Durzan, Don J

BMC Plant Biology

English

PubMed

Don J Durzan

Springer - Publisher Connector

Nitric oxide, cell death and increased taxol recovery

Durzan Don J

Directory of Open Access Journals

BioMed CentralBMC Plant Biology
ssOpen AcceMeeting abstract
Nitric oxide, cell death and increased taxol recovery
Don J Durzan*
Address: Department of Plant Sciences, University of California, One Shields Ave. Davis, CA 95616, USA
Email: Don J Durzan* - djdurzan@ucdavis.edu
* Corresponding author    
Background
With NASA support this research aimed at evaluating early
opportunities in Microgravity Sciences to commercialize
space and to develop the biotechnology facility for the
International Space Station [1]. The main task was to eval-
uate the production of taxol (generic name: paclitaxel)
with cell suspensions in bioreactors designed for the
Space Shuttle. Unexpectedly, this work led to the early
demonstration of L-arginine-dependent nitric oxide (NO)
bursts in mechanically and gravitationally stressed plant
cells, to NO-induced programmed cell death (apoptosis)
(reviewed in [2]), and to a model describing how these
factors contribute to increased taxol and taxane recovery
from conifers.
Earlier, when intermediates of the Krebs-Henseleit or urea
cycle (See Figure 1) were fed to conifers, several substi-
tuted guanidino compounds were derived from uni-
formly labeled 14C-L-arginine, and less so from 14C-L-
citrulline [3,4]. At that time, the substituted guanidines
were considered mainly as respiratory inhibitors [5,6].
Today, they are natural inhibitors of plant, animal and
human nitric oxide synthases (NOSs). NOS substrates are
L-arginine and oxygen. NOS products are L-citrulline and
NO.
Since nitrate and nitrite reductases were also known
sources of NO, we obtained an Arabidopsis nitrate reduct-
ase double mutant with the aim of finding out if cells
could produce NO in the absence of nitrate, nitrite, and
their reductases [7]. With this mutant we reaffirmed that
the source of NO was putative NOS activity. The produc-
tion of NO from L-arginine was blocked by D-arginine,
and by the NOS inhibitor, NG-monomethyl-L-arginine (L-
NMMA) ensuring that NO was produced in the absence of
any residual nitrate reductase activity. NOS-dependent
NO production in cells was inhibited by other guanidino
compounds but not by D-arginine. Subsequently, the dis-
covery by others of two plant NOS genes provided evi-
dence that plant genomes code for NOS. In our work with
Taxus cell suspensions, the substituted guanidines offered
protection against mechanically induced stress and cell-
death or apoptosis.
Taxol is an effective anti-cancer agent that was first iso-
lated from the bark of Taxus brevifolia [8]. Taxol binds to
microtubules thereby offering a novel mechanism of
blocking cell proliferation. It became the best-selling anti-
cancer drug in history. By 2000, commercial sales of taxol
were well over $1.5 billion. New models for taxol and tax-
ane biosynthesis emerged [9] so that taxol biosynthesis
could be followed at the subcellular level by immunocy-
tochemistry, and by laser confocal and scanning electron
microscopy [10-13,20]. The use of NO donors, NOS sub-
strates, products, inhibitors (substituted guanidines), and
NO traps provided new opportunities to control the cit-
rulline-NO cycle (See Figure 1), apoptosis, and taxol pro-
duction in unit gravity, simulated microgravity, and in
hypergravity.
Haploid egg cells from female trees of T. brevifolia were
selected as the experimental material [12,19]. These cells
are easily screened without the effects of dominance,
recessive, and epistatic interactions characteristic of dip-
loid cells. Lethal genes are directly expressed, and
removed by apoptosis making cell populations geneti-
cally more uniform. Diploid cell suspensions were also
established from T. cuspidata [13] needles on 3-year-old
stock obtained in 1995 from Zelenka Nursery (Grand
Haven MI), and from seeds of T. chinensis. This provided
better comparisons with all other published work with
using diploid cells.
from Cell Biology of Nitric Oxide and Cell Death in Plants
Yalta, Ukraine, 8–11 September 2004
Published: 31 May 2005
BMC Plant Biology 2005, 5(Suppl 1):S12 doi:10.1186/1471-2229-5-S1-S12
<supplement> <title> <p>Cell Biology of Nitric Oxide and Cell Death in Plants</p> </title> <note>Meeting abstracts</note> </supplement>Page 1 of 4
(page number not for citation purposes)
BMC Plant Biology 2005, 5:S12Cell suspensions were maintained in darkness at 25 ± 2°C
on semisolid media, in 125 ml Erlenmeyer flasks (60
rpm), 1 L nippled flasks on a clinostat (1 rpm simulating
2 × 10-4 × g, but with significant convective mixing of the
gaseous and liquid environment), 100 ml high-aspect
rotating vessels (HARV 12.5 cm dia.), and rotating cylin-
drical culture vessels (RCCV 7.5 cm dia.) both at ca.10-2 ×
g (Synthecon, Houston TX). The HARV and RCCV were
used by NASA in early space shuttle experiments, and in a
mini-payload integration center, designed as an in-flight
laboratory.
In all cell assays, apoptotic cells were distinguished mor-
phologically and by the TUNEL reaction [14,15]. Free
taxol, taxanes, and baccatin III in cells and the culture
medium, or bound taxol and taxanes, released after xyla-
nase activity, were determined with competitive inhibi-
tion enzyme-linked immunoassays kits from Hawaii
Biotechnology. Cells were examined by laser confocal
microscopy (Zeiss LSM 410 Invert Scan Microscope) using
single or double-labeling immunocytochemical fluores-
cence (FITC, Cy3) and colloidal gold to reaffirm the sub-
cellular locations of taxol, the taxane ring (baccatin III),
the C-13 side chain of taxol, and taxanes in general. For
samples larger than 10 g fresh biomass, these compounds
were determined by HPLC using authentic standards and
taxil columns (MetaChem Technologies Inc.) [12].
Results
With T. cuspidata suspension in RCCV bioreactors (10-2 ×
g), and in 1 L nippled flasks (3 wks in darkness at 24°C),
free taxol comprised 42 and 21 percent of the total taxanes
(1.1 and 2.4 mg taxol/kg air-dry biomass weight), respec-
tively [13]. Centrifugation of cells at 3 and 24 × g greatly
increased taxane content but reduced taxol recovery. T.
cuspidata and T. brevifolia responded similarly to mechan-
ical stresses, simulated microgravity, and hypergravity by
The reactions of the Krebs-Henseleit (urea) cycle, and their relations to NOS activityFigu e 1
The reactions of the Krebs-Henseleit (urea) cycle, and their relations to NOS activity. Enzymes: 1. ornithine carbamoyl trans-
ferase, 2. argininosuccinate synthetase, 3. argininosuccinate lyase, 4. arginase, 5. nitric oxide synthase, 6. arginine deiminase, 7. 
arginine decarboxylase, 8. numerous enzymes contributing to the formation of substituted guanidino compounds. Reactions 1 
to 4 comprise the urea cycle. Reactions 2, 3, 5, may account for NOS activity in plants (citrulline-NO cycle). Arginine deimi-
nase was reported in chloroplasts but is mostly found in microorganisms. Reactions 7 and 8 comprise decarboxylation, oxida-
tion, methylation, transamidination, phosphorylation, keeping the guanidino group intact or modifying it by methylation, 
phosphorylation, etc. They remove L-arginine as a substrate from the urea cycle, and from reactions 5 and 6. L-arginine repre-
sents an important branch point that links nitrate and ammonium nutrition to protein synthesis and turnover (not shown), to 
the urea cycle, to a postulated citrulline-NO cycle, and to the formation of guanidino compounds (substituted and non substi-
tuted). Through oxygen requirements, the stress-induced NOS activity links respiration to NO, ROS and RNS production, 
their signaling pathways and damaging reactions, e.g., the nitration of phenols and tyrosines residues in cell regulatory proteins, 
and to apoptosis.Page 2 of 4
(page number not for citation purposes)
BMC Plant Biology 2005, 5:S12producing NO bursts, apoptosis, and by the overproduc-
tion of taxol and taxanes.
T. brevifolia heat-killed cells did not produce NO. Gener-
ally 19% of the live cells (unit gravity in shaker flasks for
3 h) were always positive for NO as assayed with DAF-
2DA. With the addition of 10-4 mM SNP, a NO donor, or
after centrifugation at 150 × g for 3 h, nearly all cells pro-
duced NO. However, if 0.5 mM L-NMMA (NOS inhibitor)
was added, only 26% of the cells produced NO. When 0.5
mM D-NMMA, a non inhibitor of NOS, was added, 92%
of the cells produced NO [14].
The greater the NO produced and released by cells, the
more cells became apoptotic. Additions of SNP (10-6 to
10-2 mM) increased apoptosis from 18 to 75%. Centrifu-
gation at 150 g yielded 35% apoptosis. This was reduced
to less than 10% by the addition of the NOS inhibitor, L-
NMMA. Substituting D-NMMA for L-NMMA resulted in
apoptosis (32% cells), but this was not significantly differ-
ent from the cells exposed to 150 g [14]. The NOS require-
ment for oxygen connected NO production to the stress-
induced and damaging increases in free radicals, viz., reac-
tive oxygen species and reactive nitrogen species [2]. The
lesson learned offered guanidino compounds as counter-
measures to NO-mediated stress and apoptosis.
At unit gravity and after 10 d, egg cells exposed to SNP (10-
4 mM) produced 18 mg taxol/Kg fresh weight. This taxol
yield was now 64% higher than the controls without SNP.
The number of cells producing NO with SNP was 86.7%
higher than the controls without SNP. By contrast, the
addition of L-NMMA (0.5 mM) decreased NO production
by 87.3%. Taxol yield was reduced by 75% (4.1 mg/Kg
fresh weight) (unpublished results).
In a separate study at unit gravity with cells supported on
filter paper, the effects of SNP (10-4 mM), sodium tung-
state, (a nitrate reductase inhibitor at 1 mM), and L-
NMMA (0.5 mM) were used to evaluate taxol yields after
5 d. SNP added alone, or tungstate with SNP together,
yielded taxol at 8.0 and 8.1 mg/Kg fresh weight, respec-
tively, i.e., the differences were not significantly different.
This reaffirmed that nitrate reductase activity was not a
contributor to NO-dependent taxol production. L-NMMA
alone, or together with added tungstate, reduced taxol
recovery to 2.1 and 2.0 mg/kg fresh weight, respectively.
Here again, NOS was responsible for the NO that
increased taxol production without a significant contribu-
tion from nitrate reductase. Removing and starving the
control egg cells only slightly increased taxol recovery.
The tetracyclic diterpenoid ring of taxol is synthesized by
cyclization of geranylgeranyl diphosphate to give taxa-
4(5),11(12(-diene) in plastids and in light and darkness
[9,11]. This basic ring structure gives rise to over 300 dif-
ferent taxanes. One of these, baccatin III, can provide the
ring structure for the biosynthesis of taxol. Although the
mechanisms are not yet clear, taxol formation also
requires the addition of a C-13 side-chain originating
from phenylalanine. Additional and finishing structural
changes for taxol involve reaction in the endoplasmic
reticulum and Golgi stacks.
Using specific antibodies, the biosynthesis of the taxol
ring in plastids, and the cytoplasmic assembly of bound
taxol and related taxanes were visualized by laser confocal
and scanning electron microscopy. Gold labeling was
especially useful to identify taxol and taxanes in transport
vesicles, the plasmalemma, cell walls, and in the culture
medium. Colloidal 40 nm-gold-immunolabeled antibod-
ies to taxanes, taxol, and baccatin III were visualized with
7 to 21 nM sensitivity, with and without gold backscatter
(laser confocal and scanning electron microscopy).
In egg cells, baccatin III (taxane ring) was detected in
gravisensing amyloplasts and vesicles associated with
plastid membranes. Baccatin III, taxol, and other taxanes
were transported through the cytoplasm by these vesicles,
which then released their contents to the plasma mem-
brane. Plastids, that moved and docked at the plasma
membrane, also released bound taxol and taxanes into the
plasma membrane. The bound taxol and taxanes at the
cell-surface were deposited in newly formed cell walls or
released into the culture medium. The drug-productive
cells and materials in the culture medium containing
bound taxol, baccatin III, and taxanes were collected with
antibody-labeled paramagnetic beads [16]. Immunogold-
labeled taxol, taxanes and baccatin III were detected in
transport vesicles, plasma membrane fragments, and on
materials recovered from the culture medium.
Bound taxol and taxanes were recovered from xylanase
(commercial and purified) hydrolysates of cell biomass,
debris in the culture medium, and from the wood of trees.
Taxol and other taxanes were characterized and identified
by HPLC using authentic standards. Results indicated that
recovered taxol was bound to xyloglucan oligosaccha-
rides. Probes of cells with an antibody to xyloglucan
endotransglycosylases implied that 'touch' genes nor-
mally expressed under mechanical forces [18], could have
provided additional sites for the attachment of taxol and
other taxanes.
In hypergravity (3 to 150 × g), the taxol and taxanes
released from cells by syneresis, were recovered on hydro-
phobic PVDF (polyvinylidene fluoride) filters.
Cyclodextrins added to the culture medium enhanced
biomass yield and altered the solubility of taxanes to
improve the recovery of taxol and taxanes. The addition ofPage 3 of 4
(page number not for citation purposes)
BMC Plant Biology 2005, 5:S12Publish with BioMed Central   and  every 
scientist can read your work free of charge
"BioMed Central will be the most significant development for 
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central 
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
cyclodextrins to the culture medium improved cell bio-
mass and taxol recovery [17]. Further details are available
in US Patents [16-18,20].
Acknowledgements
This work was impossible without the support from NASA's Microgravity 
Grant No. 9-825. Special thanks go to Mr. Frank Ventimiglia for determining 
and locating taxol in the cell suspensions. Dr. Cristina Pedroso and Helena 
GarcKs carried out the NO, TUNEL and NOS- related studies. Drs. H-C 
Dong and C. Pedroso contributed to the taxol assays using the protocols 
worked out by F. Ventimiglia. A post-doctoral fellowship from China for 
Dr. Dong (China) aided in studies with NO and taxol. Dr. Dorward at the 
National Institutes of Health, Rocky Mountain Laboratory, prepared the 
scanning electron micrographs.
References
1. Durzan DJ: Metabolic engineering plant cells in a space
environment. Biotechnol Genet Eng Rev 2000, 17:349-383.
2. Durzan DJ, Pedroso MC: Nitric oxide and reactive nitrogen
oxide species in plants. Biotechnol Genet Eng Rev 2002, 19:293-337.
3. Durzan DJ: Nitrogen metabolism of Picea glauca. I. Seasonal
changes of free amino acids in buds, shoot apices and leaves,
and the metabolism of uniformly labeled 14C-L-arginine by
buds during the onset of dormancy. Can J Bot 1968, 46:909-919.
4. Durzan DJ: Nitrogen metabolism of Picea glauca. IV. Metabo-
lism of uniformly labeled 14C-L-arginine, [carbamyl-14C]-L-
citrulline, and [1,2,3,4-14C]-γ guanidinobutyric acid during
diurnal changes in the soluble and protein nitrogen associ-
ated with the onset of expansion of spruce buds. Can J Biochem
1969, 47:771-783.
5. Durzan DJ, Steward FC: Nitrogen metabolism. In Plant Physiology,
An Advanced Treatise Volume 8. Edited by: Steward FC. Academic Press,
Inc., New York; 1983:55-265.  Chapt. 2
6. Bidwell RGS, Durzan DJ: Some recent aspects of nitrogen
metabolism. In Historical and Recent Aspects of Plant Physiology: A Sym-
posium Honoring FC Steward Cornell Univ Press, College Agriculture and
Life Science; 1975:162-227. 
7. GarcKs H, Durzan DJ, Pedroso MC: Mechanical stress elicits
nitric oxide formation and DNA fragmentation in Arabidop-
sis thaliana. Annals Bot 2001, 87:567-574.
8. Goodman J, Walsh V: The story of taxol. Nature and politics in the
pursuit of an anti-cancer Drug. Cambridge Univ Press, Cambridge;
2001:282. 
9. Jennewein S, Croteau R: Taxol: biosynthesis, molecular genet-
ics, and biotechnological applications. Appl Microbiol Biotech
2001, 57:13-19.
10. Grothaus P, Bignami GS, O'Malley S, Harada KE, Byrnes JB, Waller
DF, Raybould TJ: Taxane-specific monoclonal antibodies:
Measurement of taxol, baccatin. Nat Prod 1995, 58:1003-1004.
11. Durzan DJ: Gravisensing, apoptosis, and drug recovery in
Taxus cell suspensions. Gravit Space Biol Bull 1999, 12:47-55.
12. Durzan DJ, Ventimiglia F: Free taxanes and the release of bound
compounds having taxane antibody reactivity by xylanase in
female, haploid-derived cell suspension cultures of Taxus
brevifolia. In Vitro Plant Cell Devl Biol 1994, 30P:219-227.
13. Durzan DJ, Ventimiglia F, Havel L: Taxane recovery from cells of
Taxus in micro- and hypergravity. Acta Astronautica 1998,
42:455-463.
14. Pedroso MC, Magalhaes JR, Durzan DJ: Nitric oxide induces cell
death in Taxus cells. Plant Sci 2000, 157:173-181.
15. Havel L, Durzan DJ: Apoptosis in plants. Bot Acta 1996,
109:268-277.
16. Durzan DJ, Ventimiglia F: Recovery of taxanes from plant mate-
rial. US Patent 1999. No.5,981,777. November 9. (Use of paramag-
netic beads)
17. Durzan DJ, Ventimiglia F: Cyclodextrins in plant nutrient
formulations. US Patent 2000. No.6,087,176. July 11 
18. Durzan DJ, Ventimiglia F: Taxane production in haploid-derived
cell cultures. US Patent 1996. No.5,5547,866. November 9
19. Antosiewicz DM, Purugganan MM, Polisensky DH, Braam J: Cellular
localization of Arabidopsis xyloglucan endotransglycosylase-
related proteins during development and after wind
stimulation. Plant Physiol 1997, 115:1319-1328.
20. Durzan DJ, Ventimiglia F: Recovery of taxanes from conifers. US
Patent 1999. No.5,955,621. September 21. (screening taxanes from
Coniferales)Page 4 of 4
(page number not for citation purposes)


Crossref

Cellular localization of Arabidopsis xyloglucan endotransglycosylaserelated proteins during development and after wind stimulation. Plant Physiol

Durzan DJ: Apoptosis in plants. Bot Acta

Durzan DJ: Nitric oxide induces cell death in Taxus cells. Plant Sci

Durzan DJ: Some recent aspects of nitrogen metabolism.

F: Cyclodextrins in plant nutrient formulations.

F: Taxane production in haploid-derived cell cultures.

FC: Nitrogen metabolism. In Plant Physiology, An Advanced Treatise Volume 8. Edited by: Steward FC.

Free taxanes and the release of bound compounds having taxane antibody reactivity by xylanase in female, haploid-derived cell suspension cultures of Taxus brevifolia.

Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions. Gravit Space Biol Bull

Mechanical stress elicits nitric oxide formation and DNA fragmentation in Arabidopsis thaliana. Annals Bot

Metabolic engineering plant cells in a space environment. Biotechnol Genet Eng Rev

Nitric oxide and reactive nitrogen oxide species in plants. Biotechnol Genet Eng Rev

Nitrogen metabolism of Picea glauca. I. Seasonal changes of free amino acids in buds, shoot apices and leaves, and the metabolism of uniformly labeled 14C-L-arginine by buds during the onset of dormancy.

Recovery of taxanes from conifers.

Recovery of taxanes from plant material. US Patent

Taxane recovery from cells of Taxus in micro- and hypergravity. Acta Astronautica

Taxol: biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biotech

The story of taxol. Nature and politics in the pursuit of an anti-cancer Drug.

TJ: Taxane-specific monoclonal antibodies: Measurement of taxol, baccatin. Nat Prod

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1810258

Nitric oxide, cell death and increased taxol recovery

Abstract

Similar works

Full text

Available Versions

Springer - Publisher Connector

Directory of Open Access Journals

Springer - Publisher Connector

Crossref