosteric inhibition, being
activated by 3PGA as activator and PPI as inhibitor. Its activity is regulated post translationaly by disulfide bridge formation between the two monomer of small
sub unit. When the sub unit is in monomer form, it is in reduced form. Conversely when it appears as dimmer form, it is oxidized. In potato tuber and pea leaves
chloroplast, activation thioredoxin f and m increase monomerisation of AGPase that lead to its activation by 3PGA Geigenberger et al. 2005. In leaves, AGP-
ase is regulated by light-dependent signal, by which transfer electron from photo system I to feredoxin via feredoxin: thioredoxin reductase to thioredoxin f and m
that leads to activation of the enzyme Balmer et al. 2006. AGPase also respond to sucrose level in cytosol. Trehalose has been proved to induce starch
accumulation by promoting reductive activation of AGPase in the plastid Kolbe et al
. 2005.
Figure 4. Starch breakdown in chloroplast and carbon export at night. Starch breakdown
and the fate of its derivates A, α
-amylase 1, disproportionating enzyme DPE1 2, glucose transporter 3, maltose transporter MEX1 4,
disproportionating enzyme 2 DPE2 5, Hexokinase 6 and phosphorylase 7. Cited from Chia et al. 2004. Enzyme involve in granule breakdown B.
Trehalose feeding increases T6P level within the tissue, which induces APL3 that encodes AGPase, the first enzyme of starch synthesis. Beside the induction,
T6P also reduces activation of AGPase through redox reaction. This deduced from the study of Kolbe et al. 2005, over expression of TPS1 that increase T6P
eccelerated the monomerization of AGPas the active form of AGPase. Conversely, over expression of TPP that converts T6P to trehalose reduced the
monomerization of AGPase. They further proved that adding T6P to isolated
A B
α -amylase
Branched oligosaccharides
Β -
amylase Β
- amylase
choloroplast resulted in AGPase activation. Yet when T6P was added to disrupted chloroplast, the enzyme was not activated. Addition of other sugars did
not activate the enzyme, but sucrose activated the enzyme in slowerlatter than T6P. They suggested further that T6P and not trehalose or other sugars senses
the sucrose status within cytosol to chloroplast. The increase of T6P that activates AGPase results in starch accumulation in
chloroplast. This leads to immobilization of metabolizable sugar from source to sink and caused growth arrest. Feeding 100 mM trehalose leads to the increase
of T6P level and further starch accumulation in chloroplast. When glucose is added together with trehalose, growth arrest is not shown Eastmond et al. 2004.
Starch immobilization, however, is not the only cause of growth arrest. T6P is a regulator of glucose, abcisic acid and stress signaling Avonce et al. 2004. In the
presence of metabolizable sugars, T6P down regulates ABI4, the gene encodes for enzyme involve in abcisic acid biosynthesis. Hence, growth arrest does not
appear. Conversely, in the absence of metabolizable sugars, T6P induced the expression of ABI4, hence diverse array of ABA dependent stress responses
occur Avonce et al. 2004.
Flow Chart of the Research Mycobacterium tuberculosis
Thermobifida fusca
Arabidopsis Seeds
Putative transgenic selection
Gene isolation – PCR
TfTreS
pGemT
pBIN1935S
Agrobacterium
Arabidopsis
Seeds
Putative transgenic selection
Check the presence of TfTreS
Inhetitability Chooped from pCR-
Blunt II -TOPO
MtTreS
pGemT
pBIN1935S
Agrobacterium
Preliminary test TreS – as an alternative
Selectable marker Characterization of
TreS expressing lines
No transgenic obtained
TRES - SELECTABLE MARKER TRES EXPRESSING LINES
CHARACTERIZATION
Kill curve of Wt plants of Trehalose and
Validamycin A
Grown TfTreS plant on Trehalose and
Validamycin A •
Leaf water retention test
• Leaf water
recovery test •
Drought resistance test in
planta •
Trehalose content •
Enzyme activity level
III. CLONING TREHALOSE SYNTHASE GENE from Thermobifida fusca and Mycobacterium tuberculosis
3.1 Introduction
