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First published online June 12, 2003; 10.1104/pp.103.022798 Plant Physiology 132:1631-1641 (2003) © 2003 American Society of Plant Biologists The Function of Ascorbate Oxidase in Tobacco1Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, United Kingdom (C.P., C.H.F.); Unilever Research, Colworth House, Sharnbrook, Bedford, MK44 1LQ, United Kingdom (J.M.F., J.E.W.); and Department of Agricultural and Environmental Science, Newcastle University, Newcastle Upon Tyne, NE1 7RU, United Kingdom (J.D.B.)
The function of the apoplastic enzyme ascorbate oxidase (AO) was investigated in tobacco (Nicotiana tabacum). The abundance of AO mRNA was up-regulated by light. Cytosolic ascorbate peroxidase (APX1) transcripts were also highest in the light. In contrast, L-galactono-  -lactone dehydrogenase, stromal APX, and
thylakoid APX transcripts remained constant over the day/night cycle.
Salicylic acid inhibited growth, increased expression of the
pathogenesis-related protein (PR) 1a, and decreased AO transcript abundance.
In contrast, the application of auxin enhanced growth and increased AO and PR
1a gene expression. Therefore, AO transcript abundance varied in a manner
similar to hormone-mediated changes in plant growth. To study the effects of
modified AO expression on growth, transformed tobacco plants expressing AO in
the sense and antisense orientations were generated. The resultant large
changes in apoplastic AO activity in the transformed tobacco plants had little
effect on whole leaf ascorbate (AA) content, but they had dramatic effects on
apoplastic AA levels. Enhanced AO activity oxidized the apoplastic AA pool,
whereas decreased AO activity increased the amount of AA compared with
dehydroascorbate. A relationship was observed between AO activity and plant
height and biomass. Native AO transcript levels were no longer subject to
light/dark regulation in AO sense and antisense plants. Taken together, these
data show that there is an interaction between hormone, redox, and light
signals at the level of the apoplast via modulation of ion of AA content.
Ascorbate (AA) plays a key role in defense against oxidative stress and is particularly abundant in photosynthetic tissues (Foyer et al., 1983  ;
Smirnoff, 2000). Most (over
90%) of the AA is localized in the cytoplasm, but unlike other soluble
antioxidants, a substantial proportion is exported to the apoplast, where it
is present at millimolar concentrations. Apoplastic AA is believed to
represent the first line of defense against potentially damaging external
oxidants such as ozone, SO2, and NO2
(Plöchl et al., 2000;
Barnes et al., 2002). In the
apoplast, AA is oxidized to monodehydroascorbate (MDHA) by the enzyme
ascorbate oxidase (AO). MDHA is an unstable radical and rapidly
disproportionates to yield DHA and AA. DHA is then transported into the
cytosol through the plasma membrane by a specific carrier that preferentially
translocates the oxidized form in exchange for the reduced form, ensuring a
continuous flux of reducing power to the cell wall
(Horemans et al., 2000).
Perhaps the most intriguing and poorly understood of the enzymes involved in
AA metabolism in plants is the apoplastic AO. No clear biological functions
for AO have been described to date. However, it is widely believed that AO
plays a role in cell elongation because of its extracellular localization and
its high activity in rapidly expanding tissues
(Esaka et al., 1992;
Moser and Kanellis, 1994;
Ohkawa et al., 1994;
Kato and Esaka, 1999). Recent
work has shown that tobacco (Nicotiana tabacum) Bright Yellow-2
protoplasts overexpressing a pumpkin (Cucurbita pepo) AO cDNA
elongate more rapidly than untransformed controls
(Kato and Esaka, 2000).
Several mechanisms whereby AO controls cell growth have been proposed
(Esaka, 1998;
Smirnoff, 2000). MDHA
generated from AA by AO in the apoplast, stimulates cell growth through
enhanced vacuolization (Hidalgo et al.,
1989) and ion uptake caused by depolarization of the plasma
membrane. Moreover, DHA is considered to be responsible for cell enlargement
by promoting cell wall loosening (Lin and
Varner, 1991). Local auxin-mediated production of free radicals
has also been shown to induce cell wall extension
(Joo et al., 2001). Because AA
is believed to be the most important antioxidant in the apoplast of leaves and
stems, its destruction via AO may be important in facilitating cell
expansion.
Plant growth is triggered by auxin. Exogenous applications of auxin can
trigger a variety of cellular processes such as cell elongation and lateral
root growth (Laskowski et al.,
1995
Relatively little information is available on other enzymes involved in AA
synthesis and metabolism. The last step of the pathway of AA synthesis in
plants is relatively well characterized and involves the cytochrome
c-dependent oxidation of L-galactono-1, 4-lactone by the
mitochondrial enzyme L-galactono- The present study was undertaken to elucidate the function of AO in tobacco leaves. We show that expression of AO is regulated by light and by plant hormones that modulate growth. Moreover, we demonstrate that enhanced apoplastic AO activities in transformed tobacco plants favor increased growth. Taken together, these observations provide evidence that AO has a role in the control of growth.
Diurnal Changes in Transcript Abundance of Enzymes Involved in the AA Metabolism The expression of GLDH, sAPX, and tAPX were similar in the dark and in the light (Fig. 1), giving similar values after 16 h of dark or 24 h of light. In contrast, AO transcripts fell to very low levels in the dark and increased only slowly in the light (Fig. 1). The abundance of cAPX1 mRNA was also influenced by light, being minimum in the dark and attaining maximum values in the light (>6 h; Fig. 1). Actin was used as the internal control in these and all of the following experiments after confirmation that the abundance of actin transcripts was unaffected by any of the treatments used. This was achieved by performing actin-specific RT-PCR on RNA samples extracted from leaves subjected to a wide range of light-dark transitions and hormone treatments.
Further controls were performed to rule out the possibility that the observed light-dark changes in AO and cAPX1 transcript levels were due to circadian rhythmicity. No circadian control was found in AO or cAPX1 (data not shown).
Pathogenesis-related protein (PR) 1a was used as a positive control for the
efficacy of the following treatments as its expression has previously been
shown to be triggered by SA (Durner et al.,
1998
A marked increase in AO and PR1a expression was observed 8 h after spraying with the auxin, NAA. In contrast, the expression of GLDH, sAPX, tAPX, or cAPX1 mRNA was unchanged by this treatment (Fig. 3A). SA and NAA had inverse effects on plant growth (Figs. 2B and 3B). The growth of plants sprayed with 1 mM SA was much slower than that of control plants sprayed with low SA concentrations (10 µM; Fig. 2B). In contrast, low concentrations (0.5 µM) of NAA stimulated growth (Fig. 3B).
To further explore the function of AO in tobacco leaves, two constructs for the expression of AO in the sense and antisense orientations were produced. Six independent primary transformants (T0 generation) were selected for each construct on the basis of their ability to survive growth on kanamycin, the presence of the transgene, and their leaf AO activity. Leaf AO activity was markedly different in all the transformants compared with the wild type. AO activity was five to 16 times that of PAO leaves and 0.3 to 0.5 times that of the TAO leaves than the wild type (Fig. 4). Transgene-dependent differences in leaf AO were similar whether plants were grown in tissue culture or in soil (data not shown). Genomic DNA from each of the lines was probed with 2X35S-PAO or 2X35S-TAO DNA fragments to confirm the presence of the transgene and check the copy number of the insertion. All lines tested positive for the transgene (data not shown). Three sense (PAO2-2-1, PAO1-7-2, PAO3-7-2) and two antisense (TAO2-7-1, TAO1-6-1) lines containing only one copy of the introduced sequence were selected for further analysis. These lines were grown for two additional generations (T1 and T2) and the relationship between AO and plant development was followed in each generation.
Plant growth, measured as biomass accumulation over time, was monitored in T1 and T2 populations derived from each selected transgenic line. Each population was composed of 25 individuals germinated on nonselective media, allowing simultaneous measurements of individuals that had passed through the transformation procedure but did not express the transgene and AO-expressing plants. All plants were grown under controlled environment conditions at the same time and shoot fresh weights were recorded weekly from three plants per population. Additional wild-type populations were not necessary in these experiments, given the presence of internal controls within each population. No statistically significant differences in shoot fresh weight were observed in 3-, 4-, or 5-week-old sense (PAO) or antisense (TAO) plants (Fig. 5A). However, between weeks 6 and 8, plants expressing the AO transgene in the sense orientation (PAO) accumulated greater biomass than plants expressing the AO transgene in the antisense orientation (TAO; Fig. 5A). By the end of the experiment (8 weeks), AO sense-expressing plants (PAO) showed a statistically significant increase in fresh weight (P < 0.05 at 7 weeks, and P < 0.01 at 8 weeks) compared with the AO antisense plants (TAO; Fig. 5A). The relative growth (measured as weekly biomass accumulation) rate was linear (r2 > 0.95) between weeks 6 and 8. At this point, the growth rate of AO sense plants (PAO) was 30% higher than that of AO antisense (TAO) plants. Values of 20.8 g fresh weight gain per week were measured in AO sense plants (PAO) compared with 14.1 g fresh weight gain per week in antisense (TAO) plants (Fig. 5A).
Once established that AO sense plants (PAO) showed an increased growth rate when compared with antisense (TAO) plants, growth of sense plants (PAO), measured this time as plant height, was compared with that of wild-type plants. This experiment was conducted to establish whether a correlation between AO activity and plant height could be drawn. T1 and T2 seeds from two AO sense lines (PAO2-2-1 and PAO3-7-2) that had been selected on the basis of high AO activity, were germinated on media containing kanamycin to select for plants containing the transgene. Once this was confirmed by PCR (data not shown), PAO plants were transferred to compost and were grown in controlled environment chambers together with wild-type populations. After 6 weeks, AO activities were measured in young fully developed leaves of four to five plants per line, and the height of the corresponding plants was recorded. Overall, T2 plants exhibited higher AO activity and a taller phenotype than their respective T1 progenitors (Fig. 5B). A positive correlation was demonstrated between AO activity and plant height (Fig. 5B; r2 = 0.85). Analysis of variance of the data confirmed that T2 PAO lines grew significantly taller than T1 PAO lines and that T1 and T2 PAO lines grew taller than the wild type (P < 0.01; Fig. 5, B and C).
Whole leaf AA, DHA contents, and AO activities were measured in extracts from discs excised from young fully expanded leaves of 6-week-old T1 sense (PAO lines PAO2-2-1 and PAO3-7-2) and antisense (TAO lines TAO2-7-1 and TAO1-6-1) plants. PAO plants exhibited about a 40-fold higher leaf AO activity than the wild type (Table I), whereas TAO lines exhibited 0.4-fold less leaf AO activity. Apoplastic AO activity was measured in whole leaf extracts, soluble, and ionically bound fractions (Table I). Total AO activity measured in whole leaf extracts was found to be entirely associated with the cell wall (ionically bound) and localized in the apoplast (Table I). AO activity was undetectable in soluble fractions of leaf extracts. TAO leaves contained similar amounts of AA and DHA to the wild type, but PAO leaves contained less AA and total (AA plus DHA) AA (Table I). DHA contents were higher in PAO lines, but the leaf ascorbate pool was always largely reduced, values being slightly higher for wild-type (92%) than PAO plants (85%). In contrast to whole leaf AA, modified AO expression resulted in dramatic changes in the apoplastic AA pool. The total (AA plus DHA) AA content of the apoplast was at least double that of the wild type in sense and antisense plants (Table I). However, the greatest effect was on the AA redox state (Table I). In the wild type, the AA pool in the apoplast was about 40% reduced, whereas it was 66% reduced in the antisense plants and only 3% reduced in the sense plants. The DHA content in the apoplast of sense plants was 3.5-fold greater than in wild-type or antisense plants. For apoplastic AA and DHA measurements, cytosolic contamination of the apoplast (monitored by measuring Glc-6-P in intercellular washing fluid (IWF) was maintained below 0.1% (data not shown). However, it should be noted that the presence of apoplastic acid phosphatases may lead to underestimation of the contamination of the apoplastic fraction as well.
AO transcript abundance was highest in the light and showed a marked light/dark expression pattern in wild-type plants (Fig. 1). The following experiments were performed to investigate whether such marked diurnal patterns of expression were maintained in the native wild-type AO of PAO and TAO transgenic plants. Due to the sequence similarity between the tobacco and the pumpkin AO (used for the generation of the AO sense plants), careful design of species-specific primers was necessary to allow expression patterns of native and transgenic AO to be discriminated. No expression patterns in the expression of the transgenic AO (PAO) were expected given the presence of the strong constitutive promoter 35S cauliflower mosaic virus (CaMV). The specificity of TAO (specific for tobacco AO) and PAO (specific for pumpkin AO) primers was confirmed by sequencing of the relative PCR products. They were then used to analyze by RT-PCR the expression of the native tobacco AO and the introduced pumpkin AO (PAO) in the leaves of T1 and T2 generations of transformants grown in soil under controlled environment conditions. Samples from wild type, three sense (PAO), and two antisense (TAO) lines were harvested after 16 h of light or 16 h of dark (Fig. 6). PAO was highly and equally expressed in all the sense PAO leaves in the light and in the dark. This is consistent with the constitutive expression of PAO under the control of the CaMV35S promoter. Moreover, PAO plants showed no cosuppression of the native wild-type AO because the transcript abundance of the native wild-type tobacco AO transcripts was similar in the sense PAO lines and in the wild type. In contrast to the strong dark/light pattern of AO expression observed in wild-type plants, no dark/light effects on native wild-type tobacco AO transcript abundance was detected in the PAO sense lines. In this case, the amounts of native AO transcripts were similar in the light and dark (Fig. 6). There was a large overall decrease in native AO transcripts in TAO leaves compared with the wild type (Fig. 6), demonstrating the efficacy of the antisense suppression strategy adopted. However, in the same TAO plants, there was also a decrease in the observed stimulation of native wild-type AO expression by light (Fig. 6).
Although AO activity was first described in plants many years ago, its biological function has remained elusive. Much circumstantial evidence has linked tissue AA contents to growth (Chinoy, 1984 ), but to date,
the mechanism has remained unknown and largely unexplored. Recent evidence has
provided an indication of the mechanism whereby AA is involved in the
regulation of growth (Pastori et al.,
2003). Several lines of evidence also suggest AO may be involved
in this process (for example, see Esaka,
1998). The results presented here show that the expression of
native AO is regulated by light but that this control is lost when overall AO
transcript abundance is manipulated in transgenic plants; AO transcripts are
modified by auxin and SA in a manner consistent with effects on growth, and
enhanced AO activity oxidizes the apoplastic AA pool and this can stimulate
growth in certain conditions. Taken together, these observations demonstrate
that AO activity can influence plant growth.
We report here the effects of light and dark over a 24-h period on the
expression patterns of genes involved in AA metabolism. The expression of
cAPX1 is regulated by light in tobacco
(Fig. 1), consistent with
earlier observations of APX1 mRNA abundance in tobacco
(Tabata et al., 2002
Complex transcriptional and translational controls modulate AO expression
(Esaka et al., 1992
An induction of AO by auxin, similar to that observed here, was reported in
pumpkin (Esaka et al., 1992
High concentrations of SA (1 mM) caused a pronounced inhibition
of the growth of tobacco seedlings (Fig.
2B). SA inhibition of plant growth and development is a well-known
phenomenon (Petersen et al.,
2000
The modification of AO expression in tobacco by sense
(PAO) and antisense (TAO) technologies has allowed us to
explore the relationship between AO and growth in planta. To minimize the risk
of gene silencing by cosuppression, we used a pumpkin AO cDNA for the
sense construct, which shares approximately 68% homology with the tobacco
sequence. This choice proved to be successful, as no silencing of the native
AO was found in the sense (PAO) lines. The native AO gene was
expressed at wild-type levels in PAO lines
(Fig. 6). For the antisense
suppression of AO, we used one-third of the 5' sequence of the
tobacco cDNA, as in recent years, the use of only partial cDNA sequences
proved to be very effective in antisense suppression
(Bourque, 1995 The data presented here demonstrate that our genetic approach was successful in significantly modifying apoplastic AO activity in leaves, as increases of up to 40-fold were measured in PAO sense transformed lines, whereas activities were halved in antisense TAO lines. We were unable to measure AO protein abundance in this study, as the AO antibodies that we prepared did not have sufficient specificity. Nevertheless, the parallel changes in transcripts and AO activities reported here allow us to assume that our manipulation also modified amounts of AO protein.
PAO sense lines contained less (22%–27%) total AA and AA and
more (50%) DHA than TAO antisense lines or wild-type plants
(Table I). No differences in
leaf AA content were detected between TAO antisense and wild-type
plants. The effect of high AO activity on the whole leaf AA pool is minimal,
taking into account the fact that AO activities in sense lines were increased
by up to 40-fold. The observation that such large increases in AO activity did
not lead to major changes in leaf AA may be explained by differential
localization of the two components (Lin
and Varner, 1991
In the present study, we demonstrate that increasing AO activity by 40-fold
leads to enhanced biomass accumulation and elongation in tobacco plants
(Fig. 5). In contrast,
reduction of AO activity by antisense technology results in a reduction in
growth rate (Fig. 5A). Taken
together, the data presented here suggest that enhanced AO activity can have a
positive effect on growth (Fig.
5B). Increased biomass production in PAO sense plants appears to
be mainly due to enhanced internode elongation. It is possible that AO action
alone stimulates growth through the generation of MDHA radicals in the
apoplast. This enhanced growth could occur by a chemorheological wall-loosing
reaction as described for superoxide- and hydroxyl radical-mediated extension
growth (Schopfer et al.,
2002
Plant growth is the outcome of various mechanisms of regulation, signaling,
and crosstalk. We have provided in planta evidence that AO is one of these
components and therefore participates to this broad dialogue between signaling
molecules and environmental cues. It should be noted that in another study on
modulation of AO in tobacco, no effects on growth were reported
(Sanmartin at al., 2003 In conclusion, the data presented here suggest that factors such as apoplastic redox state that is dominated by AA and regulated by AO modulate receptor function and signal transduction and that there is scope for modulation and interaction between different signals (hormone, redox, and light) in the apoplast.
Plant Material and Growth Conditions Tobacco (Nicotiana tabacum cv Petit Havana, mutant SRI) seeds were germinated in petri dishes on moistened filter paper. After 10 d, seedlings were transferred to compost (Petersfield Products, Leicester, UK) in pots. Plants were grown at 22°C day/night in controlled environment chambers supplying a photosynthetic photon flux density of 250 µmol m–2 s–1 at plant height as a 16-h photoperiod. Young fully expanded leaves from 6-week-old plants were used for AA measurements in the light and dark. Three-week-old plants were used for all treatments and RT-PCR analyses.
Three-week-old seedlings were sprayed to run-off (using an aerosol spray bottle supplied by Nalgene, Rochester, NY) with 10 µM and 1 mM SA and 0.5 µM NAA. After treatment, plants were kept in continuous light for 24 h and whole shoots were harvested for RT-PCR analysis. For the evaluation of the effects of hormones on growth, sprayed plants were subsequently returned to "growth conditions" until further analysis 1 week after the initial harvest.
Cloning of the genes encoding AO from pumpkin (Cucurbita pepo; PAO; EMBL GenBank accession no. X55779) and tobacco (TAO; accession no. D43624) was achieved from PCR amplification of full-length cDNAs using primers designed to add additional restriction sites to facilitate the cloning. For PAO the primers were 5'-ACCACTCGAGATGCTTCAGATG -3' (nucleotide position 18–29 of cDNA; added XhoI site is underlined) and 5'-ACCAGAGCTCTTAGGGGTTATTT-3' (nucleotide position 1,757–1,745; added SacI site is underlined). For TAO the primers were 5'-ACCAGAGCTCATGGCTTCCTTA-3' (nucleotide position 88–100 of cDNA; added SacI site is underlined) and 5'-ACCACTCGAGTTTGTGCCACC-3' (nucleotide position 609–559; added XhoI site is underlined). The PCR conditions were 10 cycles of 1 min at 94°C, 1 min at 40°C, and 1.5 min at 72°C, and 15 cycles of 1 min at 94°C, 1 min at 61°C, and 1.5 min at 72°C, using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA), 1 ng of cDNA, and 0.2 µM each primer in a 50-µL reaction. At the end of the cycles, the reactions were incubated at 72°C for 10 min. The identity of the 1.7-kb (PAO) and 520-bp (TAO) PCR products was confirmed by single-strand sequencing (ABI PRISM, 310 genetic analyzer; Perkin-Elmer, Warrington, Cheshire, UK).
PAO and TAO PCR products were subcloned as
XhoI-SacI fragments into the corresponding sites of pp5ln
(derived from pUC19; Frenken et al.,
1999
Sterile cultures of tobacco were transformed with cj102-PAO and
cj102-TAO constructs by A. tumefaciens leaf disc infection
(Gallois and Marinho, 1995
Genomic DNA (5 µg) from PAO sense and TAO antisense lines was digested with HindIII, which cuts once within the transgenic sequence, run on an 0.8% (w/v) agarose gel and blotted onto a nylon membrane (Hybond-NX; Amersham Life Science, Buckinghamshire, UK). The membranes were prehybridized at 55°C for 2 h in hybridization buffer (Amersham Life Science) and were hybridized overnight in the same buffer. The HindIII/SacI fragments 2x35S-PAO and 2x35S-TAO were alkaline phosphatase labeled and were used as probes (AlkPhos Direct; Amersham Life Science). Hybridization temperatures were 65°C for 2x35S-PAO and 68°C for 2x35S-TAO. The membranes were washed according to Amersham's recommendations and detection was performed with CDP-Star (Amersham Life Science) by exposing Hyperfilm enhanced chemiluminescence film (Amersham Life Science) to the membrane for 1.5 h.
Total RNA was extracted from using RNeasy plant mini kit (Qiagen, West Sussex, UK) according to the supplier's recommendation. Residual DNA was removed with DNase I, Amp Grade (Invitrogen, Strathclyde, UK). The absence of DNA contamination in the samples was confirmed by a saturating PCR of 40 cycles using actin- (X63603) specific primers (5'-CGCGAAAAGATGACTCAAATC-3' and 5'-AGATCCTTTCTGATATCCACG-3'), which give a 687-bp product with genomic DNA and a 533-bp product with cDNA. One microgram total RNA was reverse transcribed using 0.5 µg of Oligo (dT)12–18 (Invitrogen) and 200 units of Superscript II (Invitrogen) following the supplier's recommendation. cDNA samples were standardized by PCR for actin content using the gene-specific primers. On the basis of the published sequences, the following gene-specific primers were designed and used for amplification: PR-1a (X12737), 5'-GCCTTCATTTCTTCTTGTCTC-3' and 5'-TTAGTATGGACTTTCGCCTC-3'; GLDH (AB048530), 5'-TTTTAGGCTTTGACTGTGGTG-3' and 5'-TCAGATGAAGAGCTTCTCAAG-3'; cAPX1 (X59600), 5'-CTCAAGCTGTTGACAAATG-3' and 5'-AGCTTCAGCAACCAATTC-3'; sAPX (AB02 2274), 5'-TTGT TTCAGTTGGCCAGTGC-3' and 5'-CGCTGCCTTGTGTAGG-3'; tAPX (AB022273), 5'-TGTTTTCTACAGAATGGGC-3' and 5'-GTTGAGTATTTTG CTGCCAC-3'; PAO (X55779), 5'-TTGACCGGAGCAAAAACTTC-3' and 5'-AATTCAATGACGACTCCTCC-3'; and TAO (D43624), 5'-AACCAAAAACACCTCAAGGC-3' and 5'-GGTGCTTGTTTTAGGACATC-3'. For semiquantitative RT-PCR, the cycle number was kept within the linear range (30 cycles) and the conditions were 3 min at 94°C, cycle of 45 s at 94°C, 30 s at 52°C, and 45 s at 72°C, followed by 10 min at 72°C, using 0.5 µL of the RT reaction and 0.2 µM each oligonucleotide primer in a total volume of 25 µL. The identity of the PCR products was verified by single-strand sequencing (ABI PRISM, 310 genetic analyzer; Perkin-Elmer). RT-PCR products were loaded on 2% (w/v) agarose gel containing 0.5 µg mL–1 ethidium bromide and the band intensities were quantified with the Eagle Eye II (Stratagene).
Leaf samples were powdered in liquid nitrogen, then 1 mL of ice-cold 1
N HClO4 was added per 0.1-g sample (fresh weight).
Extracts were then centrifuged at 14,000 rpm for 5 min at 4°C. HEPES
buffer (0.1 M, pH 7.0) was added at a buffer:extract ratio of 1:5
(v/v). K2CO3 (5 M) was added until the
extract reached pH 5.6. The extracts were again centrifuged at 14,000 rpm,
this time for 2 min, to allow the removal of precipitated
K2ClO4, and were then assayed for AA and DHA as
described by Foyer and coworkers
(1983
IWF was prepared using a method similar to that adopted by Turcsànyi
et al. (2000
For total AO activity, leaf tissue was powdered in liquid nitrogen and then
homogenized with 0.1 M sodium phosphate, pH 6.5 (1 mL 0.1
g–1 fresh weight). The extract was then diluted
10-fold in the same buffer and 50 µL was used in the assay. Measurements of
soluble and ionically bound AO activity were performed according to Sanmartin
et al. (2003
AO activity was determined from the decrease in A265 at
25°C in a reaction mixture containing 0.1 M sodium phosphate,
pH 5.6, 0.5 mM EDTA, and 100 µM AA. One unit of AO
activity was defined as the oxidation of 1 µmol AA
min–1 at 25°C. An extinction coefficient for
AA of 14 mM–1
cm–1 at 265 nm was used in calculations
(Nakano and Asada, 1981
T1 populations were obtained by germinating T1 seeds on nonselective medium. Twenty-five plants per each transgenic line (three AO sense and two AO antisense) were used for growth experiments. Growth curves were drawn by measuring every week shoot fresh weight of three plants per line growing in compost in controlled environment. Shoots were excised at the base of the stem and were weighed for the fresh weight determination. Plant height was measured on 7-week-old plants from the basis of the stem to inflorescence.
Statistical analyses were performed using GENSTAT 5
(Peyne et al., 1993
We thank Prof. Muneharu Esaka (Hiroshima University, Japan) for the kind donation of the pumpkin and tobacco AO cDNAs and Sue Robinson and Dr. Karl Hunter (Unilever Research) for performing the large-scale greenhouse study of transgenic tobacco plants. Received February 28, 2003; returned for revision March 23, 2003; accepted April 6, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.022798.
1 This work was supported by Unilever Foods. * Corresponding author; e-mail christine.foyer{at}bbsrc.ac.uk; fax 01582–763010.
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ABSTRACT
RESULTS
-Naphthaleneacetic Acid (NAA) on AO
Transcript Abundance and Plant Growth
) and antisense
(
) AO transgenic T1 populations measured as the increase in
shoot fresh weight per week. All plants were grown under controlled
environment conditions. B, Correlation between AO activity and height in
7-week-old AO sense (PAO) and wild-type plants. Squares, wild type;
circles, T1 generation; triangles, T2 generation. Each
activity value represents the mean of three independent measurements. Mean
values for AO activities are 0.05 ± 0.00 for wild type; 1.4 ±
0.5 for T1 generation; 3.0 ± 0.4 for T2
generation. Mean values for plant heights are 33 ± 5.0 for wild type;
42 ± 9.8 for T1 generation; 50 ± 8.0 for
T2 generation. This experiment was repeated three times and similar
results were obtained. C, Phenotype of wild-type and 7-week-old AO sense
tobacco plants (PAO).
-glucuronidase
cassette. The cj102-PAO and cj102-TAO constructs obtained
were transformed into A. tumefaciens LBA4404 by electroporation
(Parry et al., 1998
