Functional Analysis of the Elaeis oleifera Sesquiterpene SynthasePromoter Reveals Non-Specific Tissue Expression and Activation under Stress Condition
Norazreen Abdul Rahman,
Chan Kok Fei,
Nik Marzuki Sidik
Che Radziah Che Mohd Zain
This research aimed to evaluate the specificity of sesquiterpene synthase promoter (SesqPro) activity in the oil palm tissues and tomato hairy roots and to determine the functional region of the promoter. The effect of jasmonic acid (JA) on the promoter activation and gene expression was also analyzed. A series of 5’ sequence deletions on the full-length SesqPro were generated and individually cloned into the pCAMBIA 1301 vector. Functional analysis was carried out on leaves, mesocarp slices and Immature Embryos (IE) of oil palm and tomato hairy roots that had been transformed with full-length SesqPro (PSPr-VF6). GUS expression was found in all the tissues and a higher activity was detected in IE and mesocarp slices. All the constructed derivatives of SesqPro were transformed into IE and mesocarp slices in order to determine the promoter regions which are responsible for gene expression. The reduction of GUS activity was found to be related to the removal of DNA sequences within the promoter region. The promoter was induced by the elicitor molecule JA, thus suggesting the presence of JA responsive elements within the promoter. Incubation with 100 μM of JA showed higher GUS activity in IE and mesocarp slices that had been transformed with PSPr-VF4 to PSPr-VF6. Nevertheless, the GUS activity was drastically reduced in IE and mesocarp slices containing the PSPr-VF3 promoter, suggesting that the presence of the G/A hybrid box located at -622 to -617 act as a specific element in response to elicitors. This study has shown that the action of SesqPro is non-specific and was influenced by JA induction.
to cite this article:
Ismanizan Ismail, Norazreen Abdul Rahman, Chan Kok Fei, Zamri Zainal, Nik Marzuki Sidik and Che Radziah Che Mohd Zain, 2009. Functional Analysis of the Elaeis oleifera Sesquiterpene SynthasePromoter Reveals Non-Specific Tissue Expression and Activation under Stress Condition. American Journal of Plant Physiology, 4: 24-37.
Plants produce a variety of secondary metabolites which are not specifically
used for plant growth and development and instead play an important role in
ecological functions for communication and defense. Terpenoids contribute to
multiple plant biochemical functions, such as the formation of quinine; the
electron transport chain, as components of membranes (prenillipid in archaeabacteria
and sterol in eubacteria and eukaryotes); regulation and subcellular targeting
(protein prenilation); photosynthesis, as a pigment component (carotenoids,
chlorophyll side chains); hormones (gibberellins, brassinosteroid, abscisic
acid); and as plant defense compounds (monoterpene, sesquiterpene, diterpene)
(Lange et al., 2001). Terpenoid biosynthesis
occurs in two kinds of metabolic pathways: the mevalonate pathway and the non-mevalonate
pathways. Isopentenyl diphosphate (IPP) is produced indirectly via the mevalonate
pathway that accumulates in the cytoplasm whereas dimethyl diphosphate is an
IPP isomer which accumulates inside the plastid via a non-mevalonate pathway.
Interestingly, sesquiterpene is only found in plants, microbes and insects
because this compound plays an important role as the intermediate for the interaction
between organisms and their environment (Kessler and Baldwin,
2001). New research has shown that sesquiterpene is important as a chemo-preventive
agent in colon and skin cancers (Picaud et al., 2006).
The primary step in sesquiterpene biosynthesis involves a group of enzymes which
are known as sesquiterpene synthases. Sesquiterpene synthases are a protein
family which are expressed ubiquitously and are able to convert the acyclic
universal precursor farnesyl diphosphate (FPP) into more than 300 different
sesquiterpene skeletons (Picaud et al., 2006).
A sesquiterpene synthase will catalyze the conversion of sesquiterpene compounds
in various plant species according to the demand and the environment. For instance,
an acyclic sesquiterpenoid such as farnesene and farnesol is produced by (E)-β-farnesene
synthase, whereas bisabolene synthase will catalyze the conversion of bisabolene
sesquiterpenoid. Germacrene and aristolechene sesquiterpenoids are catalyzed
by germacrene synthase and aristolechene synthase, respectively. Germacrene
synthase consists of a few isomers that exist naturally and are known as germacrene
A, B, C and D. The conserved amino acid sequences, intron organization and exon
size, which are almost similar in more than three dozen monoterpene, diterpenes
and sesquiterpene synthases from plants, suggest that these synthases come from
the same evolutionary origin (Trapp and Croteau, 2001).
Consequently, physical-based homology bioinformatics screening methods had successfully
been used to identify new plant terpene synthase genes. Nevertheless, there
are significant differences in the primary structures of the sesquiterpene synthases
of plants and microbes, while the crystal structure of four sesquiterpene synthases/cyclases
from different biological ancestors shows that it is highly conserved in its
three dimensional structure (Caruthers et al., 2000).
There are many studies on the uniqueness and diversity of sesquiterpene synthase,
including its genetic organization, gene regulation, biochemical properties,
phylogenetics and phytochemical properties. The primary study on sesquiterpene
synthase was conducted on aristolechene synthase and epi-aristolochene
synthase from fungi (Penicillium roqueforti) and Nicotiana tabaccum
respectively (Proctor and Hohn, 1993). Earlier molecular
biology studies on sesquiterpene synthase only involved cDNA clone characterization
which encodes for enzyme activities in certain sesquiterpene biosyntheses (Facchini
and Chappell, 1992) and its role in plant genomes. All sesquiterpene synthases
from plants will be channeled into the cytoplasm (Back and
Chappell, 1995; Chen et al., 1996) because
there is no specific plastid sequence (Chappell, 1995).
Kappers et al. (2005) has proven that cytoplasm
sesquiterpene could also be produced in mitochondria due to the availability
of farnesyl diphosphate.
Induced sesquiterpenes can also form secondary chemical defenses towards herbivorous
insects and pathogen fungi which differ from the primary defense compounds that
were produced from induced monoterpenes (Bohlmann et al.,
1998). Unlike the relationship between sesquiterpene and antimicrobe activities,
this compound is also involved in plant reproductive systems by protecting against
any attacks of insects, bacteria and fungi in vivo. In Arabidopsis,
an exclusively expressed sesquiterpene synthase is found in its flower which
catalyzes a wide variety of sesquiterpenoids in that organ (Dorothea
et al., 2005). Zea mays also produces some volatile sesquiterpenoids
when it is attacked by Spodoptera littoralis and herbivores (Schnee
et al., 2002).
There are also many studies conducted on plant responses to pathogens and abiotic
factors involving the accumulation of antimicrobes phytoalexins. Similar to
phytoalexin classes in other plant families, sesquiterpene phytoalexins could
not be detected in healthy or control plants and instead are accumulated in
response to elicitors or pathogens (Yin et al., 1997).
Phytoalexins will only accumulate in cell cultures of tobacco cells (Nicotiana
tabaccum) and Capsicum annum when these cells are being challenged
with elicitors from pathogens such as cellulose, fungi cell wall hydrolysates
and cryptogein, which is an extracellular protein from Phytophthora cryptogea
(Milat et al., 1991). Facchini
and Chappell (1992) had reported that there are 12-15 copies of sesquiterpene
synthase found in the tobacco genome. It is also possible that this gene exists
in a complex of genes which are controlled differently during development or
are regulated in response to the environment.
Sesquiterpene synthase of E. oleifera has been previously isolated (Cha,
2001) and its gene expression is very unique because it is expressed continuously
in specific mesocarp tissues. Thus, the aims of the work was to study the promoter
specificity and strength as well as to recognize which elements are essential
for the regulation of sesquiterpene synthase gene expression. The construction
of the promoter deletion series, its transformation into oil palm tissues and
tomato hairy roots and its subsequent treatment with JA as reported in this
paper will help to identify the promoter region involved in the activation of
the sesquiterpene synthase gene towards this elicitor.
MATERIALS AND METHODS
Plant Material and Growth Condition
The experiments were carried at the Plant Biotechnology Center, Institute
of System Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.
The IE and mesocarp slices used in this experiment were taken from oil palm
fruits aged between 12-15 WAA (week after anthesis) and zero frond leaves of
oil palms (unopened). Oil palm tissues were pre-cultured on an N6
OPS medium (Chu et al., 1975) at 28°C in the
dark for 7-12 days before being subjected to transformation. Tomato seeds were
cultured on an MS medium (Murashige and Skoog, 1962) for
14 days at 28°C under a photoperiod of 16:8 h (light:dark) to harvest the
cotyledons before being using these in A. tumefaciens- and A.
Generation of Promoter Deletion-GUS Constructs
The pCAMBIA-Sesq-Pro (PSP-VF) plasmids each containing the full length and
deleted sesquirtepene promoters were extracted from Escherichia coli
strain DH5α as described by Birnboim and Doly (1979).
Each of these plasmids were then digested with NcoI dan HindIII to remove the
35S CaMV promoter sequence. The resulting PSP-VF plasmid constructs which now
lack the 35S CaMV promoter then were purified using a Qiagen gel purification
kit. The fragments were made blunt-ended using Klenow enzymes and were purified
by phenol:chloroform extraction. The linearized plasmids were then ligated at
6°C overnight and transformed into E. coli strain DH5α where
50 mg L-1 of kanamycin was used as the selection antibiotic. These
new promoter constructs were designated as PSPr-VF1 to PSPr-VF6 with varying
lengths of sesquiterpene synthase promoter sequences as shown in Fig.
1. The analysis of the series of promoter deletions was carried out by confirming
the size of each construct using PCR with specific primers and digestion with
EcoRI and PstI (data not shown).
Plant Transformation and Growth Condition
The serial deletions of promoter/GUS constructs were introduced into A.
tumefaciens strain LBA 4404 and A. rhizogenes strain A4 and
subsequently were allowed to grow on LB-Glucose (5 mM) supplemented with kanamycin
(50 mg L-1) and streptomycin (100 mg L-1) and LB-Glucose
(5 mM) supplemented with kanamycin (50 mg L-1), streptomycin (500
mg L-1) and rifampicin (100 mg L-1), respectively. Both
strains were cultured for 2 days at 28°C and were then recultured to reach
an OD600 reading of 0.50.
For the transformation of tomato seedlings using A. rhizogenes, the
cotyledons were cut into 1 cm2 pieces and were then pre-cultured
for 2 days. Subsequently, they were incubated into a solution containing an
A. rhizogenes culture for 30 min and shaken gently before being transferred
to a sterile filter paper to remove excess bacteria.
||Restriction map of constructs series (pCAMBIA1301, SesqPro, r-EcoRI recombinant
and VF1-6 represent insertions of deletion derivatives). SesqPro derivatives
were cloned at EcoRI and SalI sites in the pCAMBIA 1301 vector. Arrows
indicate the orientation of the promoter. Restriction enzyme SalI, PstI,
SphI sites are located downstream of the promoter. The size for deletion
derivatives of SesqPro are indicated in the map. SesqPro contains one site
of BglII at position +11. The underlined number represents the actual size
of the deletion derivatives of the SesqPro sequence
After 2 days of co-cultivation at 28°C in the dark, the infected explants
were transferred to an MS medium supplemented with 400 mg L-1 of
cefotaxime (to inhibit bacterial growth). The transformed explants were
incubated at 28°C with a photoperiod of 18:6 h (light:dark). Hairy roots
that emerged from the cotyledons were subjected to selection by being transferring
onto a new MS medium supplemented with 100 μg mL-1 of kanamycin.
The pre-cultured oil palm tissues (IE, mesocarp slices, leaf slices) were transformed
using A. tumefaciens and were shaken gently for 30 min in an N6
6 medium (Chu et al., 1975) before being
transferred to a sterile filter paper to remove excess bacteria for a
3 day co-cultivation in the dark at 28°C. The transformed tissues were then
transferred to an N6 OPS medium and cultured at 28°C in the dark.
These tissues were used for transient analysis after 10-15 days of culturing.
Treatment with Exogenous Jasmonic Acid
To determine the promoter strength under environmental stress, the transformed
oil palm (mesocarp slices and IE) tissues were treated with 100 μM JA overnight
in an N60 (modified Chu et al., 1975)
medium at 28°C. The treated tissues were harvested and analyzed using GUS
histochemical and fluorometric assays.
GUS histochemical assays were performed 2 weeks after co-cultivation according
to the method described by Jefferson et al. (1987).
Transgenic explants were incubated in the GUS reaction buffer at 37°C for
18-24 h. To remove chlorophylls and pigments, the explants were treated with
formalin-aceto-alcohol (FAA) solution (42.5% ethanol, 42.5% glacial acetic acid,
8.5% formalin). GUS expression was studied under a Nikon light stereomicroscope.
For fluorometric assay, FluoroAce (BioRad) was used. Various tissues of hairy
roots and oil palm were homogenized in the GUS extraction buffer (50 mM natrium
phosphate, 10 mM Dithiothreitol [DTT], 1 mM EDTA, 0.1% (w/v) sodium lauryl sarcosine
and 0.1% (v/v) triton X-100). The homogenate was then centrifuged for 10 min
at 10,000 rpm at 4°C and the GUS activity of the supernatant was assessed
as described by Jefferson et al. (1987). Aliquots
of extracts (30 μg) were added to 500 μL of assay buffer (5 mM MUG
(methylumbelliferyl-β-D-glucuronide) in GUS extraction buffer ), pre-warmed
and incubated at 37°C. After 30 min of incubation, the reaction mixtures
were removed and placed in 1 mL of stop buffer (0.2 M Na2CO3,
pH 11.2). Fluorescence was measured using a FluoroAce (BioRad). The protein
concentration of the samples was determined using the procedure of Bradford
RESULTS AND DISCUSSION
Sesquiterpene Synthase Promoter (SesqPro) and Its Deletion Derivatives
The sesquiterpene synthase promoter (SesqPro) and its promoter deletion
derivatives was isolated, analyzed and were subsequently constructed and inserted
into the pCAMBIA 1301 vector (Cha, 2001). The 3
end and the 5 end primers were designed with Sal I and EcoR1 respectively
for cloning purpose. As a result, there were six constructs which differ in
size and which each carry several important putative transcription elements.
The CaMV 35S promoter was removed by digestion with Hind III and Nco 1 and the
plasmid was religated using klenow enzyme to form pCAMBIA-Sesq-Pro (PSP-VF)
The new constructs were labeled as PSPr-VF6 which is ~1336 bp in length and carries the longest SesqPro promoter, PSPr-VF5, which is ~1001 bp in length, PSPr-VF4, which is ~888 bp and PSPr-VF3 which is ~643 bp in length. PSPr-VF2 is ~430 bp in length, while the PSPr-VF1 is ~270 bp long. The presence of the promoter deletion derivatives were confirmed using PCR analysis.
Schematic diagrams of all deletion derivatives are shown in Fig. 1 which explains the location of the promoter SesqPro and its deletion series in the pCAMBIA 1301 vector backbone. The sizes of the deletion derivatives and the important putative transcription element which were present in the different promoter constructs are summarized in Table 1.
As for the SesqPro, the putative transcription initiation site, designated
as +1 was located at 33 bp upstream of the ATG translation start codon. A putative
TATA box and CAAT box, which are the general signals for eukaryotic gene expression,
were observed at positions -25 and -352, respectively and a putative GC box
was located at position -237. Both the distance of the consensus sequence, CAAT
and GC boxes in SesqPro were comparable to those found in the genome of other
plants. Usually, the eukaryotic GC box is located at position -100 (Klug
and Cummings, 1994), whereas the CAAT box is located between -80 to -300
bp from +1 (Tasanen et al., 1992).
|| Putative elements of transcription factors present in the
specified deletion derivates of SesqPro
The CAAT box is a common element found in 80% of the eukaryotic promoters and
can function in both forward and backward directions. It also plays an important
role in TATA-less promoters (Mantovani, 1999). Other
motifs present in SesqPro were the (GA)10 element located at -479
and three copies of GAGA boxes at positions -478, -677 and -695. The function
of the GAGA box/ factor in plant promoters has not been previously reported,
but the GAGA binding factor from Drosophila has been analyzed. The GAGA
factor in Drosophila promoter functions as an anti-repressor which could
inactivate the repressor protein from inactivating RNA polymerase II during
the transcription process (Kerrigan et al., 1991).
Two Dof binding factors, located at positions -411 and -450, were also present
in the SesqPro promoter. The Dof binding factor has two functions, either activating
transcription and being expressed constitutively and ubiquitously or, inactivating
transcription and being highly expressed in the stem and root, but being expressed
at a lower level in green leaves (Yanagisawa and Sheen,
Another important putative transcription factor in SesqPro is the G/A hybrid,
commonly known as the bZIP element, which is located at position -617 and has
ACGT as its core element. The CACGTA element is a homolog to the G box (CACGTG)
(Scmidt et al., 1992). An interesting characteristic
of the ACGT element is its ability to respond to elicitor induction. Therefore,
the G/A hybrid box presented in SesqPro could possibly function as a specific
element towards the elicitor induction. This is related to the native role of
SesqPro, which controls the expression of the sesquiterpene synthase gene that
is involved in the biosynthesis of phytoalexin compounds in anti-fungus and
anti-microbe infections in plant (Droge-Lase et al.,
1997). SesqPro also contains two copies of short AT-rich elements, AT1 located
at position -960, AT2 located at position -1040 and another longer AT-rich element
located at position -1180. Studies have shown that short AT elements (~20-55
bp) could enhance the level of gene expression by 6-13 folds either in normal
or reverse orientation (Sandhu et al., 1998).
The presence of three copies of AT elements in SesqPro may correlate with its
high expression of sesquiterpene synthase gene in E. oleifera mesocarp
(Cha, 2001). A (CA)n element in SesqPro which
is located at position -329 is commonly found in seed storage protein promoters
such as the napin gene promoter (Ellerstrom et
al., 1996) and β-phaseolin (Burrow et al.,
1992). Ellerstrom et al. (1996) suggested
that the (CA)n element could function as an activator or repressor
with different transcription factors in various tissues.
Analysis on Sesquiterpene Synthase Promoter Specificity Using Tomato Hairy
Roots and Oil Palm Tissues
To evaluate the specificity of the sesquiterpene synthase promoter in different
tissues, tomato hairy roots were generated from cotyledons via A. rhizogenes-mediated
transformation. Conversely, different tissues from oil palm, including the IE,
mesocarp slices and leaves had been transformed by A. tumefaciens strain
LBA 4404. In this study, PSPr-VF6, the full-length sesquiterpene synthase promoter,
was transformed into the selected tissues. Hairy roots and oil palm transformed
tissues carrying the CaMV 35S promoter were made as the positive control. Two
independent lines were analyzed; one for each tomato hairy root transformed
with either a construct containing CaMV 35S promoter or full-length SesqPro
Based on Fig. 2, two types of transformant tissues, the oil
palm tissues with transient transformation and the tomato hairy roots with stable
transformation, were used to evaluate the promoter specificity. Both indicated
different systems which were homologous for oil palms and heterologous for tomato
hairy roots. GUS histochemical assays for all tested tissues showed various
positive GUS expressions with different patterns of distribution and intensity.
Generally, both the CaMV 35S promoter and SesqPro (PSPr-VF6) were able to express
the GUS gene in all tested tissues. From close observations, it appeared that
the constitutive CaMV 35S promoter generally showed a higher expression of GUS
in the transgenic hairy roots, mesocarp and IE compared to SesqPro. The CaMV
35S promoter is a well-studied and characterized constitutive promoter derived
from the CaMV virus (Benfey et al., 1990) and
has been shown to have a high expression in dicots compared to monocots (Yang
et al., 2003).
||Distribution and intensity of gus gene expression in
the transformed immature embryos (IE), mesocarp slices and leaf slices of
oil palm (E. oleifera) (a): IE (CaMV 35) (b): IE (SesqPro); (c):
IE untransformed (d): mesocarp negative control (untransformed).) (e): mesocarp
(CaMV 35S) (f): (promoter SesqPro) (g): leaf slices (CaMV 35S) (h): leaf
slices (SesqPro) (I): hairy roots (CaMV 35S) and (j): hairy roots (SesqPro).
(k) GUS activity from fluorometric analysis used to evaluate sesquiterpene
synthase promoter specificity and CaMV 35S promoter in transformed tissues
HR: tomato hairy roots; M: mesocarp slices; IE: Immature embryos; L: Leaf
SesqPro was isolated from the promoter region of sesquiterpene synthase gene
in E. oleifera which was highly expressed in mesocarp tissues developmentally
(Cha, 2001). Natively, only the sesquiterpene synthase
gene from E. oleifera is highly expressed developmentally without induction
by a pathogen. This is in contrast to other sesquiterpene synthase genes such
as that in Elaies guineensis where its expression is only activated when
it is elicited by the invasion of a pathogen.
Previously, Salwa (2006) conducted a GUS histochemical
assay on two different sizes of sesquiterpene synthase promoter isolated from
the mesocarp of Elaies guineensis. The shorter Sesyn0.5Pro promoter only
contains a TATA box and the longer Sesyn1.2Pro contains similar putative transcription
factors as in SesqPro. Both Sesyn0.5Pro and Sesyn1.2Pro showed GUS expression
in IE and leaves but no expression was detected in the mesocarp. Sesyn0.5Pro
gave a higher GUS expression compared to Sesyn1.2Pro. The absence of the (CA)n
element in the Sesyn1.2Pro sequence may have contributed to a lower GUS
expression. In contrast, SesqPro was able to drive the GUS expression in all
tissues tested, including the heterologous tissues of tomato hairy roots.
Figure 2k shows the GUS activity from the fluorometric analysis
in evaluating the specificity of SesqPro and CaMV 35S promoters in selected
transformed tissues. Fluorometric analysis for each sample was conducted in
three replicates to increase accuracy. In comparison to different plant species,
both monocots and dicots show an obvious quantitative difference in GUS activity.
In this study, the level of GUS activity in the transgenic hairy roots was found
to be higher than that of oil palm tissues. GUS activity driven by the SesqPro
promoter in tomato hairy roots was higher compared to its activity in other
oil palm tissues and this could be related to the accumulation of higher sesquiterpenoids
in the roots (Maldonado-Bonilla et al., 2008).
Besides, the stable transformation system occurring in hairy roots could give
more accurate and consistent quantitative results compared to the transient
expression in transformed oil palm tissues. The generation of transformed oil
palm through tissue culture is time-consuming and therefore transient analysis
is the best choice and is feasible for preliminary evaluations of promoter specificity
and activity in oil palm tissues. In contrast, generation of stable transgenic
lines for tomato hairy roots is relatively easy, as it could grow very fast
in a plagiotropic manner (Nilsson and Olsson, 1997).
The level of GUS activity driven by SesqPro (PSPr-VF6) in the mesocarp and
IE of oil palms was higher than that of leaves. SesqPro drives a higher GUS
expression than the CaMV 35S promoter in leaves, but showed a lower expression
in other oil palm tissues such as the mesocarp and IE. This is because SesqPro
itself is isolated from the mesocarp and IE and it is also extracted from the
endosperm in the mesocarp. Therefore, it is possible that both tissues have
the same or similar transcription factors and communicate in line with the same
trans factors and cis elements. Conversely, the GUS activity of
the CaMV 35S promoter is lower in monocot compared to dicot plants (Basu
et al., 2003). This might be due to the CaMV 35S promoter activity
itself which involved the internal molecular interaction between cis
and trans proteins since phytoalexin activity is also active in leaf
tissues (Yedidia et al., 2003).
Based on the results obtained from both histochemical and fluorometric assays, we hypothesized that the SesqPro promoter could potentially be an active and constitutive promoter. This promoter can control and regulate gus gene expression in all tissues tested for its specificity, as can the constitutive CaMV 35S promoter, which is a well-characterized promoter.
Analysis on Sesquiterpene Synthase Promoter Strength in Mesocarp Slices
and Immature Embryos
All different sizes of SesqPro deletion derivatives gave positive GUS expression
in IE and mesocarp slices (Fig. 3a, b),
but the level of GUS activity decreased in line with the removal of 5
upstream sequences from the full-length to the shortest promoter deletion constructs
series in transformed tissues (Fig. 3c).
||(a) shows results from histochemical assays conducted on IE with different
distributions and intensities regarding its promoter deletion derivatives
constructs transformed into IE, (b) shows results from histochemical assays
conducted on mesocarps with different distributions and intensities with
respect to its promoter deletion derivatives constructs transformed into
mesocarp slices 1: PSPr-VF1; 2: PSPr-VF2; 3: PSPr-VF3; 4: PSPr-VF4; 5: PSPr-VF5;
6: PSPr-VF6. (c) GUS activity in mesocarp slices and IE of oil palm transformed
with different lengths of sesquiterpene synthase promoter and CaMV 35S promoter
Higher GUS activity was observed in PSPr-VF6-transformed tissue compared to
PSPr-VF5 which lack the AT elements in its SesqPro region. This observation
may suggest that the AT elements play an important role in regulating the expression
level of the sesquiterpene synthase gene.
GUS expression was observed to be lower in PSPr-VF4-transformed tissues compared to those transformed with PSPr-VF5 following the further deletion of both Silenser SBF and Un2 elements in the PSPr-VF4 construct. Deletion of another 200 bp sequence which contains the GAGA box and the hybrid G/A box from PSPr-VF4 gave rise to the PSPr-VF3 construct. The GUS expression observed in PSPr-VF3 was slightly lower in both IE and in the mesocarp compared to PSPr-VF4. The level of GUS expression in both PSPr-VF2 and PSPr-VF1 were still significant even though only the GC box, the CAAT box and (CA)n were left in PSPr-VF2 and TATA box in PSPr-VF1 construct.
Based on fluorometric assays shown in Figure 3c, the two
transformed tissues gave similar patterns of GUS activity accordingly. GUS activity
driven by CaMV 35S promoter was higher compared to that driven by SesqPro in
both oil palm tissues. However, there was an insignificant difference of GUS
activity driven by the CaMV 35S promoter in both the mesocarp and IE. Even though
both oil palm tissues gave similar patterns of GUS activity, the activity was
lower in IE compared to the mesocarp slices. One factor which could contribute
to this observation is the original endogenous activity because the SesqPro
was initially isolated from mesocarp tissues.
For the full-length SesqPro construct and its deletion derivatives, PSPr-VF6, which contains all of the SesqPro sequence in ~1336 bp, conferred a higher level of GUS activity in comparison to the other five deletion derivatives. As in this construct, all putative transcription motifs present and work accordingly as in its endogenous tissues, even though the GUS activity was slightly lower in IE. Overall, the GUS activity decreased according to its promoter deletion derivatives. As we can see, the GUS activity in PSPr-VF5 decreased insignificantly in both tissues. Following the deletion of a few elements from PSPr-VF5, GUS activity in PSPr-VF4 also decreased but the decrease was still insignificant compared to the other two longer promoter constructs, PSPr-VF6 and PSPr-VF5.
A significant reduction of GUS activity was detected in PSPr-VF3 followed by
PSPr-VF2 and PSPr-VF1. This observation was related to the deletion of the GAGA
box, the G/A hybrid box and also the Un4 element from the PSPr-VF4 construct.
Therefore, these three putative elements could be threshold motifs for the SesqPro
activity. However, PSPr-VF3, PSPr-VF2 and PSPr-VF1 could still confer expression
based on the GUS activity measured. This was probably due to the presence of
several basic but important transcription factors such as the TATA box, the
CAAT box and the GC box for minimal transcription activity (Butler
and Kadonaga, 2001).
As a result, we presumed that SesqPro is still able to drive the gus gene expression with its basic transcription factors. Even though SesqPro contains a few negative transcription motifs or transcription repressor motifs, it also contains activator motifs/enhancers which could counteract the effect of the repressor activity. Therefore, SesqPro is capable of driving a higher GUS activity if the enhancer motifs are within its vicinity.
Activity of the Sesquiterpene Synthase Promoter in Response to Exogenous
In order to investigate the promoter sequences of the sesquiterpene synthase
gene which are responsible for the induction of GUS activity by exogenous JA,
all the promoter constructs were tested with regard to the inducibility of GUS
expression in 15 day old transformed tissues. Sesquiterpenoid is a defensive
compound secreted in higher plants against fungal or microbial infections and
is catalyzed by the enzyme sesquiterpene synthase. To study the transcription
factor elements related to the defense activity, 100 μM of JA was added
to the liquid culture medium of transformed tissues. JA is among the exogenous
elicitors applied for the induction of defense mechanisms in plant tissue cultures
(Van Etten et al., 1994). JA and its derivatives
are believed to induce the production of secondary metabolites in plants (Sanz
et al., 2000). JA and its derivative, methyl ester (methyl jasmonate,
MJ) were proposed as the key signaling compounds in inducing the accumulation
of multiple secondary metabolites (Szabo et al.,
||GUS activity induced by 100 μM jasmonic acid in (a) mesocarp slices
and (b) IE of oil palm transformed with different lengths of the sesquiterpene
synthase promoter and the CaMV 35S promoter
As shown in Fig. 4a and b, the same pattern
of GUS activity was observed in transformed mesocarp slices and IE tissues when
100 μM of JA was applied. The GUS activity in both tissues increased upon
JA induction in the transformed tissues carrying the PSPr-VF6 construct. This
situation was similar to the other two constructs, PSPr-VF5 and PSPr-VF4. The
difference in the level of GUS activity in these three constructs was insignificant.
However, the GUS activity dropped significantly in PSPr-VF3, PSPr-VF2 and PSPr-VF1
upon induction by JA.
The G/A hybrid box were deleted from the PSPr-VF3 and the other two shorter SesqPro constructs. As discussed earlier, the presence of the G/A hybrid box in the SesqPro sequence will cause it to respond to elicitor inductions such as JA and will activate the biosynthesis of phytoalexins for plant defense mechanisms. The G/A hybrid box is categorized under the bZIP group which consists of the ACGT element and functions in response to environmental stresses and elicitor stimuli. This G/A hybrid box will interact with the accumulated exogenous JA and will activate the transcriptional machineries for plant defense mechanisms. Therefore, this will influence the transcription level of the gus gene and the proteins translated by this gene. The immediate reduction of GUS activity in PSPr-VF3 constructs was due to the loss of the G/A hybrid box in this construct. In the absence of this element, the plant defense mechanism conferred by this bZIP element is defective.
In summary, this preliminary study on the G/A hybrid box has shown that it is essential for SesqPro activation through environmental stress or elicitation by pathogen invasion. The findings reported in this study will serve as the underlying principles for future experiments further characterizing the sesquiterpene synthase promoter.
This research is funded by Agriculture Biotechnology Institute Research Grant (UKM-ABI-NBD0010-2007), Ministry of Science, Technology and Innovation of Malaysia awarded to Dr. Ismanizan Ismail. We would like to thank the Malaysian Oil Palm Board (MPOB) for providing oil palm tissue for this study.
1: Back, K. and J. Chappell, 1995. Cloning and bacterial expression of a sesquiterpene cyclase from Hyoscyamus muticus and its molecular comparison to related terpene cyclases. J. Biol. Chem., 270: 7375-7381.
CrossRef | PubMed |
2: Basu, C., A.P. Kausch, H. Luo and J.M. Chandlee, 2003. Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris). J. Plant Physiol., 160: 1233-1239.
CrossRef | PubMed |
3: Benfey, P.N., L. Ren and N.H. Chua, 1990. Combinatorial and synergistic properties of CaMV 35S enhancer subdomains. EMBO J., 9: 1685-1696.
4: Birnboim, H.C. and J. Doly, 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res., 7: 1513-1523.
CrossRef | PubMed | Direct Link |
5: Bohlmann, J., G. Meyer-Gauen and R. Croteau, 1998. Plant terpenoid synthases: Molecular biology and phylogenetic analysis. Proc. Nat. Acad. Sci. USA., 5: 4126-4133.
CrossRef | PubMed | Direct Link |
6: Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.
CrossRef | PubMed | Direct Link |
7: Burrow, M.D., P. Sen, C.A. Chlan and N. Murai, 1992. Developmental control of the β-phaseolin gene requires positive, negative, and temporal seed-specific transcriptional regulatory elements and a negative element for stem and root expression. Plant J., 2: 537-548.
8: Butler, J.E.F. and J.T. Kadonaga, 2001. Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs. Genes Dev., 15: 2515-2519.
CrossRef | PubMed |
9: Caruthers, J.M., I. Kang, M.J. Rynkiewicz, D.E. Cane and D.W. Christianson, 2000. Crystal structure determination of aristolochene synthase from the blue cheese mold, Penicilium roqueforti. J. Biol. Chem., 275: 25533-25539.
CrossRef | PubMed |
10: Cha, T.S., 2001. Characterization of genes and mesocarp-specific promoter for genetics manipulation of oil palm. M.Sc. Thesis, Faculty of Science and Technology, National University of Malaysia, Bangi, Malaysia.
11: Chappell, J., 1995. Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol., 46: 521-547.
12: Chen, X.Y., M. Wang, Y. Chen, V.J. Davisson and P. Heinstein, 1996. Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum. J. Nat. Prod., 59: 944-951.
CrossRef | PubMed |
13: Dorothea, T., F. Chen, J. Petri, J. Gershenzon and E. Pichersky, 2005. Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J., 42: 757-771.
CrossRef | PubMed |
14: Ellerstrom, M., K. Stalberg, I. Ezurra and L. Rask, 1996. Functional dissection of napin gene promoter: identification of promoter elements required for embryo and endosperm specific transcription. Plant Mol. Biol., 32: 1019-1027.
15: Facchini, P.J. and J. Chappell, 1992. Gene family for an elicitor-induced sesquiterpene cyclase in tobacco. Proc. Nat. Acad. Sci. USA., 89: 11088-11092.
CrossRef | PubMed |
16: Jefferson, R.A., T.A. Kavanagh and M.W. Bevan, 1987. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 6: 3901-3907.
Direct Link |
17: Kappers, I.F., A. Aharoni, T.W.J.M. van Herpen, L.L.P. Luckerhoff, M. Dicke and H.J. Bouwmeester, 2005. Genetic engineering of terpenoid metabolism attracts bodyguards to arabidopsis. Science, 309: 2070-2072.
CrossRef | PubMed | Direct Link |
18: Kerrigan, L.A., G.E. Croston, L.M. Lira and J.T. Kadonaga, 1991. Sequence-specific transcriptional antirepression of the Drosophila kruppel gene by the GAGA factor. J. Biol. Chem., 266: 574-582.
19: Kessler, A. and I.T. Baldwin, 2001. Defensive function of herbivore-induced plant volatile emissions in nature. Science, 291: 2141-2144.
Direct Link |
20: Lange, B.M., R.E.B. Ketchum and R.B. Croteau, 2001. Isoprenoid biosynthesis metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis. Plant Physiol., 127: 305-314.
CrossRef | PubMed |
21: Maldonado-Bonilla, L.D., M. Betancourt-Jimenez and E. Lozoya-Gloria, 2008. Local and systemic gene expression of sesquiterpene phytoalexin biosynthetic enzymes in plant leaves. Eur. J. Plant Pathol., 121: 439-449.
22: Mantovani, R., 1999. The molecular biology of the CCAAT-binding factor NF-Y. Gene, 239: 15-27.
CrossRef | PubMed |
23: Milat, M.L., P. Ricci, P. Bonnet and J.P. Blein, 1991. Capsidiol and ethylene production by tobacco cells in response to cryptogein, an elicitor from Phytophthora cryptogea. Phytochemistry, 30: 2171-2173.
24: Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Planta., 15: 473-497.
CrossRef | Direct Link |
25: Nilsson, O. and O. Olsson, 1997. Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant, 100: 463-473.
26: Picaud, S., M.E. Olsson, M. Brodelius and P.E. Brodelius, 2006. Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale. Arch. Biochem. Biophys., 452: 17-28.
27: Proctor, R.H. and T.M. Hohn, 1993. Aristolochene synthase: Isolation, characterization and bacterial expression of sesquiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti. J. Biol. Chem., 268: 4543-4548.
28: Salwa, A.S., 2006. Transient expression patterns of gus reporter gene under the induction of several constitutive and tissue-specific promoters. M.Sc. Thesis, Faculty of Science and Technology, National University of Malaysia, Bangi, Malaysia.
29: Sandhu, J.S., C.I. Webster and J.C. Gray, 1998. A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants. Plant Mol. Biol., 37: 615-622.
CrossRef | PubMed |
30: Sanz, M.K., X.E. Hernandez, C.E. Tonn and E. Guerreiro, 2000. Enhancement of tessaric acid production in Tessaria absintheoides cell suspension cultures. Plant Cell Rep., 19: 821-824.
31: Schnee, C., T.G. Kollner, J. Gershenzon and J. Degenhardt, 2002. The maize gene terpene synthase 1 encodes a sesquiterpene synthase catalyzing the formation of (E)-β-farnesene, (E)-nerolidol and (E, E)-farnesol after herbivore damage. Plant Physiol., 130: 2049-2060.
CrossRef | PubMed |
32: Szabo, E., A. Thelen and M. Petersen, 1999. Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei. Plant Cell Rep., 18: 485-489.
33: Tasanen, K., J. Oikarinen, K.I. Kivirikko and T. Pihlajaniemi, 1992. Promoter of the gene for the multifunctional protein disulfide isomerase polypeptide. Functional significance of the 6 CCAAT boxes and other promoter elements. J. Biol. Chem., 267: 11513-11519.
34: Trapp, S.C. and R.B. Croteau, 2001. Genomic organization of plant terpene synthases and molecular evolutionary implications. Genetics, 158: 811-832.
Direct Link |
35: Van Etten, H.D., J.W. Mansfield, J.A. Bailey and E.E. Farmer, 1994. Two classes of plant antibiotics: Phytoalexins versus phytoanticipins. Plant Cell, 6: 1191-1192.
CrossRef | PubMed | Direct Link |
36: Yanagisawa, S. and J. Sheen, 1998. Involvement of maize Dof zinc finger proteins in tissue-specific and light-regulated gene expression. Plant Cell, 10: 75-89.
CrossRef | PubMed |
37: Yang, M., R. Bower, M.D. Burow, A.H. Paterson and T.E. Mirkov, 2003. Genomics, molecular genetics and biotechnology: A rapid and direct approach to identify promoters that confer high levels of gene expression in monocots. Crop Sci., 43: 1805-1813.
Direct Link |
38: Yedidia, I., M. Shoresh, Z. Kerem, N. Benhamou, Y. Kapulnik and I. Chet, 2003. Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Thrichoderma asperellum (T-203) and accumulation of phytoalexins. Applied Environ. Microb., 69: 7343-7353.
CrossRef | PubMed |
39: Yin, S., L. Mei, J. Newman, K. Back and J. Chappell, 1997. Regulation of sesquiterpene cyclase gene expression. Characterization of an elicitor-and pathogen-inducible promoter. Plant Physiol., 115: 437-451.
40: Chu, C.C., C.C. Wang, C.S. Sun, K.C. Hsu, K.C. Yin, C.Y. Chu and F.Y. Bi, 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Scient. Sin., 18: 659-668.
Direct Link |
41: Droge-Laser, W., A. Kaiser, W.P. Lindsay, B.A. Halkier, G.J. Loake, P. Doener, R.A. Dixon and C. Lamb, 1997. Rapid stimulation of a soybean protein-serine kinase that phosphorylate a novel bZIP DNA-binding protein, G/HBF1, during the induction of early transcription-dependent defenses. EMBO J., 16: 726-738.
42: Klug, W.S. and M.R. Cummings, 1994. Concepts of Genetics. 2nd Edn., Prentice Hall, Inc., New Jersey.
43: Scmidt, R.J., M. Ketudat, M.J. Aukerman and G. Hoschek, 1992. Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD sein genes. Plant Cell, 4: 689-700.