Plant Science 158 2000 19 – 32
Characterization of cDNAs differentially expressed in roots of tobacco Nicotiana tabacum cv Burley 21 during the early stages
of alkaloid biosynthesis
Jianmin Wang, Moira Sheehan, Heather Brookman, Michael P. Timko
Department of Biology, Uni6ersity of Virginia, Charlottes6ille, VA
22903
, USA Received 13 March 2000; received in revised form 9 May 2000; accepted 9 May 2000
Abstract
A set of 60 cDNAs were isolated by subtractive hybridization screening of a phage library using radioactively-labeled probes generated from root mRNAs isolated from tobacco Nicotiana tabacum cv Burley 21 plants before and 3 days after topping.
Among the differentially expressed gene products were full-length and partial cDNAs encoding arginine decarboxylase ADC, ornithine decarboxylase ODC, and S-adenosylmethionine synthetase SAMS, enzymes involved in polyamine and alkaloid
biosynthesis. The other cDNAs isolated were placed into one of several categories and encode metabolic enzymes, proteins involved in transcription and translation, components of signal transduction pathways, and homologs of genes whose expression
has been shown to be regulated by phytohormones i.e. auxin, ABA, wounding or other stress responses. RNA gel blot analysis showed that the ADC and ODC transcripts were preferentially expressed in the roots and floral tissues of mature tobacco plants,
whereas SAMS transcripts were detected in all tissues examined. The steady-state levels of the ADC and ODC mRNAs increased in the roots of wild-type tobacco plants during the 24 h period after topping, whereas little change was observed in the abundance
of the SAMS transcripts in these tissues. The possible factors associated with the regulation of expression of these genes are discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Nicotine; Alkaloid biosynthesis; Arginine decarboxylase; Ornithine decarboxylase; S-adenosyl methionine synthetase www.elsevier.comlocateplantsci
1. Introduction
Alkaloids are one of the most diverse groups of secondary compounds found in plants and they
are the product of a complex biosynthesis pathway [1 – 3]. Why plants accumulate these compounds
and in so many different forms is not known. Moreover, for many alkaloids, the exact site of
synthesis and the factors that control their inter- cellular distribution and accumulation remain to
be determined [2 – 4].
Nicotine is the most abundant alkaloid present in cultivated tobacco and, like other alkaloids
found in Nicotiana and related plant species, its biosynthetic
origin begins
with the
plant polyamine putrescine [2,4]. Putrescine is formed in
plants by one of two pathways [5]. It can be synthesized directly from ornithine, in a reaction
catalyzed by the enzyme ornithine decarboxylase ODC, EC 4.1.1.17, or formed indirectly from
arginine in a reaction sequence initiated by arginine decarboxylase ADC, EC 4.1.1.19. Pu-
trescine formed by the ADC andor ODC path- way serves as precursor in the synthesis of the
higher polyamines, spermine and spermidine, cata- lyzed by the enzymes spermine synthase and sper-
midine synthase, respectively, or it is converted to N-methylputrescine by the action of putrescine
N-methyltransferase PMT, the first committed step in nicotine biosynthesis [2,4,5]. N-methyl pu-
trescine is oxidized by a diamine oxidase and cyclized to form the 1-methyl-
D
1
-pyrrolium cation,
Corresponding author. Tel.: + 1-804-9825817; fax: + 1-804- 9825626.
E-mail address
:
mpt9gvirginia.edu M.P. Timko. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 9 3 - 4
which is condensed with nicotinic acid or its derivative to form nicotine [2]. Other amino
acids, such as tyrosine, tryptophan, phenylala- nine, and related compounds e.g. anthranilic
acid, nicotinic acid, and purines can also serve as biosynthetic precursors to some classes of al-
kaloids [5].
The synthesis and accumulation of nicotine and other tobacco alkaloids are known to be
controlled by various developmental, environ- mental, and chemical cues [1,2,4]. Changes in
phytohormone e.g. auxin, cytokinin levels and or ratios as a consequence of developmental age
[2,4] or by direct manipulation of plant cell cul- ture conditions have been shown to affect the
synthesis and accumulation of nicotine and vari- ous tobacco alkaloids [2,6,7]. Various abiotic fac-
tors wounding, drought stress, pH imbalance, etc. [1,2,4], as well as biotic factors, such as
herbivory, insect feeding, and attack by various microbial and fungal pathogens, are known to
elicit increased production of nicotine and other alkaloids in the leaves of wild and cultivated to-
bacco species [8,9]. In addition, the commercial practice of topping i.e. removal of flowering
head and young leaves at the upper portions of the plant, results in increases in nicotine and the
amount and complexity of total alkaloids present in the leaves of Nicotiana tabacum [2,6]. The fac-
tors controlling the topping-induced increase in alkaloid biosynthesis are not known, but likely
involve a complex physiological response in the plant as a result of altered phytohormones and
wound induced signaling [6,10] . In this regard, considerable evidence now exists indicating that
a jasmonic acid JA-mediated signal transduc- tion pathway may play a role in regulation of
gene expression contributing to this increase in alkaloid biosynthesis [11 – 15].
The formation of nicotine and total leaf alka- loids in tobacco is known to be under the con-
trol of at least two independent genetic loci [16,17], referred to most recently in the literature
as Nic
1
and Nic
2
[6] . Nic
1
and Nic
2
are semi- dominant and operate synergistically to control
plant alkaloid content, with mutations within these genes resulting in plants with reduced lev-
els of nicotine and total leaf alkaloids wild- type \ nic
1
\ nic
2
\ nic
1
nic
2
[16,17]. Although no information is available on the nature of
their encoded products, it has been speculated that Nic
1
and Nic
2
likely encode transcriptional regulators capable of globally interacting with a
subset of
genes encoding
components of
polyamine and alkaloid biosynthesis [6]. cDNAs andor genomic DNA fragments en-
coding enzymes involved in polyamine biosynthe- sis andor the formation of nicotine and related
alkaloids have been isolated from a number of plant species [2,5,6,15,18,19]. Initially, most at-
tempts at the cloning of cDNAs for enzymes involved in alkaloid formation involved immuno-
screening of phage expression libraries with anti- bodies prepared against purified enzyme protein,
or by sequencing the protein and screening a library
with synthetic
oligonucleotide probes
based upon the defined amino acid sequence [19]. A few attempts at alternative approaches,
such as the use of differential hybridization screening [6,15,20] or the use of PCR based
strategies [21], have also been reported. In the present study, we used a subtractive hybridiza-
tion screening strategy to identify cDNAs whose encoded proteins are differentially expressed in
the roots of Burley 21 tobacco plants 3 days after topping, a developmental time known to be
active in nicotine and leaf alkaloid formation. We report here the results of our studies and
describe the nature of the gene products and their expression in wild-type tobacco plants.
2. Materials and methods
