Permpong Sriprasertsak¹, Anchalee Raweerojwiboon¹,
Tetsuji Yamada² and Thira Sutrabutra^3
1Researcher, Central Laboratory and Greenhouse Complex,
Kasetsart University Research and Development Institute, Kasetsart University, Kamphaeng Saen.
²Professor, Laboratory of Plant Pathology and Applied Genetic Engineering,
Faculty of Agriculture, Okayama University, Okayama 700, Japan.
³Professor, Department of Plant Pathology,
Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.

Abstract

        The phenylalanine ammonia-lyase is a key enzyme in phenylpropanoid synthesis, a pathway for the biosynthesis of a
widerange of natural products which play key roles in plant development and protection agianst environmental stresses
including the structural polymer lignin, flavonoids (anthocyanin pigments and UV protectants), isoflavonoids and phytoalexins
(Fig. 1). In this study, PSPAL2, a member of pea PAL gene family was determined and structurally characterized. The structure
f PSPAL2 was divided into two exons by the single intron of 90 bp. The deduced amino acid sequence of PSPAL2 showed
that the gene encode 724 amino acids with a deduced molecular weight of 79,005 Da.. In PSPAL2, we have found the
retrotransposon-like sequence in the 5^/-upstream region of PSPAL2 promoter (Fig. 2A). Moreover, we have found some
putative cis-regulatory elements of box I, box II, and box IV which are conserved among the promoter of several genes
involved in the phenylpropanoid pathway (Fig. 2B).

    To discriminate the function of PSPAL2 promoter, we have demonstrated the expression of pea PSPAL2 retrotransposon
-like sequence in the region between -406 and -2196 and the three types of sequentially deleted chimeric promoter constructs
designated as PSPAL2-FLd1, PSPAL2-FLd2 and PSPAL2-FLd3 in transgenic tobacco during developmental growth and upon
fungal ingression. The histochemical GUS expression in young seedlings and mature plants seems to be conserved in tissue
and organ-specific expressions (roots, stems, leaves, flower organs and anthers). Moreover, the levels of GUS activities in
tissues of transgenic plants depend on the 5^/-upstream region of PSPAL2 promoter were also determined. Extremely low
GUS expression was observed in healthy or undisturbed mature leaves. However, the PSPAL2 promoter activated in leaves
of transgenic tobacco plants after transfer to the greenhouse was induced upon fungal ingression, especially when leaves
inoculated with P. capsici were incubated at 22-24^oC for 48 hr. Marked expression was detected at the HR area surrounding
the inoculation site of the transformant of PSPAL2-FL. Extremely low GUS expression was observed in the transformant of
PSPAL2-FLd3. Although brown necrotic regions were observed at the cross cut. The results demonstrated that the region from
-966 to -2196 of PSPAL2 promoter played a crucial role in the regulation of induction of GUS activities in the mature leaves of
transgenic tobacco plants. It is thus clear that the 5^/-upstream region between +110 to -594 is insufficient to establish the full
capacity of defense gene response under stress even though this region contains important box sequences such as box I,
box II and box IV. The additional sequences from -594 to -2196 that include the region of retrotransposon-like sequence are
obviously  necessary for the functional expression of the gene not only during developmental growth but also in response to
fungal ingress and injuries.

Methods and Materials

     1. Construction of chimeric genes
     The pea PSPAL2 full length promoter (PSPAL2-FL, -2196 to +110) and three deleted chimeric promoters designated as
PSPAL2-FLd1 (-1486 to +110), PSPAL2-FLd2 (-966 to +110) and PSPAL2-FLd3 (-594 to +110) had been constructed into CAT
reporter gene for analyzing the transient expression in electroporated protoplasts (Yamada et al., 1994). To investigate the
expression of PSPAL2 promoters in transgenic tobacco plants, the PSPAL2-FL promoter and the three selected deletion
construct promoters in the CAT reporter gene were amplified by PCR using specific primers and subcloned into pBluescriptII
KS (+) at Hind III and Bam HI sites, then ligated with the GUS reporter gene in pBI101.2 (Fig. 3). The PSPAL2- GUS chimeric
promoter constructs were purified and transformed into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method
(Holster et al., 1978)
     2. Preparation of sterile tobacco plants
     Nicotiana tabacum was used in the transformation experiments. Seeds were surface-sterilized in 5% hypochlorite for 10
min, followed by soaking in 70% ethanol for 10 min, and rinsed in sterilized water. Sterilized seeds were germinated in a sterile
petri dish containing the MS medium (Murashige and Skoog, 1962)
     3. Leaf disk transformation
     Leaf disk of the sterilized tobacco was co-cultured in MS medium for 15-30 min with 1-5 x 10⁸ cell/ml of A. tumefaciens
LBA4404 (McCormick et al., 1986) carrying the specified chimeric genes as will be described (Fig. 4A). After drying on a
sterilized Whatman 3 MM filter paper to remove excess bacteria, inoculated leaves were placed abaxial surface down on MS
medium containing 1.0 mg/l of NAA and 1.0 mg/l of BA. The inoculated leaves were incubated at 22-24^oC for 2 days, and then,
transferred onto MS-selective medium containing the same concentration of NAA, BA, Kanamycin (100 mg/l) and claforan (500
mg/l) until shoots formed. After 2-3 weeks of incubation, adventitious shoots were transferred to new MS medium until roots
formed (Gelvin et al., 1990). All plant materials were incubated at 25-28⁰C under a 16 -hr. light (150 m E / m² /s), 8-hr dark
conditions. Young seedlings (Fig. 4B, 4C) were transplanted into soil and incubated in a greenhouse (Fig. 4D).
     4. GUS histochemical assay and PCR analysis
     GUS histochemical assay
     Mature leaves were fixed by soaking in 1% formaldehyde in 50 mM sodiumphosphate buffer (pH 7.0) for 10 min and rinsed
three times with 50 mM sodiumphosphate buffer (pH 7.0). Then, they were incubated in X-Gluc solution (1.0 mM 5-bromo-4
-chloro-3-indolyl- b -D-glucuronide, in 50 mM. sodiumphosphate buffer (pH 7.0), 0.5 mM potassium ferricyanide, 0.5 mM
potassium ferrocyanide, 10 mM EDTA) at 37⁰C for overnight as described by Jefferson et al. (1987). Straining and fixation of
X-Gluc solution into tissues was facilitated by vacuum infiltration. Staining reactions were stopped by transferring the tissues
into 70% ethanol.
     GUS-PCR analysis
     Genomic DNA extraction for GUS-PCR was performed to confirm the integration of PSPAL2-GUS fusions in the genome
of transgenic tobacco plants as described by Hosaka (1994). GUS-PCR was amplified using GUS-specific primers [primer I
(upstream) 20 mer : 5^/-TAC GTA TCA CCG TTT GTG TG-3^/; primer II (down stream) 20 mer : 5^/-GTA ATA ACG GTT CAG
GCA CA -3^/] (Fig. 5). DNA manipulation was performed according to the standard methods described by Sambrook et al.,
(1989) or  as specified by the manufacturer’s protocols.

     1. GUS expression in transgenic tobacco plants during developmental growth
     We have observed basal GUS expression of PSPAL2-FL promoter in tissues of roots, stems and leaves during
developmental growth of young seedling before transplanting to soil. The histochemical GUS expression in young
seedlingsand mature plants seems to be conserved in tissue and organ-specific expressions as observed in PSPAL1
( Kawamata et al., 1997 ) and bean PAL2 (Shufflebottom et at., 1993 and Hatton et al., 1995). PSPAL2 promoter showed
strong GUS expression in xylem, phloem elements of the vascular and endodermal tissues of lateral roots (Fig. 6), stems
(Fig. 7) and vascular tissues in veins of leaves, leaf tips, and petrioles (Fig. 8). Strong GUS activity was also found in flower
organs,especially in the pigment parts of petals, sepal tips, gland cells of trichomes (Fig. 9) and anthers (Fig. 10). However,
we could not observe the GUS expression in root hairs as found in PSPAL1. Moreover, the level of GUS activities in organ
of transgenic plants significantly declined in corresponding to the deleted 5^/-upstream chimeric PSPAL2 promoters from
-2196 to -594,especially in roots, vascular tissues in veins of leaves and stems as shown in Fig. 11.

     2. GUS expression in transgenic tobacco plants upon fungal ingression and injuries
     Histochemical GUS expression
     The leaves of transgenic tobacco plants carrying PSPAL2-FL were inoculated with Phytophthora nicotianae, a tobacco
pathogenic fungi (P) or with P. capsici, a nonpathogen (NP). The results showed that histochemically detected GUS
expression in transgenic tobacco plants was the highest at 48 hr after inoculation with P. capsici (Fig. 12B) and incubated at
22-24⁰C. Then GUS expression gradually faded away at the hypersensitive response (HR) area around the inoculation site
after 72 hr of incubation (Fig. 12C), where a plant defense system had presumably been established for blocking fungal
invasion, in a manner  similar to the expression of PSPAL1 promoter (Kawamata et al., 1997). The pattern of the GUS
xpression after inoculation with a pathogen was not as clear as one observed in the necrotic area after inoculation with a
nonpathogen and never faded until the whole leaf was colonized.
     Expression of deleted PSPAL2 promoters upon fungal infection
     To discriminate the expression of the deleted PSPAL2 promoters upon fungal infection, mature leaves of transgenic
tobacco plants carrying the PSPAL2 chimeric promoter constructs were inoculated with P. capsici. This nonpathogenic
fungus induced a very large, clear GUS expression zone around the site of the hypersensitive response (HR), especially
in the transformants of PSPAL2-FL (Fig. 13A) and PSPAL-FLd1 (Fig. 13B) at 48 hr after the inoculation. However, the responses
to fungi infection were not high in the transformants of PSPAL-FLd2 (Fig. 13C); the expression zone was restricted to the area
around the inoculation site. GUS expression in the transformant of PSPAL-FLd3 did not clearly appear (Fig. 13D). The results
showed that the pea PSPAL2 promoter expression in transgenic tobacco was strongly affected by the sequences in 5^/-
upstream region as previously shown with the transient CAT expression in electroporated pea protoplasts (Yamada et al.,
1994). The functional analysis of 5^/-nested deletions of PSPAL2 promoter in electroporated protoplasts showed that an
enhancer-like element is located at the TATA-distal region from -2196 to-406, and this promoter was activated by fungal
elicitor from M. pinodes and partially suppressed by the suppressor from the same fungus (Yamada et al., 1994). Interestingly,
the GUS expression of our constructs upon fungal ingression in transgenic tobacco leaves demonstrated the induction of
positive defense responses at the sites of infection at different levels depending on the additional sequences of the 5^/-
upstream region. Because we have observed a very low level of the GUS expression in the transformants of PSPAL2-FLd3
compared to PSPAL2-FL, the lower level of GUS expression is not due to a position effect of the integration of the chimeric
promoters. This phenomenon may be explained by the lack of some active elements that are needed for the regulation of the
PSPAL2 promoter. These elements are likely to span from -966 to -2196. Moreover, we have observed GUS expression in
mature transgenic tobacco leaves injured with sterile razor blade histochemically. Intense blue colorations were observed at
wounding sites (W) and restricted adjacent areas. The GUS expression of the deleted PSPAL2-FL promoters after wounding
also declined from a high level to an extremely low one as the deletion was extended, in a manner similar to the expression
after fungal ingression (Fig. 13E-H).
     The results demonstrated that the region from -966 to -2196 of PSPAL2 promoter played a crucial role in the regulation of
induction of GUS activities in the mature leaves of transgenic tobacco plants. It is thus clear that the 5^/-upstream region
between +110 to -594 is insufficient to establish the full capacity of defense gene response under stress even though this
region contains important box sequences such as box I, box II and box IV. The additional sequences from -594 to -2196 that
include the region of retrotransposon-like sequence are obviously necessary for the functional expression of the gene not
only during developmental } growth but also in response to fungal ingress and injuries.

References

Gelvin, S.B., Schilperoort, R.A., and Verma, D.P.S. 1990. Plant Molecular Biology Manual. Kluwer
          Academic Publishers, NY.
Hatton, D., Sablowski, R., Yung, M.H., Smith, C., Schuch, W. and Bevan, M. 1995. Two classes of cis
          sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco.
          Plant J. 7(6):859-876.
Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, N. and Schell, J. 1978.
          Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163(2):
          181-187.
Hosaka, K. 1994. Current RAPD technology. Academic booklet, Kobe University, Kobe.
Jefferson, R A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusion: b -glucuronidase as a
         sensitive and versatile gene marker in higher plants. EMBO J. 6: 3901-3907.
Kawamata, S., Shimoharai, K., Imura, Y., Ozaki, M., Ichinose, Y., Shiraishi, T., Kunoh, H and
         Yamada, T. 1997. Temporal and spatial pattern of expression of the pea phenylalanine
         ammonia-lyase gene1 promoter in transgenic tobacco. Plant Cell Physiol. 38(7): 792-803.
McCormick, S., Niedermeyer, J. and Fry, J. 1986. Leaf disc transformation of cultivated tomato
         (L. esculentum) using Agrobacterium tumefaciens. Plant Microbe Interact. 6(4): 453-466.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with
         tobacco tissue cultures. Physiol. Plant. 15: 473-497.
Sambrook, T., Fritsch, E.F. and Manitis, J. 1989. Molecular Cloning: A Laboratory Manual. Cold
         Spring Habor, NY.
Shufflebottom, D., Edwards, K., Schuch, W. and Bevan, M. 1993. Transcription of two members
         of a gene family encoding phenylalanine ammonia-lyase leads to remarkably different cell
          specificities and induction patterns. Plant J. 3(6): 835-845.
Sriprasertsak, P. 2000. Plant defense responses and control of gene expression: Structure and
          function of the promoter of PSPAL2, a pea defensive gene encoding phenylalanine
         ammonia-lyase. Dissertation Ph.D, Okayama University, Japan.
Yamada, T., Sriprasertsak, P., Kato, H., Hashimoto, T., Shimizu, H., and Shiraishi, T. 1994.
         Functional analysis of the promoters of phenylalanine ammonia-lyase genes in pea. Plant
      Cell Physiol. 35: 917-926.

Fig. 1 The phenylpropanoid biosynthesis pathway
              PAL : phenylalanine ammonia-lyase, C4H : cinnamic acid 4 - hydroxylase
             4CL : 4 - coumaric acid CoA ligase,     CHS : chalcone synthase, CHI : Chalcone isomerase
          CCR : 4 - coumaroyl CoA reductase,   CAD : coumaroyl alcohol dehydrogenase

Fig. 2A The retrotransposon-like sequence in the 5^/-upstream region of PSPAL2 promoter

Fig. 2B Conserved sequence motifs in the promoters of several phenylpropanoid biosynthesis genes
                 PAL : phenylalanine ammonia-lyase, CHS : chalcone synthase
                 Pv : Phaseolus vulgaris, Pc : Petroselinum crispum, At : Arabidopsis thaliana,
                 Am : Antirrhinum majus, Ps : Pisum sativum

Fig. 13 Expression of full-length (PSPAL2-FL : A,E) and deleted PSPAL2 promoters (PSPAL2-FLd1 : B,F; PSPAL2-FLd2 :
C,G; PSPAL-FLd3 : D,H) at 48 hr after wounding (W) and inoculation with a nonpathogen (NP, P. capsici) in transgenic
tobacco leaves. Bars equal 1 mm.