OPINION

Photoinhibition in a C4 plant, Zea mays L.: a minireview

Carlos Pimentel

Received: 1 May 2014 / Accepted: 9 June 2014 / Published online: 26 June 2014

� Brazilian Society of Plant Physiology 2014

Abstract Light and oxygen are both essential to life

but can also cause some damage to plant photosyn-

thesis and productivity, particularly excess light in the

presence of oxygen. This phenomenon is called

photoinhibition, i.e. the photooxidative damage of

photosystems due to excess light associated or not

with abiotic stresses. To tolerate photoinhibition, the

plant needs to be able to avoid or tolerate excess light

or to repair the photodamage by maintaining the de

novo D1 protein synthesis that is essential for PS II

complex activity. When the electron transport of the

photosystems is diminished because the use of final

electron acceptors, ferredoxin or NADPH2, via the

Calvin cycle is reduced, electrons from water photol-

ysis are captured by O2 forming ROS, which inhibits

protein synthesis, even in C4 plants. To tolerate excess

light or repair photodamage under photoinhibitory

conditions, several mechanisms exist to reduce ROS

and/or maintain ATP production and use for protein

synthesis and growth: the scavenging systems (reduc-

ing ROS activity), such as the xanthophyll (thermal

dissipation of excitation energy) and ascorbate cycle

(associated with the Mehler reaction, in the water–

water cycle) or the activity of antioxidants com-

pounds, such as carotenoids; the maintenance of cyclic

electron flow (only ATP production); and the photo-

respiration cycle (consuming reducing equivalents and

ATP). Photoinhibition is a phenomenon that occurs in

C3 and C4 plants, such as corn, under PPFD above

1,000 lmol m-2 s-1, and photooxidative damage

seems to be more accentuated in mesophyll chloro-

plasts than in bundle sheath agranal chloroplasts.

Keywords C4 mesophyll chloroplast � D1 protein �Photooxidative damage � Repair of photodamage �Tolerance

1 Introduction

Light, the energy source for photosynthesis to produce

energy rich compounds, i.e. carbohydrates, is required

by most life on this planet to produce the main cellular

‘‘fuel’’ ATP by photosynthesis. However, an excess of

light in the presence of O2 (a strong oxidant) can cause

damage, particularly to photosystem II (PS II) in the

thylakoid membranes of chloroplasts in plants (Poko-

rska et al. 2009). This phenomenon is called photoin-

hibition of the photosynthetic apparatus and can lead

to a reversible or irreversible photooxidative damage

of organic compounds of the photosynthesis apparatus

(Osmond et al. 1980), especially proteins that organize

chlorophylls in the light-harvesting complex II (LHC

II) associated with PS II, but also in the LHC I

associated to PS I (Nishiyama et al. 2011). When

C. Pimentel (&)

Departamento de Fitotecnia, Instituto de Agronomia,

Universidade Federal Rural do Rio de Janeiro (UFRRJ),

Km 47 BR 465, Seropedica, RJ 23897-000, Brazil

e-mail: greenman@amcham.com.br

123

Theor. Exp. Plant Physiol. (2014) 26:157–165

DOI 10.1007/s40626-014-0015-1

sunlight can be converted in chemical energy by the

photosystems, there is a minimal potential for damage.

However, when sunlight is absorbed by the photosys-

tems but cannot be fully utilized because photosyn-

thetic electron transport is saturated (Ort 2001),

photooxidative damage can occur, causing a decrease

in intrinsic PS II efficiency, often evaluated by

measuring the maximum quantum yield of PS II, i.e.

the ratio of variation in maximal fluorescence emis-

sion (Fv/Fm) obtained with chlorophyll fluorescence

equipment (Pimentel et al. 2005; Baker 2008). The

excess of absorbed excitation energy at the PS II

reaction center has the potential to be transferred with

electrons to the ever-present O2, leading to the

production of reduced and excited of oxygen species

(singlet oxygen, hydrogen peroxide, hydroxyls and

others radicals), called reactive oxygen species (ROS).

The ROS can cause damage to the photosystems on

thylakoids and, ultimately, leaf death (Demmig-

Adams and Adams 2006). Thus, on most sunny days,

plants encounter a photosynthetic photon flux density

(PPFD) that exceeds their photosynthetic capacity and

photoinhibition can occur (Ort 2001). Plant evolution

has had to tread a path between maximizing light

interception for photosynthesis and minimizing the

potential for damage arising from the over-excitation

of the photosynthetic apparatus (Adams et al. 2013),

especially in marginal areas for agriculture submitted

to high PPFD and others environmental stresses

(Pimentel 2006).

Species with their center of origin in these marginal

areas under high PPFD have adapted their photosyn-

thetic capacity (Harlan 1992) and the ability to avoid

photoinhibition (by chloroplast and leaf movement,

increased leaf reflectance or anthocyanin accumula-

tion, for example) or to tolerate it by dissipation of this

intercepted excess light energy and other mechanisms

of tolerance to photoinhibition or repair of photodam-

age (Long et al. 1994). On the other hand, species like

corn (a C4 plant evolved in the altiplanes of Central

America), with a center of origin that presents more

favorable conditions, high precipitation and tempera-

tures adequate to the species (Harlan 1992), have not

evolved with avoidance, tolerance or repair mecha-

nisms against environmental stresses (Osmond et al.

1980). Thus, they are more sensitive to environmental

stresses that are nonexistent in their center of origin;

for example, corn shows photoinhibition associated

with cold, heat or droughts. During their evolution,

other grasses originating from the Fertile Crescent

region (wheat, oat and barley, all C3 plants) or from the

Sahel region (sorghum, a C4 plant) developed mech-

anisms of avoidance, tolerance or repair of the effects

of photoinhibition and the environmental stresses

(Ludlow and Powles 1988) that occur in these regions

(Harlan 1992), and this adaptation is independent of

the C3 or C4 photosynthetic pathway (Pimentel 1998).

Therefore, photoinhibition is a phenomenon

defined as the reversible or irreversible light-induced

decrease in photosystems activity, as a result of

exposure to high PPFD that exceeded the photosyn-

thetic apparatus ability to fully utilize the energy

absorbed, in the presence or absence of other envi-

ronmental stresses (Osmond et al. 1980). When the

relative consumption rates of ATP and NADPH2 are

lower than their production rates, under high PPFD,

photoinhibition can occur (Powles 1984; Adams et al.

2013). The presence of other environmental stresses,

including: high vapor pressure deficit (VPD) and

drought, since high VPD and drought induce stomatal

closure, reducing CO2 availability; salinity, which

induces stomatal closure and osmotic stress, and can

cause more damage than drought; nutrient deficien-

cies, particularly nitrogen, because more than 50 % of

leaf nitrogen is used for the synthesis of ribulose-1,5-

bisphosphate carboxylase/oxygenase, i.e. Rubisco;

high and low temperatures for the crop, which reduces

the enzymatic activity of the Calvin cycle, all increase

the photoinhibitory effect of high PPFD. All these

environmental stresses can promote a reduction in the

Calvin cycle activity, photosynthetic carbon assimila-

tion and growth, increasing ROS production and

photodamage (Pimentel 2006). When the environ-

mental conditions restrict plant growth and photosyn-

thesis, under high PPFD, then a truly massive level of

excess excitation energy is encountered and photo-

damage due to ROS production increases. Photooxi-

dative damage can be reversible, through mechanisms

of avoidance, tolerance or repair of photodamage,

even at the end of the day (Fig. 1b); or it can be

irreversible depending of the intensity of PPFD and

abiotic stresses (Demmig-Adams and Adams 2006).

Therefore, the extent of photoinhibition is associated

with a balance between the rate of photodamage,

which depends on avoidance and tolerance mecha-

nisms, and the repair of PS II. In addition, photodam-

age and inhibition of PS II repair can also be prevented

by avoiding light absorption by the manganese cluster

158 Theor. Exp. Plant Physiol. (2014) 26:157–165

123

in the oxygen-evolving complexes (OEC), because

recent studies have shown that photodamage to PS II is

associated with light absorption by OEC, and effec-

tively consuming (or dissipating) the light energy

absorbed by photosynthetic pigments, respectively

(Takahashi and Badger 2011).

Frequently, over half of the light absorbed by

photosystem II chlorophylls in healthy and fully

functional leaves need to be redirected to discharge

the excess absorbed light energy. Thus, PPFD equiv-

alent to about one half of full sunlight (around

1,000 lmol m-2 s-1) frequently produces a mild

diurnal photoinhibition, although full recovery occurs

within a few hours (Ort 2001), as observed for corn

irrigated in the field (Fig. 1b). Photoinhibition causes

a reduction in photosynthesis and/or photosynthetic

efficiency (e.g. in Fv/Fm), which is due to photodam-

age of the photosynthetic apparatus, which in turn

induces a decrease in plant growth and yield (Pimentel

et al. 2005). At least 10 % of the yield potential is lost

due to photoinhibition of crops in full sunlight. In

addition, high PPFD (above 1,000 lmol m-2 s-1), in

combination with other environmental stresses

A (

mo

l m-2

s-1

)

10

20

µ

30

40

gs

(mo

l m-2

s-1

)

0,2

0,4

0,6

0,8

1,0

Time of the day

6 8 10 12 14 16 18 20

Fv/

Fm

0,0

0,2

0,4

0,6

0,8

1

NP

Q

0,0

0,2

0,4

0,6

0,8

1,0

A

B

Fig. 1 The diurnal gas

exchange and chlorophyll

fluorescence of Zea mays L.,

in an irrigated field:

a photosynthetic CO2

assimilation (A, filled

square) and stomatal

conductivity (gs, open

square); b maximum

quantum yield of PS II (Fv/

Fm, filled square) and non-

photosynthetic energy

quenching (NPQ, open

square)

DFFF ( mol m-2 s-1)

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200

A (

mo

l m-2

s-1

)

-10

0

10

µ

20

30

40

50

µ

Fig. 2 A/Q curve of Zea mays L.: the relation between

photosynthetic CO2 assimilation (A) and photosynthetic photon

flux density (PPFD)

Theor. Exp. Plant Physiol. (2014) 26:157–165 159

123

affecting photosynthetic carbon assimilation (A),

increases photodamage and can lead to leaf death,

even for a C4 plant like corn (Long et al. 1994). In

corn, A is not limited by an excess of PPFD, as shown

by the tendency line in Fig. 2, but the values of A

above 1,000 lmol m-2 s-1 of PPFD do not increase

to a sufficient extent to consume the increased

reducing equivalent and ATP produced at high PPFD

and thus to prevent ROS production and accelerate

the repair of PS II. Therefore, PPFD above

1,000 lmol m-2 s-1 can cause photoinhibition, even

in a C4 plant like corn (Pokorska et al. 2009), due to the

reduction in Fv/Fm while under 1,000 lmol m-2 s-1

even for only three hours exposure (Fig. 3), with slow

recovery following dark adaptation.

2 Photodamage

Photoinhibition can also be considered a consequence,

rather than a cause, of limited plant productivity, when

more sugars are produced in leaves rather than can be

utilized by the rest of the plant (Adams et al. 2013),

which will down regulate photosynthesis and photo-

system activity due to an excess of sugars in the cell

(Pimentel 1998). When this phenomenon occurs in an

ambient with high PPFD, ROS production and photo-

damage increases (Demmig-Adams and Adams 2006).

Photoinhibition of PS II occurs when the rate of

photodamage to PS II exceeds the rate of repair of the

photodamaged PS II. The rate of photodamage of PS II

is directly proportional to PPFD and the repair of PS II

is particularly sensitive to inactivation by ROS; thus

the activity of PS II under high PPFD depends on the

balance between the rates of photodamage, which in

turn depends on avoidance and tolerance mechanisms

and the repair of PS II (Takahashi and Badger 2011).

Recent studies showed that photoinhibition is

related to D1 protein damage (rate of dephosphoryl-

ation and degradation of D1 protein and its de novo

synthesis) from the reaction center of PS II in the OEC

of the PS II–LHC II super complex (Pokorska et al.

2009), and D1 protein damage leads to a decrease in

electron transfer (Long et al. 1994). The rate of

photodamage to PS II is directly proportional to PPFD

and this effect is due mainly to the suppression of de

novo synthesis of proteins, such as D1 protein,

inactivated by ROS produced under photoinhibitory

conditions. Under photoinhibition, acidification of the

lumen occurs, due to reduced electron transfer in

photosystems and ATP synthase (ATPase) activity,

which transfer protons from the lumen to stroma and

could inactivate the OEC and increase ROS produc-

tion, thus reducing de novo D1 protein synthesis

(Nishiyama et al. 2011). Cell growth and protein

synthesis are both processes that are highly sensitive to

environmental stresses, reducing plant productivity

(Pimentel 2004) caused by photodamage because the

repair of D1 protein is reduced by the suppression of

de novo protein synthesis (Adams et al. 2013). In

addition, the thylakoid fatty acid composition can also

influence photodamage intensity and species with a

high level of saturated fatty acid and phosphatidyl-

glycerol (PG) in the thylakoid membranes are more

tolerant to photoinhibition and abiotic stresses (Pi-

mentel 2004). However, others studies using inhibitors

of plastid-encoded protein synthesis, such as linco-

mycin and chloramphenicol (which block the de novo

synthesis of the plastid-encoded D1 protein), have

shown that direct photodamage to PS II cannot be

directly associated with excessive light energy

absorbed by photosynthetic pigments or with the

production of ROS and thus, it remains controversial

whether photodamage to PSII can be exclusively

attributed to the effects of excessive light energy and

ROS on the PS II reaction centre (Takahashi and

Badger 2011).

Time (minutes)

0 20 40 60 80 100 120

F v/F

m

0,0

0,4

0,5

0,6

0,7

0,8

Fig. 3 Photoinhibition of healthy and fully functionally leaves

of Zea mays L. plants (with three leaves completely expanded, at

28 days after sowing). The control plants were maintained in the

dark for 3 h (filled square), and the photoinhibited leaves were

maintained under PPFD of 1,000 lmol m-2 s-1 (open circle) or

under PPFD of 2,000 lmol m-2 s-1 (filled triangle) for 3 h,

before measuring the Fv/Fm of dark-adapted leaves at 10 min

interval over 2 h, for the three treatments

160 Theor. Exp. Plant Physiol. (2014) 26:157–165

123

In addition, UV and blue light are much more

energetic than light in other parts of the spectrum and,

for this reason, they are more effective at damaging PS

II by inducing manganese ion release from the PS II in

parallel with the inactivation of this photosystem

(Baker 2008). Photodamage of PS II can happen in a

two step process: in the first step, upon absorption of

light energy, damage occurs in the OEC, and this step

is particularly sensitive to UV and blue light; and in

the second step, damage occurs in the PS II–LCH II as

a consequence of excessive absorption of light energy

by chlorophylls (Nishiyama et al. 2011).

3 Repair of photodamage

Photoinhibition is related to D1 protein damage and

recovery and D1 protein damage leads to a decrease in

electron transfer in photosystems. The recovery from

photoinhibition is dependent on de novo D1 protein

synthesis (Adams et al. 2013). The repair cycle for the

PS II consists of dephosphorylation and degradation of

photodamaged D1 protein and de novo synthesis of

this protein of the PS II–LHC II super complex,

followed by activation of the PS II reaction center.

Rapid synthesis of the D1 protein and the repair of the

photodamaged PS II subunits require a large amount

of ATP and reducing equivalents (Pokorska et al.

2009). However, PS II damage reduces the non-cyclic

electron flow and the rate of ATP synthesis (Ort 2001).

One possible alternative pathway for ATP synthesis is

the cyclic electron flow around PS I during the repair

of PS II, but if PS I is severely photodamaged, the

repair of PS II, through de novo D1 protein synthesis,

will be reduced (Adams et al. 2013).

Therefore, repair of PS II activity requires the rapid

de novo synthesis of the D1 protein and the light-

dependent de novo synthesis of this protein is dom-

inantly regulated at the elongation step of translation

of the polypeptide, which depends on ATP and

reducing equivalents of ferredoxin and thioredoxin

from photosystems for its activation (Takahashi and

Badger 2011). Reducing equivalents generated by the

photosynthetic transport of electrons are transmitted to

an elongation factor G (EF-G), via thioredoxin-

dependent redox pathway, which control the de novo

synthesis of D1 protein and cotranslational insertion of

D1 protein into the PS II complex. High levels of ROS,

produced by photosynthetic machinery, interrupt the

redox signal by maintaining EF-G in an oxidized state

and, as a consequence, they suppress the protein

synthesis that is required for the repair of PS II

(Nishiyama et al. 2011). Thus, under conditions of

excess light for photosynthesis, the rate of repair is

depressed due to inhibition of the de novo synthesis of

D1 protein, and ROS produced under this condition

increases photoinhibition not only through photodam-

age, but also by inhibiting the repair of photodamaged

PS II by the de novo synthesis of D1 protein

(Takahashi and Badger 2011). Under high PPFD,

accumulation of ROS and inactivation of the repair of

PS II can be observed, especially when the Calvin

cycle is suppressed (Long et al. 1994).

4 Photoprotection (mechanisms to mitigate

photoinhibition)

Photoinhibition is due to photooxidative damage

associated with the impairment of various mecha-

nisms that protect the integrity of the PS II complex

against photoinhibition, including the thermal dissi-

pation of excitation energy via the xanthophyll cycle

(Ort 2001; Baker 2008), photorespiration, which

consumes O2, ATP and NADPH2 (Takahashi and

Badger 2011), the cyclic transport of electrons,

helping to maintain the trans-thylakoid membrane

proton gradient and favors the synthesis of ATP

(Nishiyama et al. 2011), or the Mehler-peroxidase

reaction, i.e. the water–water cycle, associated with

the ascorbate/glutathione cycle (Baker and Rosenqvist

2004), which uses NADPH2 to reduce H2O2 to H2O

(electron flow from water to water), and maintain the

photosystem compounds oxidized (‘‘open state of

photosystem’’) to receive electrons from water pho-

tolysis. All these photoprotective mechanisms reduce

ROS production or its effects on the photosystems

(Demmig-Adams and Adams 2006).

Actually, there is compelling evidence that excess

PPFD conditions are sensed or signaled by a large

DpH, i.e. low-lumen pH compared to stroma pH,

which forms when reducing equivalents and ATP use

is restricted by CO2 availability or by stress-induced

dysfunction in the enzymology of carbon reduction,

leading to reduced electron transport on the photosys-

tems and transthilakoid DpH (Ort 2001). Only when

the lumen pH is driven to very low values the

photoprotective thermal energy dissipation within PS

Theor. Exp. Plant Physiol. (2014) 26:157–165 161

123

II initiate by activating violaxanthin de-epoxidase,

which in turn converts violaxanthin, a xanthophyll

pigment bound to the LCH II of the PS II, to

zeaxanthin thereby initiating thermal energy dissipa-

tion at the LCH II (Baker 2008). The DpH-dependent

accumulation of zeaxanthin results in the reversible

oligomerization of LCH II, leading to an increase in

heat quenching of the energy of chlorophyll in the

excited state, i.e. an increase in the thermal dissipation

of absorbed light energy and thus, the xanthophyll

cycle has an indirect role in this process by mediating a

critical conformational change within the PS II

antenna to induce the thermal dissipation of the

absorbed energy by chloroplasts. Chlorophylls can

transfer or dissipate the absorbed energy as chemical

energy (producing ATP and reducing equivalents in

the photosystems), fluorescence (proportional to the

chemical energy used in the photosystems) and heat

(Takahashi and Badger 2011). Therefore, thermal

dissipation appears to play a role in preventing the

generation of ROS and ROS-induced suppression of

protein synthesis resulting in the maintenance of the

repair of PS II, when the PSII-mediated transport of

electrons is reduced (Nishiyama et al. 2011). Fre-

quently over half of the light absorbed by PS II

chlorophylls in healthy, fully functional leaves can be

redirected and excess PPFD energy can be harmlessly

discharged as heat (Ort 2001). This phenomenon is

also referred as the non-photochemical quenching

(NPQ), which can be monitored in terms of chloro-

phyll fluorescence (Fig. 1b), as reported by Baker

(2008). Under conditions of excess of PPFD, the

production of ROS is accelerated both at PS II and at

PS I (Baker and Rosenqvist 2004).

Cyclic electron flow (CEF) around PS I enhances

the generation of a DpH across the thylakoid mem-

brane through increased electron transfer from PS I to

plastoquinone (PQ), via both NADPH2 dehydrogenase

(NDH) complex-dependent and ferredoxin (Fd)-

dependent electron transport pathways (Takahashi

and Badger 2011). CEF has been demonstrated to be

important in the generation of a DpH across the

thylakoid membrane, which helps to prevent destabi-

lization of the OEC complex, and to produces ATP,

which is required for the repair of PS II by de novo D1

protein synthesis (Nishiyama et al. 2011). In addition

to thermal dissipation of LCH II absorbed light energy,

to avoid oxidative stress by ROS, chloroplasts scav-

enge ROS effectively using multiple antioxidant

systems: enzymes, such as superoxide dismutase,

ascorbate peroxidase and peroxiredoxin, or antioxi-

dant compounds, such as the water-soluble ascorbate

(vitamin C), the membrane-bound tocopherol (vitamin

E) and carotenoids, such as zeaxanthin, neoxanthin

and lutein in chloroplasts (Takahashi and Badger

2011). Antioxidants have a crucial role in redox

sensing and signaling for plant response to abiotic and

biotic stresses, and redox balance plays a key role both

in the modulation of growth and development and in

the control of photosynthetic events via oxidative

modifications and redox modulation (Demmig-Adams

and Adams 2006).

Other processes involved in protecting against

photooxidative damage of PS II are the Calvin cycle

and photorespiration (Baker and Rosenqvist 2004;

Osmond et al. 1980). The accumulation of excess

electrons, inducing inactivation of the repair of PS II,

has been observed when the Calvin cycle is sup-

pressed. Thus, appropriate flow of electrons from the

photosystems to the Calvin cycle, through reducing

equivalents used to reduce CO2 to sugars appears to

protect PS II from photoinhibition, by preventing

ROS production and its induced deceleration of PS II

complex repair (Nishiyama et al. 2011). In addition,

photorespiration protects PS II from photoinhibition

due to its effective elimination of excess electrons in

the glycolate/glycerate cycle, which uses ATP and

reduced equivalents (Osmond et al. 1980), maintain-

ing the photosystems ‘‘open’’ (oxidized) to receive

electrons from water photolysis, thus preventing

ROS production and its effects on the repair of PS II.

During the photorespiratory carbon cycle, ammonia

and CO2 are produced by the mitochondrial glycine

decarboxylase and ammonia is subsequently refixed

into glutamate, by chloroplastic isozymes of gluta-

mine synthetase and Fd-dependent glutamate syn-

thase in the photorespiratory nitrogen cycle

(Takahashi and Badger 2011). Photorespiration is

maintained even under conditions where the supply

of CO2 is limited due to stomatal closure (high VPD,

drought or salinity) or reduced CO2 solubility (high

temperatures, above the optimum for the crop), but

O2 is still available and photorespiration is increased

in these stressful conditions (Ludlow and Powles

1988), but it is a limited electron sink in C4

photosynthesis (Pimentel 2004).

162 Theor. Exp. Plant Physiol. (2014) 26:157–165

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5 Response of corn, A C4 plant, to photoinhibition

The kinetics of PS II photodamage is similar in C3 and

C4 plants (Long et al. 1994; Osmond et al. 1980).

Nevertheless, corn is a NADP-ME (NADP-malic

enzyme) type, like sugar cane and sorghum, and their

bundle sheath chloroplasts are agranal and do not

synthesize ATP and NADPH2, while the mesophyll

chloroplasts are granal producing ATP and NADPH2.

Thus reducing equivalents and ATP are necessary for

the C4 cycle in mesophyll tissue, but they are also

transported to the bundle sheath, where the Calvin cycle

is restricted, by trioses or organic acid shuttles (Pimen-

tel 1998). However, PS II and the LHC II complex exist

in the thylakoids of the bundle sheath chloroplasts, but

there is no electron transport and ATP synthesis, which

depends on the formation of the lumen of the granum to

create a DpH between lumen and stroma, for ATPase

activity (Pokorska et al. 2009). In corn under photoin-

hibitory conditions, it has been shown that the D1

protein was dephosphorylated (dephosphorylation of

the D1 protein precedes its degradation) in mesophyll

and bundle sheath chloroplasts, but it was rapidly

degraded only in the bundle sheath agranal chloro-

plasts, to be de novo synthesized for the repair of

photodamage in the non functional bundle sheath

photosystem. In these mesophyll chloroplasts, PSII

monomers accumulated and minimal degradation of D1

protein was observed, thus repair of photosystems

through de novo D1 protein synthesis presents greater

reduction in mesophyll cells than in bundle sheath cells.

Perhaps the low content of the degradation enzyme

(Deg1) observed in mesophyll chloroplasts isolated

from corn grown under moderate light retards the D1

repair processes in this type of plastid, increasing

photodamage (Pokorska et al. 2009).

Thus, under high PPFD and CO2 deprivation due to

stomatal closure (or other stress that reduces photo-

synthetic enzymes activity), corn is photoinhibited

(Hichem et al. 2009) and its mesophyll cells present

greater photodamage than bundle sheath cells (Poko-

rska et al. 2009). The mesophyll cells lack Rubisco and

numerous enzymes of the Calvin cycle and of the

photorespiratory pathway and hence are unlike to

produce internal CO2 to maintain A (Pimentel 1998),

such that the mesophyll cells of corn are particularly

sensitive to photoinhibition (Osmond et al. 1980).

Under water deficit, the mesophyll chloroplast ultra-

structure of corn was disorganized rather more readily

than that of bundle sheath chloroplasts. In addition,

under heat stress, photosynthesis in corn is completely

inactivated before the symptoms of any other high

temperature injury can be detected (Osmond et al.

1980). The observed thermal inactivation of corn

photosynthesis under high PPFD is caused primarily

by a direct effect of temperature on the photosynthetic

machinery, inactivating PS II electron transport. An

increase in the thermal stability of thylakoid mem-

brane reactions plays a key role in the superior

performance of heat-adapted corn genotypes. These

different responses could represent variable stages of

photoinhibition and its effects on protein synthesis

(Osmond et al. 1980).

Even for corn plants irrigated in the field, when

growing under high PPFD on days of high VPD, leaf

photosynthesis reach a maximum in the late morning

and then decreases gradually as the day progresses,

though the soil was well irrigated (Hirasawa and Hsiao

1999). The same response was detected in a corn

irrigated in the field and cultivated at the University of

Illinois, under high PPFD and VPD (Fig. 1). The

reduction of A after late morning is due in part to

stomatal closure (Fig. 1a), as affirmed by Hirasawa

and Hsiao (1999), but it is also due to a transient

photoinhibitory effect, which can be detected by the

reduction in Fv/Fm and increase in NPQ at noon,

recovering in the late afternoon (Fig. 1b).

Furthermore, several chloroplast enzymes are

known to be activated through reduction by the

photosynthetic electron transfer chain, via the ferre-

doxin–thioredoxin system. One of these enzymes is

NADP-malate dehydrogenase, also known as the

NADP-malic enzyme (NADP-MDH or NADP-ME

enzyme), which is very sensitive to photoinhibition in

corn; its activity is inhibited when a reduction in its S–

S groups, via the ferredoxin–thioredoxin system, does

not occur in this enzyme, such that a reduction of more

than 50 % is required for activity to appear (Miginiac-

Maslow et al. 1990). NADP-ME can be fully active

under conditions where the reduction state inside the

chloroplast is maximal, and a partial decrease in the

electron transfer efficiency can result in complete

inhibition of NADP-ME activity (Miginiac-Maslow

et al. 1990). This enzyme is responsible for the malate

decarboxylation to pyruvate plus CO2, furnishing CO2

for Rubisco in the Calvin cycle that occurs in the

agranal chloroplast of the bundle sheath of corn, a

NADP-ME C4 type (Pimentel 1998).

Theor. Exp. Plant Physiol. (2014) 26:157–165 163

123

Most of the studies with corn under photoinhibitory

conditions have been conducted under chilling-

induced photoinhibitory conditions, because corn is

an important crop for Northern European and North

American agriculture, where the temperature in early

spring is between 4 and 15 �C. However, the optimal

corn growth temperature are between 20 and 30 �C

and the crop is considered sensitive to low and high

temperature, as well as to drought (Kingston-Smith

and Foyer 2000; Osmond et al. 1980). Crops originat-

ing from the subtropics, like corn, appear to be

particularly sensitive to chilling-induced photoinhibi-

tion and corn is considered one of the most sensitive

plant among those grown in a temperate climate

(Aguilera et al. 1999). The association of high PPFD

and these environmental stresses can causes irrevers-

ible photodamage to young corn seedlings (Pimentel

et al. 2005). Consequently, stress tolerance has

become a major selection criterion for corn breeding

programs (Kingston-Smith and Foyer 2000). There-

fore, even the healthy, functional leaves of Zea mays

L., a C4 plant, are sensitive to photoinhibition under

half of full sunlight (around 1,000 lmol m-2 s-1), as

reported by Ort (2001) (see Fig. 3), with a reduction in

Fv/Fm after three hours of exposure to a PPFD of

1,000 lmol m-2 s-1, which is accentuated when

exposed to a PPFD of 2,000 lmol m-2 s-1. As stated

before, chilling-induced photoinhibition (tempera-

tures below 15 �C and high PPFD) causes an accen-

tuated decrease in its photosynthetic capacity, which is

a major barrier to the extension of viable corn

production in cooler climates and numerous studies

have been conducted to evaluate corn chilling toler-

ance (Aguilera et al. 1999). Although corn recovery

from photoinhibition on cool mornings may occur

within a few hours of warm weather, this recovery is

too low to prevent significant losses (around 10 %) in

potential carbon assimilation, growth and yield (Long

et al. 1994). Growth and protein synthesis are partic-

ularly sensitive to photoinhibition conditions when

corn seedlings are in transition between heterotrophic

and autotrophic growth at the 2nd to 3rd mature leaf

stage (Pimentel et al. 2005) (see Fig. 3), but also when

the plants are in the pollination stage (Pimentel 2004).

However, selection for improved chilling-depen-

dent photoinhibition in corn should concentrate on

selection for not only tolerance (mechanisms to

mitigate photoinhibition), but also for the capacity

for repair and recovery from photodamage (de novo

D1 protein synthesis), which seems to be at least

partially independent (Long et al. 1994). There is

evidence of an intraspecific variation in susceptibility

to chilling-dependent photoinhibition and the capacity

for recovery in corn for the selection of improved corn

genotypes with increased photosynthetic capacity at

low temperatures and high PPFD (Pimentel et al.

2005). Kingston-Smith and Foyer (2000) showed that

molecular manipulation to over-express Mn-superox-

ide dismutase in transformed corn leaves induced an

increase in the activity of monodehydroascorbate

reductase, dehydroascorbate reductase and glutathi-

one reductase enzymes from the antioxidant system

ascorbate–glutathione, which is important for reduc-

ing photodamage in mesophyll chloroplasts of corn,

but the increase in their activity did not change the

content of H2O2, ascorbate or glutathione in corn

leaves. These antioxidant enzymes are located in

mesophyll cells and are absent in bundle sheath cells

(Kingston-Smith and Foyer 2000).

Photoinhibition is most easily diagnosed as a

decrease in Fv/Fm (Hichem et al. 2009), measured

with chlorophyll fluorescence equipment (Baker

2008), which is linearly related to the maximum

quantum yield of carbon assimilation (Long et al.

1994). Some variation in corn tolerance in chilling-

dependent photoinhibition has been detected (Pimen-

tel et al. 2005), which has been associated with a wide

range of traits in comparative physiological studies.

Three quantitative trait loci (QTL) were detected to be

closely associated with genes that have been related to

photoinhibition tolerance and repair, using Fv/Fm

measurements in a QTL analysis. Therefore, Fv/Fm

measurements by chlorophyll fluorescence is an

approach that could be used in establishing marker-

assisted breeding for improved tolerance to chilling of

corn’s chilling (Pimentel et al. 2005). However,

whereas chlorophyll fluorescence provided a rapid

method to assess the occurrence of photoinhibition, it

is not as effective as direct gas exchange measure-

ments, but the latter requires more time to perform the

measurements (at least 20 min), in discriminating

genotypes of corn with respect to photoinhibition of

the photosynthetic apparatus, especially in the most

vulnerable genotypes (Aguilera et al. 1999). To select

corn genotypes, a plant with open pollination, screen-

ing of a large population (around 200 families) is

needed and gas exchange measurements cannot be

performed, because the time required for such

164 Theor. Exp. Plant Physiol. (2014) 26:157–165

123

measurements would be excessive, thus rapid chloro-

phyll fluorescence measurements can be used to

screen these families (Pimentel et al. 2005).

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