Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002
25
Original Article
Anthocyanins, photoinhibitory risk and photosynthesis in maize at low temperatureF. Pietrini et al.
Plant, Cell and Environment (2002) 25, 1251–1259
Anthocyanin accumulation in the illuminated surface of
maize leaves enhances protection from photo-inhibitory
risks at low temperature, without further limitation to
photosynthesis
F. PIETRINI, M. A. IANNELLI & A. MASSACCI
Istituto di Biochimica ed Ecofisiologia Vegetali, CNR, Via Salaria Km 29·3, I-00016 Monterotondo Scalo (Roma), Italy
ABSTRACT
At suboptimal temperatures, anthocyanins accumulate
in the illuminated leaf surface of some maize genotypes
and, if the anthocyanins shade chloroplasts, they can effectively reduce the risk of photo-inhibition but also photosynthesis. To investigate this phenomenon, gas exchange,
fluorescence, superoxide dismutase activity and xanthophyll composition of anthocyanin-containing HOPI and
anthocyanin-deficient W22 maize genotypes were measured in either white or red light, where the latter is not
absorbed by anthocyanins. Despite differences in light
absorption in chloroplasts, photosynthesis did not differ
between HOPI and W22 under either light source, suggesting that neither CO2 supply nor photochemistry were more
limiting in red leaves than in green leaves. In fact, no major
differences in transpiration were detected. The ∆F/Fm (photosystem II quantum yield) of HOPI in white light was
higher than in red light and higher than ∆F/Fm of W22 with
either light source. This probably compensated for the
lower white light absorption of HOPI chloroplasts compared with W22 because of the presence of anthocyanins
and led to similar rates of calculated electron transport for
both genotypes. After exposure to high white light at 5 °C,
xanthophyll de-epoxidation and superoxide dismutase
activity were lower in HOPI than in W22. Further, HOPI
could be exposed to a much higher irradiance than W22
before Fv/Fm was reduced to that of W22.
Key-words: electron transport; photosystem II quantum
yield; pigments; superoxide dismutase; transpiration;
xanthophylls.
INTRODUCTION
Tropical maize is not suitable for growth in the cold climates at high latitudes or altitudes, where daily tempera-
Correspondence: Angelo Massacci. Fax: + 39 6 9064492; e-mail:
[email protected]
© 2002 Blackwell Publishing Ltd
tures around zero and bright sunlight are common (Long
et al. 1990; Baker et al. 1994). Such conditions predispose
the plants to a series of events that can ultimately lead to
destruction of the photosynthetic apparatus of exposed
leaves (Wise 1995). Photogeneration of active oxygen species (AOS), following the formation of chlorophyll triplets
and singlet oxygen in the light-harvesting antennae when
light in excess of that usable for photosynthesis is absorbed,
is the key event for this oxidative destruction (Wise 1995).
Under optimal or mild stress conditions, xanthophylls can
contribute to efficient dissipation, as heat, of this excess
energy, with a high proportion of de-epoxitated xanthophylls (zeaxanthin and antheraxanthin) indicating
enhanced energy dissipation (Demmig-Adams & Adams
1996). A well-organized antioxidant system can also efficiently scavenge the AOS eventually formed (Asada 1996).
However, these defence mechanisms need to be efficiently
regulated and photosynthesis is likely to be involved in the
regulatory mechanism (Massacci et al. 1995; Fryer et al.
1998; Fracheboud et al. 1999).
Under severe stress conditions, however, the capacity of
the protective mechanisms can be overwhelmed. An effective strategy for protection under such conditions could be
to reduce the amount of light arriving at the chloroplasts
rather than to have the defence mechanisms working at full
activity for an unpredictable length of time. An interesting
example of such a strategy comes from some highland races
of maize from Mexico that grow successfully where lowland
tropical maize cannot be grown (Eagles & Lothrop 1994).
Among the phenotypic characteristics shown by highland
races is that they tend to accumulate anthocyanin pigments
in stems and leaves (Chong & Brawn 1969). These pigments
are water-soluble flavonoids that are concentrated in cell
vacuoles and absorb visible light in the range 400–550 nm
(Harborne 1976). Their synthesis is determined by inherited factors and enhanced by conditions such as a combination of low temperature and high light (Mancinelli
1983; Holton & Cornish 1995 for a review). According to
Gould et al. (2000), the role of anthocyanins is not clear and
may depend on whether their location is in the vacuoles
of the abaxial or adaxial leaf epidermis, in the cytosol of
1251
1252 F. Pietrini et al.
mesophyll cells, in roots, or in stems. Gould et al. (1995)
suggested that in, Begonia pavonina and Triolena hirsuta,
anthocyanin accumulation in leaves prevented photoinhibition by shading chlorophyll b and, as a result, photosynthesis of red leaves was higher than photosynthesis
of green leaves. In contrast, anthocyanin accumulation
seems to reduce photosynthesis in many other species
(Chalker-Scott 1999). Recently Havaux & Kloppstech
(2001) have shown that, in flavonoid Arabidopsis mutants,
anthocyanin exerts very little protection against photoinhibition and or photo-oxidation, giving flavonoids
absorbing UV and blue light a more important role for
photoprotection. These observations suggest that the role
of anthocyanins should be further investigated, particularly
with respect to its effects on photosynthesis and other protective mechanisms.
In this article we address the question of how anthocyanin accumulation in maize leaves may reduce the risk of
photo-inhibition at low temperature by enhancing the
photoprotection capacity, without further limitation to
photosynthesis.
MATERIALS AND METHODS
Plant material and growth conditions
Seeds of two maize (Zea mays L.) genotypes, the anthocyanin-accumulating HOPI (cod. 97-T850) and the anthocyanin-deficient W22 r-g (cod. 93-T420) (Petroni et al. 2000),
were planted in 2 L pots containing 1/1/1 soil/sand/peat. As
reported by Taylor & Briggs (1990), genotypes with r-g
pigmentation patterns produce no measurable anthocyanin
pigments, but they can produce chalcone synthase, a crucial
enzyme for biosynthesis of all classes of flavonoids, including aurones, flavones, flavonols and proanthocyanidins, as
well as anthocyanins.
The plants were grown in a growth cabinet at a day/night
temperature of 25/20 °C, a light regime of 800 µmol m−2 s−1
for 16 h and a relative humidity of 60%. Plants were
watered twice a week to soil saturation and once with halfstrength Hoagland's nutrient solution. Fourteen plants of
each line were grown under these conditions for 2 weeks
and then the cabinet temperature was lowered to 18/16 °C
for 2 d to enhance anthocyanin accumulation, before the
temperature was returned to 25/20 °C.
Anthocyanin localization
Fresh leaves of HOPI were sectioned by hand in the presence of 10% sucrose, and photographed immediately with
a Zeiss (Jena, Germany) light microscope equipped with a
DC120 Zoom Digital Camera (Eastman Kodak Company,
New York, USA), as reported by Gould et al. (2000). The
images were analysed with KODAK software (Microscopy
Documentation Software 120; Eastman Kodak Company)
to evaluate the intensity of red, anthocyanin-containing
patches.
Pigment analysis
Two square centimetres of the youngest fully expanded leaf
were ground under dim light in a mortar containing liquid
N2. When the leaf was reduced to a fine powder, 10 mL of
acetone-water (80% v: v) were added to extract the pigments. The samples were centrifuged at 12 000 × g and 5 °C
for 10 min, and the supernatant was removed and used for
pigment determinations. Absorbance was measured at 470,
646·8 and 663·2 nm with a spectrophotometer (Perkin
Elmer, Norwalk, CT, USA). The extinction coefficients and
the equations reported by Lichtenthaler (1987) were used
to calculate the pigment amounts. A second 2 cm2 area of
leaf was sampled and homogenized in 1 mL of acidified
(1% HCl) methanol and maintained at 4 °C for 4 h to avoid
degradation of chlorophylls, whose products may interfere
with the absorbance of anthocyanins at 530 nm. Particulates were removed by centrifugation at 10 000 × g for
30 min and the supernatant was passed through a 0·45 µm
syringe filter (Acrodisc CR PTFE; Gelman Sciences,
Ann Arbor, MI, USA). The optical density of the cleared
supernatant was scanned between 400 and 700 nm and
anthocyanins were measured as described by Mancinelli
(1984) using OD530 nm − 0·25 OD 657 nm, to account for interference from chlorophylls. Anthocyanin content was calculated as cyanidin-3-glucoside using 29600 (E 1 cm 1%) as
extinction coefficient and 445 as the molecular weight
(Wrolstad 1976). The total UV-absorbing flavonoid content
was estimated as described by Havaux & Kloppstech
(2001).
Gas exchange and fluorescence analysis
The central part of the leaf was enclosed in the cuvette of
a gas exchange system (HCM 1000, Walz, Effeltrich,
Germany), configured for simultaneous measurement of
chlorophyll fluorescence (PAM 101 modulated fluorometer; Walz, Effeltrich, Germany). The relative humidity of air
entering the cuvette was set at 50%, CO2 partial pressure
at 350 µbar bar−1 and air and cuvette temperatures at 25 °C
or 7 °C. The transparent top of the cuvette was masked to
reduce the area from 5 to 1·2 cm2 and the incoming air flow
was reduced to 250 mL min−1. The cuvette surface area
reduction allowed illumination with the fibre-optic guide of
only that portion. A white light source (KL 1500; Schott,
Mainz, Germany), a red LED light source (consisting of
six ultrabright LEDs; Toshiba, Tokyo, Japan; 20 nm halfbandwidth and emission peak at 650 nm), the modulated
light and fluorescence detector were all connected to the
multiple ends of the light guide. The light flux was varied
using neutral density filters inserted between the light
sources and the light guide ends. Photosynthesis and stomatal conductance were calculated according to von Caemmerer & Farquhar (1981). The quantum yield of electron
transport through photosystem II (∆F/Fm) was estimated by
dividing the difference between the maximum fluorescence
(Fm) and the steady state fluorescence (Fs) in the illuminated leaf (∆F = Fm − Fs) by Fm, as reported in Genty,
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
Anthocyanins, photo-inhibitory risk and photosynthesis in maize at low temperature 1253
Briantais & Baker (1989). The leaf absorptance (Aleaf) and
leaf anthocyanin absorptance (Aant) of HOPI and W22
leaves were measured with a spectroradiometer (Li-1800;
Licor Inc., Lincoln, NE, USA), as reported by Pietrini &
Massacci (1998). Linear electron transport through photosystem II (PSII) was estimated as Jf, and obtained from ∆F/
Fm × PPFDinc × (Aleaf − Aant)/2, where PPFDinc is the incident photosynthetic photon flux density (PPFD) in
µmol m−2 s−1 and the value Aleaf − Aant is the percentage difference between leaf absorptance and leaf anthocyanin
absorptance to obtain an estimation of the actual flux of
radiant energy absorbed at the chloroplasts and used for
photochemistry. Division by 2 assumes that the efficiency
of the two photosystems is equal and that light is equally
distributed between them, as reported in Loreto et al.
(1994).
High light treatments
Fully expanded leaves within the temperature-controlled
cabinet were enclosed in the gas exchange cuvette containing the terminal part of the fibre optic. The cuvette and
cabinet temperature were set to 7 °C and the leaf surface
was illuminated with white light of 2400, 1800, 1200 or
600 µmol photons m−2 s−1 or with red light of 1200, 900, 600
or 300 µmol photons m−2 s−1 for 2 h. The cuvette was supplied with ambient air, but with the relative humidity and
CO2 controlled to 40% and 350 µbar bar−1, respectively. At
the end of the illumination period, the first part of the
treated leaf was sampled and stored in liquid nitrogen at −
80 °C for measurements of superoxide dismutase (SOD)
activity and xanthophyll content. The second part of the
leaf remained in darkness for 30 min and then the basal (F0)
and maximum (Fm) fluorescence emission (stimulated by
applying a saturating light pulse) were recorded. The ratio
Fv/Fm was calculated, obtaining Fv from Fm − F0.
In a different experiment to investigate the responses to
mild and severe photo-oxidative conditions, leaves were
exposed for 2 h at 5 °C to 1000 or 2000 µmol photons
m−2 s−1 white light and then returned for 2 h to 25 °C in the
same gas exchange conditions as described above. A control experiment at 5 °C, 2000 µmol photons m−2 s−1 in red
light was also carried out under the same conditions. In this
experiment, only non-destructive measurements of photosynthesis and fluorescence were taken.
Antioxidant analysis
Frozen leaves (0·2–0·5 g fresh weight) were ground to a fine
powder in a mortar and pestle under liquid nitrogen.
The proteins were then extracted at 4 °C by grinding in a
cold 50 mM phosphate (pH 7·8) buffer containing 0·1%
ascorbic acid, 10 mM dithiothreitol, 0·1% Triton X-100, 1%
polyvinyl-polypyrrolidone. The homogenate was centrifuged at 4 °C for 20 min at 12 000 × g. The clear supernatant
fraction was used for the enzyme assays. Protein concentration was quantified as described by Bradford (1976), using
bovine serum albumin as a standard.
Total SOD activity (EC 1·15·1·1) was assayed according
to its ability to inhibit ferric cytochrome c reduction under
a constant flux of O2– generated by the xanthine–xanthine
oxidase system (McCord & Fridovich 1969). The reaction
mixture contained 10 µM KCN to inhibit cytochrome c oxidase. One unit of SOD was defined as the quantity of
enzyme required to inhibit the reduction of cytochrome c
by 50% in a 1 mL reaction volume. All the reagents used
were of analytical grade and were obtained from Aldrich
(Steinheim, Germany) and Sigma (St. Louis, MO, USA).
Xanthophyll analysis
Xanthophyll extraction and high-performance liquid chromatography analysis were conducted on two leaf discs
(1·5 cm2) taken from the central part of the exposed
leaf, as described by Thayer & Björkman (1990). The deepoxidation state (DEPS) of the zeaxanthin cycle was determined as the ratio of (zeaxanthin + 0·5 antheraxanthin)/
(violaxanthin + antheraxanthin + zeaxanthin) (Schindler &
Liechtenthaler 1996).
Statistical analysis
In any one experiment, photosynthesis, fluorescence and all
biochemical analyses were measured on a minimum of four
samples. Standard errors were calculated by pooling data
of three different experiments made under the same conditions with similar material. The Student–Newman–Keuls
test was performed for the leaf pigment data.
RESULTS
Pigments
Chlorophylls, carotenoids, anthocyanins, and flavonoids
were quantified in leaves of HOPI and W22 (Table 1),
grown at 800 µmol m−2 s−1 and 25 °C for 2 weeks and then
exposed for 2 d to 18 °C, to enhance anthocyanin synthesis
and accumulation in vacuoles of the illuminated leaf
surface. The two maize lines had similar contents of chlorophyll a and flavonoids, although chlorophyll b and carotenoids were slightly higher in HOPI and anthocyanins
were 8·1 µg cm−2 in HOPI and undetectable in W22. Crosssections of HOPI leaves (Fig. 1) showed that anthocyanins
accumulated mostly in the vacuoles of the upper illuminated surface. A comparative estimation, using image analysis software, of red colour intensity indicated that this
upper surface accumulated about 70% of total anthocyanins. The leaf absorptance of HOPI, containing anthocyanins, was significantly higher than the absorptance of W22
(89 versus 80%) (Table 1).
Gas exchange and fluorescence
The effect of anthocyanin shading on chloroplasts was analysed using white and red light. The red LED light, as proposed by Smillie & Hetheringhton (1999) for pods of
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
1254 F. Pietrini et al.
Table 1. Chlorophyll (Chl a and Chl b), carotenoids (Car), flavonoids (Flav) and anthocyanin (Ant) contents, leaf absorptance before
(Aleaf) and after (Aleaf − Aant) the correction for the anthocyanin absorptance and leaf anthocyanin (Aant) absorptance (400–700 nm) in
HOPI and W22 genotypes grown for 2 weeks at 25 °C and for 2 d at 18 °C. Means in the same column followed by the same letter are not significantly different (P < 0·05) according to the Student–Newman–Keuls test
HOPI
W22
Chl a
(µg cm−2)
Chl b
(µg cm−2)
Car
(µg cm−2)
Ant
(µg cm−2)
Flav
(A350 × cm−2)
Aleaf
(% of incident
PPFD)
Aant
(% of incident
PPFD)
Aleaf − Aant
(% of incident
PPFD)
19·0 a
18·9 a
5·1 a
4·5 a
4·0 a
3·3 a
8·1 a
n.d b
2·98 a
2·86 a
89·0 a
80·0 b
28 a
0b
61·0 a
80·0 b
Figure 1. Cross-sections of fresh, fully developed HOPI leaves grown for 2 weeks under a PPFD of 800 µmol m−2 s−1 at 25 °C and transferred for 2 d to 18 °C to induce anthocyanin synthesis and accumulation in vacuoles of epidermal cells. (A) indicates (arrow direction) a leaf
illuminated on the adaxial side and (B) on the abaxial side.
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
Anthocyanins, photo-inhibitory risk and photosynthesis in maize at low temperature 1255
Table 2. Photosynthesis (Ph), quantum yield of photosystem II (as ∆F/Fm) and minimal fluorescence yield (F0) of red HOPI and green W22
leaves in response to mild, 5 °C and at a photosynthetic photon flux density of 1000 µmol m−2 s−1 (PPFD) and severe, 5 °C and at a PPFD of
2000 µmol m−2 s−1, photo-inhibitory conditions and to recovery (rec) at 25 °C and a PPFD of 1000 µmol m−2 s−1. A control under severe
photo-inhibitory condition was carried out with a red light (R). Means ± standard errors are reported for n = 4
Ph (µmol m−2 s−1)
∆F/Fm (relative units)
F0
PPFD (µmol m−2 s−1)
Temp, time
(°C, min)
HOPI
W22
HOPI
W22
HOPI
W22
0
1000
1000
1000 (rec)
0
25, 30
25, 30
5, 120
25, 120
25, 30
–
28·2 ± 1·2
7·2 ± 1·2
27·8 ± 0·7
–
–
32·7 ± 2·7
1·2 ± 0·2
27·5 ± 1·5
–
0·70 ± 0·01
0·39 ± 0·02
0·09 ± 0·01
0·39 ± 0·01
0·67 ± 0·03
0·71 ± 0·01
0·37 ± 0·01
0·04 ± 0·01
0·34 ± 0·02
0·65 ± 0·02
22 ± 1
20 ± 1
18 ± 2
20 ± 1
22 ± 1
24 ± 2
20 ± 1
17 ± 2
20 ± 1
24 ± 2
0
1000
2000
1000 (rec)
0
25, 30
25, 30
5, 120
25, 120
25, 30
–
30·6 ± 2·2
–
26·8 ± 3·0
–
–
29·7 ± 2·7
–
9·6 ± 2·4
–
0·66 ± 0·01
0·44 ± 0·01
–
0·40 ± 0·03
0·61 ± 0·03
0·66 ± 0·03
0·36 ± 0·02
–
0·14 ± 0·02
0·38 ± 0·02
22 ± 2
20 ± 1
15 ± 2
20 ± 1
25 ± 3
24 ± 1
19 ± 2
14 ± 2
14 ± 1
18 ± 1
0
1000 (R)
2000 (R)
1000 (R) (rec)
0
25, 30
25, 30
5, 120
25, 120
25, 30
–
30·2 ± 2·4
–
12·0 ± 2·8
–
–
30·0 ± 2·5
–
10·6 ± 2·2
–
0·67 ± 0·02
0·38 ± 0·02
–
0·16 ± 0·03
0·36 ± 0·04
0·65 ± 0·03
0·36 ± 0·03
–
0·14 ± 0·02
0·30 ± 0·03
24 ± 2
19 ± 1
17 ± 2
17 ± 1
21 ± 2
22 ± 1
16 ± 2
16 ± 1
13 ± 2
16 ± 1
1.0
5
0.5
-1
W m-2 nm (red light)
-1
W m-2 nm (white light)
1.0
0.8
a
4
0.6
3
2
0.4
c
b
0.2
1
0
0
400
Absorptan ce (%)
1.5
500
600
700
0
800
Wavelength (nm)
Figure 2. (a) Absorptance spectrum of pure anthocyanin standard in acidified methanol; (b) emission spectrum of white and (c)
red light used for the light treatments, fluorescence and photosynthesis measurements.
Bauhinia variegata, was used because of its narrow emission spectrum (20 nm half-bandwidth) with a peak at
650 nm, that is not absorbed by anthocyanins. In Fig. 2 the
emission spectra of red and white light and the absorptance
spectrum of pure anthocyanin standard in acidified methanol are shown. The 1000 µmol photons red light-induced
photosynthesis of both HOPI and W22, measured at 25 °C,
was around 30 µmol m−2 s−1 and remained similar over the
whole range of irradiances used (Fig. 3A). The same measurements, made at 7 °C, showed only a slight difference
between the two lines, with photosynthesis around
4 µmol m−2 s−1 at 1000 µmol photons m−2 s−1 (Fig. 3A) How-
ever, when measurements were carried out under white
light, the photosynthesis of HOPI was comparable with the
photosynthesis of W22, even though one would have
expected anthocyanin absorption to have reduced the
energy available for photochemistry (Fig. 3A). Transpiration responses to light followed those of photosynthesis
(Fig. 3B). The quantum yield of photosystem II (∆F/Fm) at
either 25 or 7 °C was much higher over the whole PPFD
range for HOPI in white than in red light, and was higher
than W22 in either white or red light (Fig. 3C). This is
probably the major consequence of the light attenuation by
anthocyanins. The linear electron transport flux through
PSII, Jf, calculated using actual light absorptance by chloroplasts (Aleaf − Aant) in the presence of anthocyanins and
estimated as 28% of incident light (Table 1) by an indirect
method (Pietrini & Massacci 1998), was similar between
genotypes and irradiances at 25 °C. At 7 °C, it was slightly
higher for leaves with anthocyanins measured in white light
(Fig. 3D). To show the effect of anthocyanins on the linear
PSII electron transport in HOPI leaves at 7 and 25 °C, the
values of Jf calculated using the leaf absorptance (Aleaf) are
also reported in Fig. 3D (grey triangles).
Anthocyanins and protection of chloroplasts
from high light at low temperature
High light treatment of leaves reduced the Fv/Fm ratio in
the dark with respect to the initial value of the untreated
leaf (Fig. 4A). A 50% Fv/Fm reduction was found in HOPI
at a photon flux density of 2400 µmol m−2 s−1, whereas in
W22, a 50% reduction occurred at a photon flux density of
1200 µmol m−2 s−1. In contrast, under red light and with all
light treatments (closed symbols), the reduction in Fv/Fm
was exactly the same for HOPI and W22, and equal to the
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
1256 F. Pietrini et al.
5
Transpiration (mmol m-2s-1)
Photosynthesis (µmol m-2s-1)
35
30
25
25˚C
20
A
15
10
7˚C
5
0
4
25˚C
3
B
2
1
7˚C
0
0
200
400
600
800
1000
0
200
PPFD (µmol m-2s-1)
200
0.7
175
0.6
150
800
1000
0.4
25˚C
D
125
100
Jf
∆F/Fm
C
25˚C
600
PPFD (µmol m-2s-1)
0.8
0.5
400
0.3
75
0.2
7˚C
50
7˚C
0.1
25
0
0
0
200
400
600
800
1000
PPFD (µmol m-2s-1)
0
200
400
600
800
1000
PPFD (µmol m-2s-1)
Figure 3. Responses to incident PPFD of (A) photosynthesis; (B) transpiration; (C) ∆F/Fm, and (D) calculated photosynthetic electron
transport, Jf, using Aleaf (grey tringles) and Aleaf − Aant (white tringles) for anthocyanin-containing HOPI (triangles) and anthocyanin-free
W22 (circles) leaves. Dashed lines represent measurements obtained at 25 °C and dotted lines measurements at 7 °C. PPFDs were produced
by a white light source (open symbols) or a red LED source (closed symbols). See Material and Methods for more details. Bars represent ±
standard errors (n = 4).
reduction for W22 under white light. The xanthophyll
pool was higher in green than in red leaves (not shown).
The DEPS was also always higher in the green leaves
and in both leaf types it reached maximum values at
900 µmol m−2 s−1 (Fig. 4C).
The activity of SOD in leaves exposed to high light treatments was always significantly lower in HOPI than in W22
(Fig. 4B). A control treatment with red light (closed symbols) showed, as expected, no differences in the SOD activities between the two maize lines (Fig. 4B)
Anthocyanins, photo-inhibition and recovery
of photosynthesis
Table 2 shows the effects on photosynthesis and quantum
yield of PSII of mild or severe photo-inhibitory treatments
at low temperature, and recovery at 25 °C. Under mild
white light (1000 µmol photon m−2 s−1 and 5 °C for 2 h)
photo-inhibition, photosynthesis and ∆F/Fm were less inhibited in HOPI than in W22 (7·2 and 1·2 µmol CO2 fixed
m−2 s−1, and 0·09 and 0·04 µmol CO2 fixed m−2 s−1, respec-
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
Anthocyanins, photo-inhibitory risk and photosynthesis in maize at low temperature 1257
tively). Recovery was complete after 2 h at 25 °C.
Under more severe photo-inhibition treatments
(2000 µmol m−2 s−1 and 5 °C), photosynthesis was completely inhibited and ∆F/Fm was close to zero after 2 h of
exposure in both lines. Recovery of photosynthesis and of
∆F/Fm after 2 h at 25 °C were greater in HOPI than in W22.
A control treatment with red light showed that, without
anthocyanins, the pattern of response to treatment and
recovery did not differ between genotypes.
Fv/Fm (rel.units)
0.8
0.6
0.4
DISCUSSION
0.2
A
0
SOD (U mg prot -1)
150
Anthocyanin accumulation in the
illuminated leaf surface
100
50
B
0
0.8
0.6
DEPS
There have been several attempts to explain anthocyanin
accumulation in leaves (Lee, Brammeier & Smith 1987;
Krol et al. 1995; Gould et al. 1995, 2000; Chalker-Scott
1999). Nevertheless, we consider that the present results
support the suggestion that accumulation primarily
enhances protection of the photosynthetic apparatus by
screening it from the detrimental effects of high light, without limiting photosynthesis in red leaves.
0.4
0.2
C
0
0
As anthocyanins occur not only in vacuoles of the abaxial
leaf epidermis but also in roots, stems and seeds, it is clear
that their role must depend on their position (Gould et al.
2000). The data presented are consistent with the idea that
anthocyanins capture part of the incoming light before
chlorophylls are excited (Fig. 1A & B). In maize, anthocyanin synthesis in the cytoplasm is stimulated by high light
(Mancinelli 1983; Chalker-Scott 1999) and they are then
glycosylated and transferred into vacuoles of the epidermal
cells (Marrs et al. 1995). The reason for this transfer is not
yet well understood. Our results confirmed that anthocyanins did accumulate strongly in the naturally light-exposed
leaf surface (Fig. 1A), but they could also accumulate in the
lower leaf surface when the leaf was inverted (Fig. 1B). This
suggests that a light-dependent mechanism directs the
anthocyanins to accumulate strongly in the more illuminated leaf surface. Gould et al. (2000) showed that anthocyanins are largely absent from cells within the mesophyll
of red leaves of many species. This could support our conclusion that, in our red leaf (Fig. 1), most anthocyanins are
in the red-coloured areas and thus in that position their
principal function is to screen from incoming light.
300 600 900 1200 1500 1800 2100 2400
PPFD (µmol m-2s-1)
Figure 4. Responses of (A) Fv/Fm,, the variable to maximum
chlorophyll fluorescence (B) superoxide dismutase (SOD), and
(C) de-epoxidation status (DEPS) to PPFD of anthocyanin-containing HOPI (triangles) and anthocyanin-free W22 (circles)
leaves after being held at 5 °C for 2 h. PPFDs were produced using
either a white light source (open symbols) or a red LED source
(closed symbols). See Material and Methods for more details. Bars
represent ± standard errors (n = 4).
Anthocyanins and other mechanisms
protect photosynthesis from high light
at low temperature
Chloroplast protection from high light involves specialized
mechanisms, such as the dissipation of excess energy as heat
and, eventually, the efficient scavenging of active oxygen
species thus formed. Despite anthocyanins seems to potentiate antioxidant activities in red leaves of Elatostema rug-
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
1258 F. Pietrini et al.
osum (Neill et al. 2002), we consider that high anthocyanin
synthesis is not justified by the requirement for this pigment
as a minor antioxidant. Our results demonstrate that the
role of anthocyanins, at least in maize leaves, is integrated
with the role of other protection mechanisms, reducing the
need for antioxidants and for non-radiative heat dissipation
when leaves of HOPI and W22 were exposed at 5 °C to
increased white and red irradiances. Under these conditions, lower SOD activity and lower zeaxanthin + 0·5
antheraxanthin contents were found in the red leaves compared with the green leaves. However, these differences
were no longer measurable when the light absorption by
anthocyanins was circumvented using red illumination
(Fig. 4B & C). This indicates that anthocyanins might
enhance the protective capacity of leaves against photoinhibitory damage. In fact, a 50% reduction in Fv/Fm
occurred at a white irradiance that was much higher in
HOPI than in W22. A protective role for anthocyanins in
preventing photo-oxidation and maintaining the efficiency
of nutrient uptake, was also suggested by Feild, Lee &
Holbrook (2001) for senescing leaves of red-osier dogwood. A photoprotective role for anthocyanins has been
also suggested in apple fruits (Merzlyak & Chivkunova
2000) and in pods of Bawhinia variegata (Smillie &
Hetheringhton 1999). Further, Table 2 shows that anthocyanin protection probably causes higher photosynthesis of
HOPI as compared to photosynthesis of W22, under both
mild and severe photo-inhibitory conditions. Moreover,
this experiment demonstrate that no flavonoids, other than
anthocyanins, are involved in photoprotection from visible
radiation in our maize genotypes in contrast to that shown
by Havaux & Kloppstech (2001) in Arabidopsis. This contrasting result could perhaps be attributed to differences in
the treatment conditions or even in different localization of
anthocyanins in Arabidopsis with respect to maize.
increase in leaf temperature of red leaves that absorb more
light energy than green leaves (Table 1). It seems likely
that, in our experiment, linear electron transport was also
not limiting, as the decreased light reaching the chloroplasts
in red leaves was compensated by a higher quantum yield
of PSII (∆F/Fm) (Genty et al. 1989; Pietrini & Massacci
1998). Similarly, Di Marco et al. (1989) argued that a yellow-green mutant of wheat and its wild type, absorbing 60
and 77% of incident light, respectively, did not differ in
photosynthetic capacity because electron transport was not
limiting.
CONCLUSIONS
Maize lines capable of accumulating high amounts of
anthocyanins in the illuminated leaf surface may present
an adaptive response to harmful conditions of low temperature associated with high light. High anthocyanincontaining leaves can be exposed to much higher ambient
illumination than green leaves before Fv/Fm (an indicator
of intrinsic PSII efficiency) is strongly inhibited. Further,
experimental evidence is provided here that this particular
pigment localization is no more limiting to photosynthesis
in red leaves compared to green leaves. We have shown that
this is possible, first, because the light attenuation at the
chloroplast increases ∆F/Fm thus producing a similar electron transport rate for anthocyanin-containing and anthocyanin-free leaves and, second, transpiration is not altered
because stomata are not shaded and the light-dependent
main control of their aperture (Jones 1992) is not affected.
The reduction in the risk of photo-inhibition, without further photosynthetic limitation, provides advantages that
might explain the natural occurrence of anthocyanin-rich
plant species at high altitudes or in northern latitudes.
ACKNOWLEDGMENTS
Effects of anthocyanin accumulation on
photosynthesis at 7 and 25 °C
Anthocyanins absorb light at the same wavelengths as chlorophyll b, thus reducing damage and leading to high rates
of photosynthesis of red leaves as compared with green
leaves under high light stress (Gould et al. 1995). Our
results show no limitation to photosynthesis (Fig. 3A)
under normally non-photo-inhibitory conditions, such as at
25 °C and 1000 µmol photons flux m−2 s−1, even though one
might expect such conditions of anthocyanin accumulation
would reduce photosynthesis by absorbing some of the incident photons (Bjorkman 1981). Control illumination with
red light at 650 nm (not absorbed by anthocyanins) showed
that there was no inherent photosynthetic difference
between the two maize lines. The same or slightly higher
photosynthesis in the high anthocyanin plants, both at 25
and 7 °C, implies that neither CO2 supply nor photosynthetic electron transport rate are more limited in red leaves
than in green leaves. Indeed, no major differences in transpiration were detected at 25 or 7 °C (Fig. 3B), with the
slight enhancement in red leaves possibly caused by a small
We thank Hamlyn G. Jones for critical reading of this
manuscript and Chiara Tonelli for providing the HOPI and
W22 seeds. This work was supported by a CNR grant.
REFERENCES
Asada K. (1996) Production and scavenging of radicals. In
Photosynthesis and the Environment (ed. N.R. Baker), pp.
123–150. Kluwer Academic Publishers, Dordrecht, The
Netherlands.
Baker N.R., Farage P.K., Stirling C.M. & Long S.P. (1994) Photoinhibition of crop photosynthesis in the field at low temperatures. In Photoinhibition of Photosynthesis from Molecular
Mechanisms to the Field (eds N.R. Baker & J.R. Bowyer), pp.
349–364. Bios Scientific Publisher, Oxford, UK.
Bjorkman O. (1981) Responses to different quantum flux densities.
In Physiological Plant Ecology I. Responses to the Physical Environment. Encyclopaedia of Plant Physiology. New Series (eds
O.L. Lange, P.S. Nobel, C.B. Osmond, & H. Ziegler) Vol. 12a,
pp. 57–107. Springer-Verlag, Berlin, Germany.
Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle
of protein-dye binding. Analytical Biochemistry 72, 248–254.
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259
Anthocyanins, photo-inhibitory risk and photosynthesis in maize at low temperature 1259
von Caemmerer S. & Farquhar G.D. (1981) Some relationships
between the biochemistry of photosynthesis and the gas
exchange of leaves. Planta 153, 376–387.
Chalker-Scott L. (1999) Environmental significance of anthocyanins in plant stress responses. Photochemistry and Photobiology
70, 1–9.
Chong C. & Brawn R.I. (1969) Temperature comparisons of purple
and dilute sun red anthocyanin colour types of maize. Canadian
Journal of Plant Science 49, 513–516.
Demmig-Adams B. & Adams W.W. III (1996) Xanthophyll cycle
and light stress in nature: uniform response to excess direct
sunlight among higher plant species. Planta 198, 460–470.
Di Marco G., D'Ambrosio N., Giardi M.T., Massacci A. & Tricoli
D. (1989) Photosynthetic properties of leaves of a yellow green
mutant of wheat compared to its wild-type. Photosynthesis
Research 21, 117–122.
Eagles H.A. & Lothrop J.E. (1994) Highland maize from central
Mexico – its origin, characteristics, and use in breeding programs. Crop Science 34, 11–19.
Feild T.S., Lee D.W. & Holbrook N.M. (2001) Why leaves turn
red in autumn. The role of anthocyanins in senescing leaves of
red-osier dogwood. Plant Physiology 127, 566–574.
Fracheboud Y., Haldimann P., Leipner J. & Stamp P. (1999) Chlorophyll fluorescence as a selection tool for cold tolerance of
photosynthesis in maize (Zea mays L.). Journal of Experimental
Botany 50, 1533–1540.
Fryer M.J., Andrews J.R., Oxborough K., Blowers D.A. & Baker
N.R. (1998) Relationship between CO2 assimilation, photosynthetic electron transport, and active O2 metabolism in leaves of
maize in the field during periods of low temperature. Plant Physiology 116, 571–580.
Genty B., Briantais J.M. & Baker N.R. (1989) The relationship
between the quantum yield of photosynthetic electron transport
and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990, 87–92.
Gould K.S., Kuhn D.N., Lee D.W. & Oberbauer S.F. (1995) Why
leaves are sometimes red. Nature 378, 241–242.
Gould K.S., Markham K.R., Smith R.H. & Goris J. (2000) Functional role of anthocyanins in the leaves of Quintilia serrata A.
Cunn. Journal of Experimental Botany 51, 1107–1115.
Harborne J.B. (1976) Functions of flavonoids in plants. In Chemistry and Biochemistry of Plant Pigments (ed. T.W. Goodwin),
pp. 736–778. Academic Press, New York, USA.
Havaux M. & Kloppstech K. (2001) The photoprotective functions
of carotenoids and flavonoids pigments against excess visible
radiation at chilling temperature investigated in Arabidopsis
npq and tt mutants. Planta 213, 953–966.
Holton T.A. & Cornish E.C. (1995) Genetics and biochemistry of
anthocyanin biosynthesis. Plant Cell 7, 1071–1083.
Jones H.G. (1992) Stomata. In Plants and Microclimate. Cambridge University Press, Cambridge, UK.
Krol M., Gray G.R., Hurry V.M., Öquist G., Malek L. & Huner
N.P.A. (1995) Low-temperature stress and photoperiod affect
an increased tolerance to photoinhibition in Pinus banksiana
seedlings. Canadian Journal of Botany 73, 1119–1127.
Lee D.W., Brammeier S. & Smith A.P. (1987) The selective advantages of anthocyanins in developing leaves of mango and cacao.
Biotropica 19, 40–49.
Lichtenthaler H.K. (1987) Chlorophylls and carotenoids: pigments
of photosynthetic biomembranes. In Methods in Enzymology
Vol. 148, pp. 350–382. Academic Press Inc, London, UK.
Long S.P., Farage P.K., Aguilera C. & Macharia J.M.N. (1990)
Damage to photosynthesis during chilling and freezing, and its
significance to the photosynthetic productivity of field crops. In
Current Research in Photosynthesis (ed. M. Baltscheffsky) Vol.
IV, pp. 471–474. Kluwer Academic Publishers, Dordrecht, The
Netherlands.
Loreto F., Di Marco G., Tricoli D. & Sharkey T. (1994) Measurements of mesophyll conductance, photosynthetic electron transport and alternative sinks of field grown wheat leaves.
Photosynthesis Research 41, 397–403.
Mancinelli A. (1983) The photoregulation of anthocyanin synthesis. In Encyclopaedia of Plant Physiology (eds A. Pirson & M.H.
Zimmermann) Vol. 16B, pp. 640–661. Springer-Verlag, Berlin,
Germany.
Mancinelli A. (1984) Photoregulation of anthocyanin synthesis.
VIII. Effects of light pre-treatments. Plant Physiology 75, 447–
453.
Marrs K.A., Alfenito M.R., Lloyd A.M. & Walbot V. (1995) A
glutathione-S-transferase involved in vacuolar transfer encoded
by the maize gene Bronze-2. Nature 375, 397–400.
Massacci A., Iannelli M.A., Pietrini F. & Loreto F. (1995) Photosynthetic characteristics and mechanisms of protection on contrasting maize genotypes grown at low temperature. Journal of
Experimental. Botany 46, 119–127.
McCord J. & Fridovich I. (1969) Superoxide dismutase: an enzymatic function for erythrocuprein. Journal of Biological Chemistry 22, 6049–6055.
Merzlyak M.N. & Chivkunova O.B. (2000) Light-stress-induced
pigment changes and evidence for anthocyanin photoprotection
in apples. Journal of Photochemistry and Photobiology 55, 155–
163.
Neill S.O., Gould K.S., Kilmartin P.A., Mitchell K.A. & Markham
K.R. (2002) Antioxidant activities of red versus green leaves in
Elatostema rugosum. Plant, Cell and Environment 25, 539–548.
Petroni K., Cominelli E., Consonni G., Gusmaroli G., Gavazzi G.
& Tonelli C. (2000) The development expression of the maize
regulatory gene Hopi determines germination-dependent
anthocyanin accumulation. Genetics 155, 323–336.
Pietrini F. & Massacci A. (1998) Leaf anthocyanin content changes
in Zea mays L. grown at low temperature: significance for the
relationship between the quantum yield of PSII and the apparent quantum yield of CO2 assimilation. Photosynthesis Research.
58, 1–8.
Schindler C. & Liechtenthaler H.K. (1996) Photosynthetic CO2assimilation, chlorophyll fluorescence and zeaxanthin accumulation in field grown maple trees in the course of a sunny and
cloudy day. Journal of Plant Physiology 148, 399–412.
Smillie R.M. & Hetheringhton S.E. (1999) Photoabatment by
anthocyanin shields photosynthetic systems from light stress.
Photosynthetica 36, 451–463.
Taylor L.P. & Briggs W.R. (1990) Genetic and photocontrol of
anthocyanin accumulation in maize seedlings. Plant Cell 2, 115–
127.
Thayer S.S. & Björkman O. (1990) Leaf xanthophyll content and
composition in sun and shade determined by HPLC. Photosynthesis Research 23, 331–343.
Wise R.R. (1995) Chilling enhanced photoxidation: the production, action and study of reactive oxygen species produced during chilling in the light. Photosynthesis Research 45, 79–97.
Wrolstad R.E. (1976) Color and pigment analyses in fruit products.
Oregon State University Agriculture Experimental Station Bulletin 624, 1–17.
Received 1 April 2002; received in revised form 9 May 2002;
accepted for publication 20 May 2002
© 2002 Blackwell Publishing Ltd, Plant, Cell and Environment, 25, 1251–1259