Journal of Experimental Botany, Vol. 52, No. 357, pp. 811±820, April 2001
Responses to bleaching herbicides by leaf chloroplasts
of maize plants grown at different temperatures
F. Dalla Vecchia1, R. Barbato2, N. La Rocca1, I. Moro1 and N. Rascio1,3
1
Dipartimento di Biologia, UniversitaÁ di Padova, Via U. Bassi 56uB, I-35131, Padova, Italy
Dipartimento di Scienze e Tecnologie Avanzate, UniversitaÁ del Piemonte Orientale `Amedeo Avogadro',
Corso Borsalino 54, I-15100, Alessandria, Italy
2
Received 19 July 2000; Accepted 10 October 2000
Abstract
The effects of growth temperature on chloroplast
responses to norflurazon and amitrole, two herbicides inhibiting carotenogenesis, at phytoene desaturation and lycopene cyclization, respectively, were
studied in leaves of maize plants grown at 20 8C
and 30 8C in light. At the lower temperature both
chemicals caused severe photo-oxidative damage
to chloroplasts. In organelles of norflurazon-treated
leaves neither carotenoids nor chlorophylls were
detectable and the thylakoid system was dismantled.
In organelles of amitrole-treated leaves lycopene was
accumulated, but small quantities of b-carotene and
xanthophylls were also produced. Moreover, some
chlorophyll and a few inner membranes still persisted, although these latter were disarranged, lacking essential protein components and devoid of
photosynthetic function. The increase in plant growth
temperature to 30 8C did not change the norflurazon
effects on carotenoid synthesis and the photooxidative damage suffered by chloroplasts. By contrast, in organelles of amitrole-treated leaves a large
increase in photoprotective carotenoid biosynthesis
occurred, with a consequent recovery of chlorophyll
content, ultrastructural organization and thylakoid
composition and functionality. This suggests that
thermo-modulated steps could exist in the carotenogenic pathway, between the points inhibited by the
two herbicides. Moreover it shows that, unlike C3
species, C4 species, such as maize, can express
a strong tolerance to herbicides like amitrole, when
3
supplied to plants growing at their optimum temperature conditions.
Key words: Amitrole, carotenoid biosynthesis, chloroplast
photo-oxidation, norflurazon, Zea mays.
Introduction
Carotenoids are integral components of photosynthetic membranes, involved in different tasks required
for plastid organization and function. They contribute, as
antenna pigments, to light harvesting for photosynthesis
(Young et al., 1998), and recently it has been ascertained
that carotenoids can also operate in thylakoid membranes
as stabilizers of the lipidic phase (Havaux, 1998). However,
the most important role of these pigments is the protective
one against photo-oxidative damage of photosynthetic
apparatus, played by quenching triplet chlorophyll and
singlet oxygen (Young, 1991).
The necessity for carotenoid photoprotection is underlined by chlorophyll bleaching and photodamage suffered
by membranes and other chloroplast components of lightgrown seedlings when deprived of these pigments due
to herbicide treatments (Feierabend et al., 1979; Sagar
and Briggs, 1990) or mutations (Faludi-Daniel et al.,
1968; Walles, 1972; Nielsen, 1974).
Despite the essential role of carotenoids in preserving
an ef®cient photosynthetic apparatus, on which plant
survival depends, the knowledge of their biosynthesis in
leaf chloroplasts is still rather scarce, most information
about the carotenogenic pathway coming from studies
To whom correspondence should be addressed. Fax: q39 49 8276280. E-mail: [email protected]
Abbreviations: AM, amitrole; amitrole, (2-amino-1,2,4-triazole); BSA, bovine serum albumin; CP43, chlorophyll-protein 43; CP47, chlorophyll-protein
47; cyt, cytochrome; HEPES, N-2-hydroxyethylpiperazine-N9-2-ethanesulphonic acid; HPLC, high performance liquid chromatography; LHCII, light
harvesting chlorophyll aub-protein complex of PSII; NF, norflurazon (4-chloro-5-(methylamino)-2-(a,a,a-trifluoro-m-tolyl)-3-(2H)-pyridazinone); PBS,
phosphate buffer saline; PSII, photosystem II; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate; TBS, TRIS buffer saline;
TRIS, (hydroxymethyl); aminomethane buffer saline; Tricine, N-(tris-hydroxymethyl)glycine.
ß Society for Experimental Biology 2001
812
Dalla Vecchia et al.
carried out on chromoplasts of ripening fruits. Very little
is known, moreover, regarding mechanisms regulating
carotenoid synthesis in chloroplasts, which might be
different from those operating in chromoplasts (Fraser
et al., 1994).
However, previous research carried out on leaves
of the lycopenic tigrina 034 mutant of barley (Casadoro
et al., 1983) and of barley plants treated with amitrole,
a bleaching herbicide inhibiting lycopene cyclization
(Agnolucci et al., 1996), suggested that temperature could
be a factor affecting carotenoid biosynthesis. In both
cases, in fact, chloroplasts of plants grown at 20 8C
accumulated lycopene and produced very low quantities
of protective carotenoids, suffering chlorophyll photooxidation and great damage of thylakoid membranes. By
contrast, the organelles of plants grown at 30 8C showed
a notable increase in carotenoid biosynthesis, with
recovery of their structure and function.
Thus, it was deemed important to widen the research
by studying the leaf behaviour of maize plants treated
with amitrole and grown at 20 8C and 30 8C. This was in
order to clarify whether, in a C4 species which requires
growth temperatures (30±35 8C) higher than a C3
(20±25 8C), changes in carotenoid production would
occur in the same range of temperature in which they were
noticed in barley. Moreover, in addition to amitrole,
nor¯urazon, which inhibits phytoene desaturation, was
used in order to analyse if plant responses to this latter
herbicide would also be affected by plant growth temperature. The plant supply with the two chemicals affecting
different points of the carotenogenic pathway might be
useful to provide some information on the location of
eventual thermo-modulated steps.
Carotenoid content and composition, chlorophyll
amounts and chloroplast membrane organization and
functionality were analysed with biochemical, ultrastructural and immunological methods in leaves of maize
plants treated with herbicides and grown at the two
different temperatures.
Materials and methods
Plant material
Grains of maize (Zea mays L. cv. Dekalb DK 300) were germinated and seedlings were grown at 20 8C or 30 8C in a growth
chamber with a 12 h photoperiod, light of 200 mmol m 2 s 1
and relative humidity of 80%, on an inert substrate (vermiculite)
moistened with water (control plants), 200 mM AM or 100 mM
NF. A plant series was also grown in the same way and in the
same chamber on water or on the two herbicide solutions for 5 d
at 20 8C and then moved to 30 8C.
All the analyses were carried out on the second leaf of
9±10-d-old plants.
Total pigment analysis
Total chlorophylls and carotenoids from leaf tissues were
analysed in a spectrophotometer (GBC UVuVIS 918) after
extraction with N,N-dimethylformamide. The extinction
coef®cients proposed earlier (Lichtenthaler, 1989) were used to
determine the pigment concentrations.
Reverse phase HPLC
One gram of fresh leaf tissue was ground in a mortar with liquid
nitrogen and 200 mg of NaHCO3, in green safe light. The ®ne
powder was transferred with 50 ml of acetone to a Buchner ®lter
funnel and washed with 10 ml of ethyl acetate. After evaporation up to a ®nal volume of 1 ml, 50 ml of the extract was
injected into an Ultrasphere C18 reverse phase ion pair column
(ODS 250 3 4.6 mm I.D., 5 mm) with a guard column. Elution
was performed by using a concave gradient of 80% methanol to
100% ethyl acetate for 16 min. The gradient was generated by
two pumps controlled by a system controller, with a ¯ow rate of
0.750 ml min 1. The eluted pigments were detected at 430 nm
and identi®ed by comparing the obtained retention times with
those of published chromatograms (Casadoro et al., 1983;
Agnolucci et al., 1996).
Assay of leafuenvironment oxygen exchanges
Oxygen release or uptake was measured with an oxygen
electrode (YSI Model 53, Yellow Springs Instruments) on small
pieces of leaf tissues (according to the method adopted by Ishii
et al., 1977).
Electron microscopy
Samples from leaf tissues were ®xed overnight at 4 8C in 3%
glutaraldehyde in 0.1 M sodium cacodylate (pH 6.9) and then
processed for the electron microscopy (Rascio et al., 1991).
Ultrathin sections, cut with an ultramicrotome (Ultracut,
Reichert-Jung), were observed with a transmission electron
microscope (TEM 300, Hitachi) operating at 75 kV.
All the observations were focused on chloroplasts of
mesophyll cells, because in maize leaves only the organelles of
these cells show an inner membrane system with grana and
stroma thylakoids and contain photosystem II.
Isolation of thylakoid membranes
Thylakoids were isolated by grinding leaf tissues in ice-cold
buffer containing 0.1 mol l 1 Tricine (pH 7.8), 5 mmol l 1
MgCl2, 15 mmol l 1 NaCl, and 0.33 mol l 1 sorbitol. After
centrifugation at 10 000 g for 20 min, the pellet was resuspended
in 50 mmol l 1 HEPES (pH 7.2), 5 mmol l 1 MgCl2 and
centrifuged as before. Thylakoids were then resuspended in
the same buffer containing 0.1 mol l 1 sorbitol, recentrifuged at
40 000 g for 10 min and ®nally resuspended in a solution
containing 50 mmol l 1 NaHCO3, 50 mmol l 1 dithiothreitol
and 20% sucrose.
SDS-PAGE and immunoblotting
Gel electrophoresis in the presence of 0.1% SDS and 6 mol l 1
urea was carried out as described earlier (Laemmli, 1970), with
modi®cations (Gounaris et al., 1988), using a 12±18% linear
acrylamide gradient. Forty micrograms of protein per line were
loaded. Protein concentration was measured using the Bio-Rad
Protein Assay method (Bio-Rad) (Bradford, 1976). Proteins
were electroblotted onto a nitrocellulose column (Sartorius,
0.45 mm) (Dunn, 1986). Before immunodetection, proteins were
visualized by staining the ®lters with 0.2% Ponceau S in 3%
Herbicide effects on maize chloroplasts
813
trichloracetic acid. Filters, blocked with 10% skimmed milk
in TBS, were incubated with rabbit primary antibody and
biotinylated goat-antirabbit IgG and then with streptavidin
alkaline phosphatase-conjugated (Sigma). Coloured bands were
observed upon the addition of nitroblue tetrazolium and
4-chloro-5-bromo-p-indolylphosphate.
Polyclonal antibodies against some polypeptides of PSII were
used in this study: anti D1 (Barbato et al., 1991) and anti D2
(Barbato et al., 1992a) of the reaction centre, anti CP43 and anti
CP47 of the inner antennae (Barbato et al., 1992b), anti LHCII
(Barbato et al., 1992b) and anti 22 kDa (Barbato et al., 1995).
Antibody against cyt f belonging to the cyt b6uf complex, was
also used. This last antibody was a gift of Dr E Bergantino
(Department of Biology, University of Padova).
Results and discussion
Plants grown at 20 8C
Phenotypes: Maize plants submitted to the distinct
experimental treatments showed very different phenotypes (Fig. 1). Compared to the green control plants, the
leaves of plants supplied with AM were pale yellow,
whereas the NF-treated ones exhibited completely white
leaves. Moreover, plants treated with both herbicides
were shorter than the control ones.
This reduced growth could be explained by curtailed
availability of organic nutrients due to lack of green
photosynthetic tissues. However, it could also depend on
an abscisic acid (ABA) de®ciency, related to inhibited
carotenogenesis (Gamble and Mullet, 1986; Neill et al.,
1986; Zeevaart and Creelman, 1988), xanthophylls being
involved in ABA biosynthetic pathway (Liotenberg et al.,
1999; Cutler and Krochko, 1999). A slower growth rate
has also been noticed in plants of ABA-de®cient `wilty'
mutant of pea (De Bruijn et al., 1993) and ascribed to the
disturbed water relations caused by the diminished
hormone production.
Photosynthetic pigments: The pigment analyses (Fig. 2a)
and their identi®cation by HPLC (Fig. 2b), showed
the presence of some coloured carotenoids and chlorophylls in organelles of AM-treated leaves, although very
reduced with respect to those of the control ones
(Fig. 2a). Moreover, the HPLC analyses revealed a
peculiar carotenoid spectrum where, besides small quantities of main xanthophylls and b-carotene, the lycopene
peak was also recognizable (Fig. 2b).
In maize chloroplasts, as already shown in barley ones
(Agnolucci et al., 1996), the AM-treatment, inhibiting
lycopene cyclization, still permitted the synthesis of
detectable carotenoid amounts, and this could account
for some chlorophyll photoprotection. Interestingly, in
organelles of barley lycopenic mutants a comparable situation, with lycopene accumulation and the presence of
small quantities of photoprotective carotenoids and
chlorophylls was noticed (Casadoro et al., 1983).
Fig. 1. Phenotypes of 10-d-old plants of maize grown on water (C),
200 mM amitrole (A) or 100 mM nor¯urazon (N) at different temperatures. (a) Plants grown at 20 8C. (b) Plants grown at 30 8C. (c) Plants
grown for 5 d at 20 8C and then moved to 30 8C.
Differently, the absence of any coloured carotenoids
and chlorophylls was noticed in leaf plastids of NFtreated plants (Fig. 1a, b), thus showing that inhibition
of phytoene desaturase and interruption of photoprotective pigment biosynthesis brought about the complete
photo-oxidation and disappearance of chlorophylls.
Chloroplast ultrastructure: In the mesophyll cells of
control leaves the mature chloroplasts showed an abundant and well organized inner membrane system and
814
Dalla Vecchia et al.
Fig. 2. Photosynthetic pigments in leaves of control (C), amitroletreated (A) or nor¯urazon-treated (N) plants grown at 20 8C. (a) Total
carotenoid and chlorophyll contents. The values represent the mean of
three measurements. NDˆnot detected. (b) HPLC chromatograms of
carotenoids and chlorophylls. Key: 1, neoxanthin; 2, violaxanthin; 3,
lutein; 4, chlorophyll b; 5, chlorophyll a; 6, lycopene; 7, b-carotene.
Fig. 3. Plastids of mesophyll cells from leaves of control or herbicidetreated plants grown at 20 8C. The organelle of a control leaf (a) exhibits
a well-organized inner membrane system, with grana thylakoids (gt) and
stroma thylakoids (st) and a stroma rich in ribosomes. The plastids of
an amitrole-treated leaf (b) contain masses of irregularly distributed
thylakoids (arrows) and long electron-dense membrane stacks (double
arrows). A reduced number of ribosomes are recognizable in the stroma.
The plastid of a nor¯urazon-treated leaf (c) shows only two membrane
pro®les (arrow) in the stroma deprived of ribosomes (barsˆ1 mm).
a ribosome-rich stroma (Fig. 3a), while in the corresponding tissues of AM-treated leaves severe damage was
suffered by chloroplasts (Fig. 3b), in which loss of part
of the ribosomes and reduction and alteration of the
thylakoid system occurred. This latter consisted of a few
membranes irregularly distributed in the stroma or tightly
pressed to form very electron-dense stacks. These ultrastructural features, seem to be characteristic of lycopeneaccumulating organelles which have been described by
other authors in damaged chloroplasts from maize
Herbicide effects on maize chloroplasts
Fig. 4. Immunoblotting of thylakoids isolated from plastids of control
(lanes C) and amitrole-treated (lanes A) plants grown at 20 8C, with anti
D1, D2, CP43, and CP47 (a), anti 22 kDa and LHCII (b), and anti cyt f (c).
(HudaÂk, 1998) and barley (Nielsen, 1974; Casadoro et al.,
1983) lycopenic mutants, and also noticed in organelles of
AM-treated barley plants (Agnolucci et al., 1996). Thus,
in chloroplasts of AM-treated plants, the maintained
ability to synthesize small quantities of photoprotective
carotenoids led to the preservation of some chlorophyll,
as well as a certain number of inner membranes.
In the mesophyll cells of NF-supplied leaves, the
plastids looked impressively damaged. They were almost
devoid of thylakoids and with few ribosomes recognizable
in a rather empty stroma (Fig. 3c). Thus, the total
carotenoid-de®ciency gave rise to photo-oxidative events
which, besides chlorophyll degradation, brought about
dismantling of thylakoids and demolition of most 70S
ribosomes.
815
Thylakoid membrane composition: The analysis of thylakoid composition, carried out on chloroplasts of control
and AM-treated plants showed that, besides organization, thylakoid composition was also dramatically
altered in the organelles of the herbicide-supplied leaves.
Western analysis, accomplished with antibodies against
essential components of PSII, particularly sensitive to
photo-oxidative damage (Aro et al., 1993), revealed that
these membranes lacked chlorophyll a-binding polypeptides encoded by the organelle DNA, such as D1 and
D2 of the reaction centre and the inner antennae CP43
and CP47 apoprotein (Fig. 4a). However, in the altered
thylakoids the same analysis showed a certain amount,
although reduced with respect to that of control chloroplasts, of the cyt f apoprotein (Fig. 4c), which is also
encoded by the plastid DNA.
The ®nding of this latter polypeptide showed that, in
contrast to the assumption by Feierabend et al., ribosomes still present in damaged organelles were functional
and able to sustain a certain, though limited, protein
synthesis (Feierabend et al., 1979). This suggested that the
total lack of chlorophyll a-binding polypeptides could be
due to degradation of their newly-synthesized molecules,
as a consequence of chlorophyll photo-oxidation,
preventing the pigment±protein cotraductional linkage,
necessary for apoprotein stabilization (Mullet et al.,
1990).
PSII polypeptides encoded by nuclear DNA, such as
22 kDa, and the chlorophyll aub-binding apoproteins of
LHCII were found in the anomalous membranes
(Fig. 4b). Their quantities were also lower than those of
control chloroplasts. However, it has to be considered
that in the photo-oxidized organelles the protein insertion
anduor stability in the impaired membranes might be
negatively affected. As regards the LHCII proteins, their
stabilization in altered thylakoids might plausibly depend
on the ability to bind the limited but available quantities
of chlorophylls, maintained by pigment turnover. In fact,
even though chlorophylls are photo-oxidized afterward,
their biosynthesis still occurs in carotenoid-de®cient
plastids (Frosch et al., 1979).
Leafuenvironment oxygen exchanges: As expected, no
emission, but only oxygen absorption occurred in leaf
tissues of both AM- and NF-treated plants (Fig. 5).
Moreover, the values of oxygen uptake were similar
when measured in light or in darkness and comparable
with the respiratory consumption of control leaves. This
indicated that leaf tissues supplied with herbicides lacked
any trace of photosynthetic activity.
The speci®c targets of bleaching herbicides are indeed
chloroplasts, while it is generally assumed that they do
not affect the structure and functionality of other cellular
components, such as mitochondria (Feierabend and
Shubert, 1978; Feierabend et al., 1979; Taylor, 1989;
816
Dalla Vecchia et al.
Fig. 5. Oxygen releaseuuptake by leaf tissues of control (C), amitroletreated (A) or nor¯urazon-treated (N) plants grown at 20 8C, measured
in either light (L) or darkness (D). The values represent the mean of
three measurements.
Sagar and Briggs, 1990; Zito et al., 1995; Agnolucci et al.,
1996). However, it has been noticed that some of these
chemicals, including amitrole, can also inhibit the activity
of the peroxisomal catalase (Feierabend and Shubert,
1978; Feierabend et al., 1979)
Plant grown at 30 8C
Phenotypes: The increase in growth temperature led, in
AM-treated plants, to an astonishing recovery of leaf
pigmentation as well as of growth rate, both of which
now approached those of control plants (Fig. 1b). In
contrast, it did not lead to a change in the appearance of
NF-treated plants, which were reduced in height and with
totally white leaves (Fig. 1b).
Photosynthetic pigments: Pigment analyses (Fig. 6a) and
chromatograms obtained by HPLC (Fig. 6b) showed that
in AM-treated leaves both carotenoid and chlorophyll amounts (Fig. 6a) underwent a great improvement,
reaching values approaching those of control leaves.
Moreover, HPLC chromatograms (Fig. 6b) showed a
normalization of the carotenoid spectrum, with the disappearance of lycopene and an increase of the b-carotene
and xanthophyll peaks. In this case, the rise in plant
growth temperature drastically reduced the inhibitory
effects of herbicide on carotenogenesis, leading to a substantial recovery of pigment biosynthesis and to a consequent chlorophyll protection against photo-oxidation.
These events, occurring in maize plants at 30 8C,
con®rmed what had already been noticed, at the same
temperature, in barley plants treated with amitrole (Zito
Fig. 6. Photosynthetic pigments in leaves of control (C), amitroletreated (A) or nor¯urazon-treated (N) plants grown at 30 8C. (a) Total
carotenoid and chlorophyll contents. The values represent the mean
of three measurements. (b) HPLC chromatograms of carotenoids
and chlorophylls. Key: 1, neoxanthin; 2, violaxanthin; 3, lutein; 4,
chlorophyll b; 5, chlorophyll a; 7, b-carotene. ND, not detected.
et al., 1995; Agnolucci et al., 1996) as well as in barley
lycopenic mutant tigrina 034 (Casadoro et al., 1983), which
did not undergo treatment with exogenous herbicide.
Herbicide effects on maize chloroplasts
In contrast, the complete absence of carotenoids and
chlorophylls in NF-treated leaves (Fig. 6a, b) demonstrated that the rise in growth temperature did not
interfere with the inhibitory effect of herbicide on
photoprotective pigment biosynthesis.
Plastid ultrastructure: The large increase in carotenoid
biosynthesis gave rise to a good recovery of plastid ultrastructure in AM-treated leaves. As in the well organized
chloroplasts of control leaves (not shown), the organelles
of mesophyll cells (Fig. 7a) exhibited a rather abundant
membrane system with grana and stroma thylakoids
regularly distributed in a rather ribosome-rich stroma. By
contrast, the total lack of photoprotective pigments was
responsible for the dramatic damage still suffered by
plastids of NF-treated leaves, where, in the stroma devoid
of most ribosomes, vesicles and membrane pro®les
could occasionally be found (Fig. 7b).
817
AM-treated chloroplasts, by normalization of their protein composition. The immunoblotting analyses (Fig. 8)
revealed comparable amounts of all the polypeptides
tested, encoded by both nucleus (Fig. 8b) and plastid
(Fig. 8a, c) DNA, in membranes of control and herbicidesupplied organelles, including the chlorophyll a-binding
proteins of the PSII core, not found in the altered
thylakoids of AM-treated plants grown at 20 8C.
Leafuenvironment oxygen exchanges: Likewise, the
increase in plant growth temperature led to a substantial recovery of photosynthetic activity in AM-treated
leaves, which, evaluated as oxygen emission in light,
Thylakoid membrane composition: The achievement of
the correct thylakoid organization was paralleled, in
Fig. 7. Plastids of mesophyll cells from leaves of herbicide-treated plants
grown at 30 8C. Note in the chloroplasts of an amitrole-treated leaf (a)
the rather abundant and well-organized inner membrane system, with
grana thylakoids (gt) and stroma thylakoids (st). The plastid of a
nor¯urazon-treated leaf (b) shows few vesicles (arrow) and two
membrane pro®les (double arrow) in the stroma (barsˆ1 mm).
Fig. 8. Immunoblotting of thylakoids isolated from plastids of control
(lanes C) and amitrole-treated (lanes A) plants grown at 30 8C, with anti
D1, D2, CP43 and CP47 (a), anti 22 kDa and LHCII (b), and anti cyt f (c).
818
Dalla Vecchia et al.
Conclusions
Fig. 9. Oxygen releaseuuptake by leaf tissues of control (C), amitroletreated (A) or nor¯urazon-treated (N) plants grown at 30 8C, measured
in either light (L) or darkness (D). The values represent the mean of
three measurements.
exceeded 80% of that of control plants (Fig. 9). By
contrast, only oxygen uptake was still recorded in
NF-treated leaves.
Plants grown at 20±30 8C
In a further experiment, plants grown for 5 d at 20 8C
were moved to 30 8C. As shown in Fig. 1c, tissues of
AM-treated leaves differentiated from the basal meristem
at the two temperatures exhibited a very different
pigmentation.
The older leaf regions, which had grown at 20 8C and
contained damaged carotenoid-de®cient plastids, were
pale yellow, whereas the younger leaf regions, grown at
30 8C, looked green, due to the greatly improved carotenoid biosynthesis and the previously described normalization of the other chloroplast parameters. Moreover,
the persistence of pale yellow leaf tissues at 30 8C revealed
that photodamage suffered by chloroplasts at 20 8C was
irreversible and did not permit organelle recovery with
the rise in plant growth temperature. On the other hand,
the ability of the leaf to produce green tissues at the
higher temperature showed that herbicide-damaging effects
at 20 8C did not affect the proplastids of meristematic
cells. These organelles, still devoid of chlorophylls, did
not undergo photo-oxidative damage due to carotenoidde®ciency and could give rise to green chloroplasts
when the temperature rise led to improved carotenoid
biosynthesis.
The experimental results show that at the lower plant
growth temperature, both amitrole and nor¯urazon
cause dramatic damage to maize leaf chloroplasts, due
to their inhibitory effects on carotenoid production. The
NF-treatment, which interrupts the biosynthetic pathway
at the level of phytoene desaturation, brings about a
complete chlorophyll photo-oxidation and photosynthetic membrane demolition. The treatment with
amitrole, hampering lycopene cyclization, still permits
reduced production of photoprotective pigments, with
preservation of small chlorophyll quantities as well as of
some inner membranes. These latter, however, are greatly
altered and totally deprived of photosynthetic activity
due to the photo-oxidative degradation of essential
components of the electron transport chain.
As far as the responses to herbicides are concerned,
plant behaviour drastically changes with the rise in
growth temperature. At 30 8C the NF-treatment still
causes the chloroplast damage noticed at 20 8C, with total
carotenoid and chlorophyll loss and thylakoid dismantling. By contrast, in organelles of AM-treated leaves the
damage found at 20 8C is quite subdued. Carotenoid
synthesis is greatly improved and quite normalized,
with the disappearance of lycopene and the production
of b-carotene and xanthophyll amounts near to those
of control plants. Consequently, the chlorophyll content,
the correct thylakoid organization and composition and
the photosynthetic functionality are all recovered.
The essential event, occurring with the increase of
plant growth temperature and accounting for normalization of chloroplast parameters is the regained ability of
AM-treated organelles to carry out carotenoid synthesis,
as also shown by plant leaves grown ®rst at 20 8C and
then at 30 8C.
The hypothesis that this event might be due to
a herbicide thermo-instability, with its faster degradation
at the higher temperature was excluded by previous
analyses on barley, which showed comparable AM
quantities in leaves grown at 20 8C or 30 8C (Zito et al.,
1995; Agnolucci et al., 1996). Moreover, the same
temperature effect on carotenogenesis occurred in barley
lycopenic mutant tigrina 034 (Casadoro et al., 1983),
where the lycopene cyclization was inhibited by mutation
and not by herbicide supply.
Thus, in maize, as already found in barley, the
carotenoid biosynthetic pathway seems to be sensitive
to temperature. This might be due to the existence of
thermo-modulable step(s) in which some changes in
membrane environment, such as increased ¯uidity or
modi®ed interaction between lipidic components and
enzymatic proteins, as well as conformational changes
of these latter might play a role. The plant responses to
herbicides suggest that this thermo-modulability might
Herbicide effects on maize chloroplasts
involve reactions between the NF-inhibited step, not
affected by temperature increase, and the AM-inhibited
one.
Recently, b-carotene and xanthophyll synthesis
through an alternative way, bypassing the lycopene
cyclization, has been proposed for tomato chromoplasts
(Pecker et al., 1996) and for Arabidopsis chloroplasts
(Cunningham et al., 1996). Thus the possibility might be
considered that this way is common in leaf chloroplasts
and that its activity and rate depends on plant growth
temperature.
Information worthy of note emerging from this experimental work is that in maize chloroplasts the temperature
range, in which most of the amitrole inhibitory effect
on carotenogenesis occurs, is the same as that noted in
barley. This is of particular interest, considering that
barley is a C3 plant growing well at 20 8C, whereas maize,
as a C4, prefers higher growth temperatures of 30±35 8C.
This means that plants such as barley, or other C3
species with the same temperature requirements, are
sensitive to amitrole-damaging effects and cannot survive
its supply at their usual growth temperature. By contrast,
plants such as maize or other C4 species can express
a strong tolerance to the same herbicide, and probably
to other chemicals operating in the same way, when
supplied to the plants growing at their best temperature
conditions.
Acknowledgements
The authors are grateful to Dr Elisabetta Bergantino for the gift
of the antibody against cyt f. This research was supported by
grants from National Research Council of Italy and from
MURST.
References
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