Long - 2013 - Toward Cool C4 Groups

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First published online as a Review in Advance on photosynthesis, Miscanthus, maize, chilling, cold, RuBisCO
March 4, 2013
The Annual Review of Plant Biology is online at Abstract
plant.annualreviews.org
C4 photosynthesis under optimal conditions enables higher-efficiency
This article’s doi:
10.1146/annurev-arplant-050312-120033
use of light, water, and nitrogen than the C3 form used by many crops.
It is associated with the most productive terrestrial plants and crops but
Copyright c 2013 by Annual Reviews.
All rights reserved
is largely limited to the tropics and subtropics. It has been argued that
the C4 photosynthetic apparatus is inherently limited to warm environ-
ments. A small group of C4 species appear to have overcome this, and
in contrast to the major C4 crop, maize, these species are able to accli-
mate their photosynthetic apparatus to chilling conditions. This review
explores the mechanisms underlying this difference as well as the po-
tential of introducing these changes into maize and other warm-climate
C4 crops.
701
energy capture by a leaf to the synthesis of

Contents glucose shows that, at today’s atmospheric CO2
concentration, C4 photosynthesis is potentially
INTRODUCTION . . . . . . . . . . . . . . . . . . 702
30% more efficient than the ancestral C3 form
What Could Agriculture Gain from
(129). The highest recorded annual dry-matter
Cold-Adapted C4 Crops? . . . . . . . . 703
productivity for any terrestrial vegetation cur-
The C4 Process . . . . . . . . . . . . . . . . . . . . 704
rently known is for monotypic natural stands of
IS C4 PHOTOSYNTHESIS
the C4 grass Echinochloa polystachya growing on
INHERENTLY UNABLE TO
the Amazon floodplain, which have averaged
COPE WITH CHILLING
100 tonnes per hectare per year (t ha−1 year−1 ;
TEMPERATURES? . . . . . . . . . . . . . . 705
productivity is given throughout as mass of
WHY DOES MAIZE SUFFER THE
oven-dry matter) (88). The highest recorded
CHILLS? . . . . . . . . . . . . . . . . . . . . . . . . . 707
annual dry-matter yield for a crop is for the C4
WHAT MIGHT WE INFER FROM
forage grass Pennisetum purpureum growing in

THE PHYSIOLOGICAL
El Salvador, which produced 88 t ha−1 year−1
ECOLOGY OF C4 SPECIES IN
(9). This superior yield potential associated
THE COLD? . . . . . . . . . . . . . . . . . . . . . 709
with C4 photosynthesis and realized in the trop-
MISCANTHUS AND SPARTINA . . . . . 710
ics has prompted a major effort to introduce C4
Miscanthus × giganteus:
photosynthesis into the major C3 staple crop,
Field Trials . . . . . . . . . . . . . . . . . . . . . 710
rice (54). Although C4 photosynthesis clearly
Miscanthus × giganteus:
confers production advantages in the tropics
Physiological and Molecular
and subtropics, there has been far less certainty
Analysis of Acclimation to
as to whether it confers any advantage in cooler
Chilling . . . . . . . . . . . . . . . . . . . . . . . . 711
climates, where the process has been suggested
Spartina . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
to be inherently disadvantaged relative to C3
PROSPECTS FOR ENGINEERING
photosynthesis (24, 25, 100).
COLD TOLERANCE INTO C4
What are the cooler climates of relevance
CROPS . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
to this review? Following the Köppen-Geiger
With C3 Crops Already Available,
system, these are defined as (a) maritime or
Why Attempt to Adapt C4 Crops
cool temperate climates, such as Britain and
to the Cold? . . . . . . . . . . . . . . . . . . . . 713
the northwestern coastal United States, and
How Might We Make C4 Crops
(b) continental-microthermal climates, defined
Chilling Tolerant? . . . . . . . . . . . . . . 714
by a mean winter temperature of the coldest
Interspecific Breeding for
month of ≤−3◦ C and a mean summer temper-
Chilling Tolerance . . . . . . . . . . . . . . 715
ature of the warmest month of ≥22◦ C, such
as the US corn belt, Ukraine, and Quebec
(55). These encompass many of the major
grain-producing areas of the globe. With a few
INTRODUCTION exceptions, C4 plants are largely absent from
C4 photosynthesis is theoretically the most the first group of climates and are limited in
efficient method of assimilating carbon in the second group to growth in the few months
higher plants with respect to light, water, with mean temperatures of ≥14◦ C. In this
and nitrogen use. C4 plants differ from their climate they are classed as warm-season species
C3 ancestors by fixing CO2 first into a four- (60, 61, 100), as is the current case for Zea mays
carbon (C4 ) carbohydrate, which combined (maize) in the US Midwest (21). Ironically,
with their unique anatomy allows them to because some cool temperate climates, such
minimize photorespiration (41, 95). Analysis as that of western Scotland, may be frost free,
of the energy-transduction steps from sunlight they can allow the cultivation of tropical C3
702 Long · Spence

plants, such as palms and tree ferns, yet the 60

summer temperatures are usually too cold to Miscanthus × giganteus (25°C)
Miscanthus × giganteus (14°C)
allow cultivation of C4 crops (13, 60). This 50 Spartina anglica
Leaf CO2 uptake (µmol m –2 s –1)

highlights the fact that it is the growing season Zea mays (field)
temperature, rather than the winter temper- Zea mays (14°C)
40 Seslaria albicans (C3)
ature, that appears critical to the survival and
production of C4 species. In their dormant
form, some C4 perennial grasses can survive 30
winter temperatures of ≤−20◦ C, yet an average

minimum temperature of the warmest month 20
of the year of ≥9◦ C appears critical for the
presence of any C4 species (60, 61). The focus 10
of this review is the impact of and adaptation to

chilling temperatures, i.e., temperatures above

0
freezing but below 15◦ C (66, 94). 5 10 15 20 25 30 35 40 45 50
Leaf temperature (°C)
Figure 1
What Could Agriculture Gain from
Temperature responses of light-saturated leaf CO2 uptake (Asat ) of four
Cold-Adapted C4 Crops? grasses. The C4 grasses are Miscanthus × giganteus (79), Spartina anglica (71),
C4 photosynthesis originated in the tropics and Zea mays (4, 79); the arctic-alpine C3 grass Sesleria albicans (68) has been
added for comparison. Curves are included for M. × giganteus and Z. mays
and subtropics, where its advantages in terms
grown under either near-optimal temperatures (i.e., warm field conditions or
of increased production potential, water use, 25◦ C in a controlled environment) or chilling conditions (14◦ C in a controlled
and nitrogen use are greatest. So why concern environment) (79).
ourselves with adaptation to colder climates?
Although diminished, as shown below, a the- or seed produced. In 2010, 844 million tonnes
oretical productivity advantage remains even (Mt) of maize was produced, compared with
in cold climates (61). Further, a narrow group 696 Mt of rice and 654 Mt of wheat, which
of C4 grasses have evolved an ability not only ranked second and third in global production,
to survive but also to be highly productive in respectively (30). The light-saturated leaf
cool temperate and continental-microthermal CO2 uptake rates (Asat ) achieved by maize
climates. There is also an emerging molecular under optimal temperatures are approximately
understanding of how this adaptation in 50–60 μmol m−2 s−1 (Figure 1), among the
photosynthesis has been achieved. So what highest recorded and almost double the max-
application might this knowledge provide? imum rates for rice and wheat (77). No doubt
Although most crops are C3 , maize, Sorghum this difference is one major factor explaining
bicolor (sorghum), and Saccharum officinarum why average global maize yields per hectare of
(sugarcane) are C4 , as are several tropical and land are currently 42% higher than the average
warm-season forage grasses, some minor grain for rice and wheat (30). In 2010, 316 Mt of
crops, and the emerging temperate bioen- maize was produced in the United States,
ergy crops Miscanthus × giganteus, Panicum representing more than one-third of the global
virgatum, and Spartina pectinata (23, 43, 61, total; 178 Mt was produced in China; and 58
95). Increasing their ability to tolerate chilling Mt was produced in the European Union (30).
conditions could both expand their potential As such, the three largest producers accounted
growing seasons and increase the latitudes at for 65% of the global total (30). So although
which they could be grown. The significance maize is tropical in origin, the majority of
of this with respect to maize is explained below. world production today is in cool temperate
Maize is today the most important of all and continental-microthermal climates (30).
crops globally in terms of total mass of grain Rising world population and changing diets are
www.annualreviews.org • Toward Cool C4 Crops 703

estimated to require a 70% increase in global chilling conditions, with particular reference to
grain production by 2050 if shortages are to the comparison of chilling-sensitive maize with
be avoided (62). World production of rice and its chilling-tolerant relative M. × giganteus.
wheat has been stagnating, which could place
even more pressure on maize (70). The United The C4 Process
States is not only the largest producer but also C4 species are characterized by their light-
the largest exporter (30). Could its production energy-driven mechanism of concentrating
be increased? In 2011, 87% of US maize CO2 at the site of ribulose-1,5-bisphosphate
grain was produced in the Midwest, with Iowa (RuBP) carboxylation via RuBP carboxy-
and Illinois accounting for almost half of this lase/oxygenase (RuBisCO) (reviewed in 95,
total (118). The growing season in this area is 103). This elevation of CO2 concentration
limited by temperature and averages just four ([CO2 ]) up to approximately 10 times the
months (21). In a side-by-side comparison, current atmospheric level is sufficient to largely

the cold-adapted and related C4 grass M. × inhibit the oxygenase reaction catalyzed by
giganteus was 59% more productive than a RuBisCO (which would otherwise lead to
modern cultivar of maize in Illinois owing to photorespiration) and the inefficiency in net
a longer growing season permitted by superior carbon gain that photorespiration causes in
cold tolerance (21). If similar cold tolerance released CO2 and consumption of ATP and
of C4 photosynthesis could be introduced NADPH in competition with photosynthesis
into maize, a close relative of Miscanthus, then (32, 63). By avoiding photorespiration, C4
significant gains could be made in global grain plants have on average an approximately 30%
productivity and future food security. This may higher potential photosynthetic productivity
also become important to the major effort in (129) and higher efficiencies of water and
engineering C4 photosynthesis into rice (54). nitrogen use (34) compared with C3 plants.
Significant areas of current global rice produc- This concentration mechanism requires the
tion are subject to chilling conditions during isolation of RuBisCO from the intercellular air
early growth (47). Understanding how maize space, which is achieved, with very few excep-
could be adapted to chilling may therefore be tions, by two distinct layers of photosynthetic
equally important in achieving effective C4 rice tissue arranged concentrically around the leaf
crops. vascular bundles. This arrangement, termed
The photosynthetic C4 dicarboxylate Kranz (i.e., wreath-like) anatomy, contains the
cycle and its distribution between dimor- mesophyll cells on the outside in contact with
phic photosynthetic cells are the only major the intercellular air space. The mesophyll cells
photosynthetic differences between a C4 and surround the bundle sheath cells, which are
C3 plant, and are thus key to understanding isolated from the air space but in contact with
why C4 plants appear less productive in the inner surfaces of the mesophyll cells via
cold conditions. Of particular interest is the multiple plasmodesmata (41, 80, 99, 121). The
functioning of the C4 process under chilling bundle sheath cells, as the name implies, typ-
conditions. Tolerance of these conditions is ically form a ring around the vascular bundle.
critical to growth in the late spring and early The mesophyll cells assimilate CO2 from the
autumn in continental-microthermal climates air space into the four-carbon acid oxaloacetate
and throughout the growing season in cool (OAA) via phosphoenolpyruvate carboxylase
temperate climates. Physiological and molecu- (PEPc), which is localized in the mesophyll cell
lar mechanisms of damage by and acclimation cytoplasm. The phosphoenolpyruvate (PEP)
to chilling temperatures that are common to is synthesized in the mesophyll chloroplasts
all plants have been recently reviewed in depth from pyruvate, catalyzed by pyruvate inorganic
(98). The objective of this review is to examine phosphate dikinase (PPDK), at the expense of
the potential for adapting the C4 process to two ATPs per PEP molecule formed. OAA is
704 Long · Spence

either reduced to malate or aminated to aspar- of all three pathways may be found in individu-
tate. The four-carbon dicarboxylate diffuses als to varying degrees, with the balance varying
to the bundle sheath cells that surround the not just with clade and species but also with
vascular bundle and constitute the inner layer development and environment (32). All types
of the C4 Kranz anatomy. In the bundle sheath, are believed to use the same pathway of PEP
the dicarboxylate is decarboxylated by one of carboxylation and (with the possible exception
three pathways, directly or indirectly providing of some PEP-CK types) PEP synthesis by
pyruvate to complete the C4 dicarboxylate PPDK within the mesophyll. In each case, the
cycle (22, 32, 103). These three pathways have C4 cycle may be viewed as an accessory to the
been used to categorize C4 species: Calvin-Benson cycle, which utilizes additional
1. NADP-ME species reduce OAA to ATP to concentrate CO2 at the site of Ru-
BisCO in the bundle sheath, thereby inhibiting
malate in the mesophyll chloroplasts.
This malate diffuses to the bundle sheath the oxygenation of RuBP (22, 103, 121).
chloroplasts, where it is decarboxylated This common difference from C3 plants

by NADP-dependent malic enzyme to explains how C4 plants are more efficient in
pyruvate, which is then returned to the warm climates. Grown under cool conditions of
mesophyll. ≤14◦ C, however, most C4 plants lose their pho-
tosynthetic capacity at all measurement tem-
2. NAD-ME species aminate OAA to
peratures, as exemplified by maize when grown
aspartate in the mesophyll cytoplasm.
at chilling temperatures (Figure 1). Among
Aspartate diffuses to the bundle sheath
the most obvious environmental features relat-
and via transamination is converted to
ing to the distribution of C4 species is their
OAA, which is then reduced to malate
rarity in cool temperate climates and tempo-
and decarboxylated in the mitochondria
ral limitation of growth to the warm season in
by NAD-dependent malic enzyme.
continental-microthermal climates (60, 61).
The resulting pyruvate is converted
to alanine via transamination with the
incoming aspartate and diffuses back to IS C4 PHOTOSYNTHESIS
the mesophyll, where it is converted back INHERENTLY UNABLE TO
to pyruvate. COPE WITH CHILLING
3. Phosphoenolpyruvate carboxykinase TEMPERATURES?
(PEP-CK) species similarly aminate C4 photosynthesis avoids the energetic cost of
OAA to aspartate, which diffuses to the photorespiration under most circumstances but
bundle sheath as in NAD-ME species; uses additional energy in the C4 dicarboxylate
it is then converted back to OAA, but
cycle. Even so, under optimal temperatures,
this takes place in the cytoplasm rather the maximum quantum yield—i.e., the max-
than in the mitochondria. The OAA imum number of CO2 molecules fixed per
is decarboxylated in the cytoplasm by
absorbed photon (CO2,max )—is greater
PEP-CK, yielding PEP. This PEP is in C4 plants because the additional energy
converted to pyruvate, aminated to expended in photorespiration exceeds that
alanine, returned to the mesophyll, and used in the C4 dicarboxylate cycle. However,
converted back to pyruvate (22). It has photorespiration relative to photosynthesis
been argued that, in some instances, this declines in C3 plants with decreasing tem-
PEP formed in the bundle sheath might perature, so the net energy expended per
diffuse back to the mesophyll, bypassing molecule of CO2 assimilated declines, and as
the need for PPDK (105). a result CO2,max rises. C4 photosynthesis
Although species might be described as belong- largely avoids photorespiration, so CO2,max
ing to one of these three groups, combinations is constant with variation in leaf temperatures,

uptake rate is independent of the maximum

3.0
efficiency of light use at saturation, where
Leaf or canopy CO2 uptake (C4/C3 ratio)
there is still a substantial benefit in avoiding

2.5
photorespiration, resulting in a higher Asat (61).
Asat Figure 2 shows the higher theoretical Asat of
C4 compared with C3 leaves at all temperatures,
2.0 along with actual data from two cold-adapted
grasses, one C3 and one C4 . Below 25◦ C,
Ac does the higher light-saturated capacity of C4
1.5 plants, described by Asat , offset their lower
ΦCO2,max
light-limited capacity, described by CO2,max ?
A plant or plant canopy gains carbon by
1.0
a mixture of light-limited and light-saturated
photosynthesis, with the leaves at the upper

0 5 10 15 20 25 30 35 surface light saturated and the lower leaves
Leaf or canopy temperature (°C) light limited in sunlight and all leaves light
Figure 2 limited around dawn and dusk. To address the
Predicted C4 -to-C3 ratios of maximum quantum yield of CO2 uptake question of whether the higher Asat offsets a
(CO2,max ), leaf-saturated CO2 uptake (Asat ), and daily canopy CO2 uptake lower CO2,max , a biophysical model of light
(Ac ) at different temperatures. Predictions were made from established distribution within a canopy of leaves over a
steady-state biochemical models of C3 and C4 leaf photosynthesis and
integrated into a canopy microclimate model to predict Ac . The data points
diurnal cycle was combined with predictions of
(dots) indicate the ratio of measured Asat of the chilling-tolerant C4 grass leaf photosynthesis based on steady-state bio-
Miscanthus × giganteus to that of the arctic-alpine C3 grass Sesleria albicans, as chemical models of C3 and C4 photosynthesis
given in Figure 1. The canopy simulation assumes a leaf area index of 3, a (61) (Figure 2). The simulation shows a clear
spherical distribution of leaf angles, a latitude of 52◦ N (e.g., London, England), theoretical advantage of C4 photosynthesis
a midsummer day (180th day of the year), and an atmospheric transmissivity of
0.85. For all simulations, the C4 model assumes an 11% leakage of CO2 from
at all temperatures in terms of daily canopy
the bundle sheath and a minimum quantum requirement of 14.2 absorbed carbon gain. For simplicity, this simulation also
photons per CO2 molecule assimilated (41). Details of the models are as given assumed a uniform temperature throughout
previously (61). the canopy; in reality, leaf temperature on the
sunlit leaves would be higher, giving a further
within its permissible growth temperature advantage to the C4 canopy.
range. Both this theory and observations show However, if C4 plants have an advantage
that at approximately 25◦ C, the energy saved at all temperatures, then why are they so rare
by avoiding photorespiration approximately in cold climates? Two reasons have recently
equals that expended in the C4 dicarboxylate been proposed. First, C4 plants evolved largely
cycle (24, 61). Below approximately 25◦ C, from tropical and subtropical C3 ancestors and
CO2,max is greater in C3 photosynthesis, thus carry all of the disadvantages that tropi-
and above this temperature it is greater in C4 cal plants face in cooler climates, regardless of
photosynthesis (Figure 2). This observation, photosynthetic type. For example, crops such
combined with the close correlation between as banana and cassava are C3 but cannot be
the 25◦ C isotherm and the geographical distri- cultivated in cool temperate or continental-
bution of C4 species on the Great Plains, led to microthermal climates. Second, RuBisCO ex-
the suggestion that C4 plants are competitively erts a strong metabolic control on C4 photo-
excluded from colder climates because of their synthesis. Transgenic reduction of RuBisCO in
inferior CO2,max at temperatures below 25◦ C the C4 plant Flaveria bidentis suggested that it
(24, 25). However, CO2,max determines the accounts for 70% of the Asat limitation at opti-
rate of CO2 assimilation only under strictly mal temperature (33). Given a Q10 of 2.2, i.e.,
light-limiting conditions. By definition, CO2 an activity loss of approximately 55% for every
706 Long · Spence

10◦ C decrease in leaf temperature, RuBisCO gen use efficiency (NUE) of C4 plants. NUE
is therefore likely to become even more limit- does indeed decline in C4 plants relative to C3
ing at suboptimal temperatures, so preventing plants with decreased growth temperature (60).
expression of the advantages in Asat and Ac , pre- However, even when grown at 52◦ N, the C4
dicted in Figure 2 (61, 100). grasses M. × giganteus and Spartina cynosuroides
This could be overcome if RuBisCO in- show a superior NUE to C3 species (12).
creased during acclimation to chilling or were
already present at a high enough concentration
to avoid such limitation at low temperatures. It WHY DOES MAIZE SUFFER
has been argued, however, that C4 plants may THE CHILLS?
be physically unable to accommodate more Figure 2 shows that C4 plants are theoret-
RuBisCO. Because C4 photosynthesis requires ically superior in carbon assimilation at all
dimorphic photosynthetic cells, of which only temperatures. As shown in Figure 1, maize

half contain RuBisCO, effectively only half the achieves remarkably high rates of leaf CO2 up-
volume is available to accommodate RuBisCO take when growing at optimal temperatures.
relative to C3 leaves (100). In C3 leaves, However, leaves grown at 14◦ C have almost
chloroplasts commonly hug the mesophyll no photosynthetic capacity at any measurement
wall adjacent to the intercellular air space (38). temperature (Figure 1). Similarly, when plants
This is assumed to be because diffusion of are transferred from warm conditions to 14◦ C,
CO2 in liquid is very slow, and concentration mature leaves lose much of their photosyn-
diminishes greatly with distance from the air thetic capacity (123)—in contrast, for example,
space (27), which appears to limit the number to wheat (49). Given the considerable interest
and therefore volume of chloroplasts in C3 in extending the growing season of maize and
leaves. In the C4 bundle sheath, elevation extending its planting into colder climates, the
of CO2 concentration avoids this diffusional basis of its intolerance to cold temperatures has
restriction, and so more plastids could be received much attention (36, 74). Although low
included, in contrast to the C3 situation. This spring temperatures may limit germination and
is consistent with the observation that plastid root growth into the soil (48), through the sep-
distributions within C4 bundle sheaths can be arate chilling of roots and shoots in controlled
both centrifugal (next to the mesophyll) and environments, the most critical factor was
centripetal (next to the bundle sheath). They found to be shoot temperature during the light
may also be seen to visibly occupy significantly period (74). This conclusion was subsequently
more of the cross-sectional leaf area than their supported by field experiments showing that
mesophyll counterparts in the cool-tolerant C4 shading during periods of chilling protects pho-
grass Spartina anglica (68). As discussed below, tosynthetic capacity and production (5).
improved immunological methods for detect- Mature maize leaves exposed to temper-
ing changes in RuBisCO have now shown atures of 17◦ C and below in the light lose
significant increases in this protein during photosynthetic capacity through slow or irre-
acclimation to chilling in two cool-tolerant C4 versible photoinhibition of photosynthesis (65,
grasses (107). Further, one C4 species in partic- 66). The primary cause of this damage appears
ular, M. × giganteus, has provided proof that to be decreased capacity for photosynthetic car-
the high productivity efficiencies associated bon assimilation at low temperatures, which in
with C4 photosynthesis can be achieved in a turn decreases the rate of photochemical
cool temperate climate and that the photosyn- utilization of absorbed light energy, increasing
thetic apparatus can be acclimated to function excitation pressure within the thylakoid mem-
efficiently at chilling temperatures. Increased brane and the probability of photoinhibition
investment in photosynthetic enzymes would (66). This is consistent with the fact that the
be expected to offset the normally higher nitro- less vigorous and photosynthetically less active

dissipation of excess absorbed light energy,

100
chilling-dependent photoinhibition, impaired
Miscanthus × giganteus synthesis of key proteins of photosystem II
Initial Asat at time 0 (%)
and the light-harvesting complex (LHC), and

80 damage to key proteins, notably the D1 protein
of photosystem II (66, 81, 109).
A critical process for the maintenance of
photosynthesis during chilling is the ability of
60 the chloroplast to degrade damaged D1, a par-
ticularly labile component of photosystem II,
and then synthesize and assemble D1 back into
Zea mays photosystem II (31, 98). This repair function
40
is inhibited in maize during chilling, which
has been attributed to decreased expression

and translation of psbA, the plastid gene that
200
encodes D1 (2, 37). Simultaneously, Asat is
Initial mRNA PPDK at time 0 (%)
depressed, corresponding to 50% and 30%

Miscanthus × giganteus
decreases in PPDK and RuBisCO, respectively,
150 which occur within a few days of the transfer
of plants from optimal temperature to 14◦ C
chilling conditions (122) (Figure 3).
Figure 4 shows that leaves of maize grown
100 at 14◦ C day/10◦ C night have approximately
80% lower Asat relative to those grown at 25◦ C
Zea mays day/20◦ C night, which corresponds to a simi-
lar decline in capacity for regeneration of PEP
50
(Vpr ). This, in turn, corresponds to a similar-
0 2 4 6 8 magnitude loss of PPDK protein and a smaller
Days after transfer to 14°C
loss of RuBisCO, whereas PEPc is only slightly
reduced. Although stomatal conductance is also
Figure 3
greatly decreased, it fails to keep pace with the
Changes in light-saturated leaf CO2 uptake (Asat , measured at 14◦ C) and the
loss of photosynthetic capacity, as shown by a
quantity of mRNA transcripts for PPDK during acclimation of leaves to
chilling growth conditions (14◦ C day/10◦ C night) following transfer of plants significant increase in intercellular CO2 con-
from warm growth conditions (25◦ C day/20◦ C night). Data points are means centration (Ci ). As a result, the intrinsic water
of three plants of maize ( filled blue circles) and of M. × giganteus (open red use efficiency declines.
circles), and bars represent ± 1 SE from the data of Reference 123. What remains unclear is whether the inabil-
ity to form a fully functional photosynthetic
inbred parent lines are more vulnerable to apparatus during chilling, in terms of both
chilling-induced photoinhibition than their key thylakoid and stromal proteins, is a direct
hybrids (45). Although young leaf tissue is effect of temperature or an indirect effect of
less vulnerable to this damage (64), leaves photoinhibition. If the former, then multiple
that develop at suboptimal temperatures show genetic changes would be necessary to over-
impaired synthesis of the photosynthetic appa- come this barrier in maize; if the latter, then
ratus and a depressed photosynthetic capacity changes might be necessary only in the ability
(81, 82, 109), which correlates with depressed of the photosynthetic apparatus to harmlessly
expression of key genes (107, 117, 125, dissipate excess energy during chilling, which
127, 128). These changes cause a decreased could be as simple as upregulating concen-
CO2,max resulting from a combination of the trations of zeaxanthin to nonphotochemically
708 Long · Spence

dissipate excess energy or upregulating path-

ways involved in dissipating oxidizing radicals Asat Miscanthus × giganteus
Vpr Zea mays
in the plastids. Chilling impairs the capacity of
the photosynthetic apparatus to dissipate excess ΦCO2, max
light energy, leading to increased production of θ

gs
oxidizing radicals. Oxidizing radicals, in partic-
Ci
ular hydrogen peroxide, are now known to be
mRNA PPDK
important in retrograde signaling between the
PPDK
plastid and nucleus (83). For example, overpro- PEPc
duction of hydrogen peroxide in the chloroplast RuBisCO
through diffusion to the nucleus downregulates RuBisCO activation
the Lhc genes, which code for the LHC-binding Zeaxanthin
proteins (83). Indeed, this might explain the ob-

–75 –50 –25 0 25 50 75 100 125 225 250
served decrease in these proteins and their gene

Change with growth at 14°C relative to 25°C (%)
transcripts during chilling of maize in the light
Figure 4
(81, 117). Further evidence of such a link comes
from transgenic upregulation of superoxide dis- Changes in measures of photosynthesis and components of the photosynthetic
apparatus observed in plants grown and measured under chilling conditions
mutase within maize chloroplasts, which would (14◦ C day/10◦ C night) relative to plants grown and measured at 25◦ C
accelerate the removal of oxidizing radicals. day/20◦ C night. Absent bars indicate that no statistically significant differences
This upregulation increases protection against between the two temperature regimes were observed. Measures and sources:
chilling-induced reduction in photosynthetic light-saturated leaf CO2 uptake (Asat ) (78); maximum rate of PEP regeneration
capacity relative to untransformed lines (119). calculated from Asat responses to intercellular CO2 concentration (Vpr ) (78);
maximum quantum yield of CO2 uptake (CO2,max ) (78); rate of transition
Chilling-dependent photoinhibition and between light-limited and light-saturated CO2 uptake of the nonrectangular
impaired development of the photosynthetic hyperbola fitted to the measured response of photosynthesis to light (θ) (78);
apparatus are not inherent or unique to C4 pho- stomatal conductance ( gs ) (78); intercellular CO2 concentration (Ci ) (78);
tosynthesis. The C4 grasses Miscanthus spp., S. number of mRNA transcripts determined by real-time polymerase chain
anglica, and Echinochloa crus-galli, for example, reaction for PPDK (123); amounts of PPDK, PEPc, and RuBisCO
large-subunit protein per unit leaf area determined by western blotting (107,
have evolved forms that can acclimate to chill- 123); RuBisCO activation level determined in vitro (122); and zeaxanthin
ing at 14◦ C, and in the case of M. × giganteus, content per unit leaf area (28, 39).
symptoms similar to those observed in maize at
14◦ C are not apparent until temperatures are because they have other adaptive features
<10◦ C (10, 28, 67, 93). The C3 crops Phaseolus (notably salinity tolerance) and avoid interspe-
vulgaris (common bean) and tomato, however, cific competition. Further, leaves of isolated
appear equally vulnerable to photoinhibition at individuals would be exposed to more direct
chilling temperatures as maize, with which they sunlight than those in a community of plants,
share the same center of domestication (37, 94). giving them a greater photosynthetic advantage
than is illustrated in Figure 2. However, C4
plants at high latitudes and altitudes are not
WHAT MIGHT WE INFER FROM limited to open or saline habitats. Although
THE PHYSIOLOGICAL ECOLOGY once thought to be absent from cool temper-
OF C4 SPECIES IN THE COLD? ate and continental-microthermal climates,
Among the first C4 species discovered in cool many C4 species have now been shown to
temperate climates were S. anglica, which be present in such regions (61, 100). The
extends in distribution to 58◦ N in western C4 sedge Cyperus longus is found in wetland
Europe, colonizing bare mudflats, and Salsola communities in southern England, and the C4
kali, which occurs at 61◦ N, colonizing bare grass Muhlenbergia glomerata is found in fen
sand on coastal fore-dunes (68, 69). It could communities in Canada (52, 56). Miscanthus
be argued that these species are present only sinensis and Miscanthus sacchariflorus are

dominant components of natural grasslands ing section explores the productivity of these
throughout Japan and eastern China, extending species and how they acclimate to chilling when
into Siberia (108, 110). Similarly, S. pectinata maize does not.
occurs in prairie and mixed grasslands to 61◦ N
in Canada, often dominating wetter microsites
(76). MISCANTHUS AND SPARTINA
These C4 plants in cold climates may be Although they include tropical species, these
divided into two groups: those that avoid two genera of rhizomatous perennial C4 grasses
chilling conditions and those that can accli- also include species and ecotypes that not only
mate to chilling conditions. Avoidance may extend into cold climates but have proved
be as seen in warm-season grasses, where highly productive in cool temperate climates.
growth is limited to the summer months, when
chilling temperatures are unlikely, as in maize

Miscanthus × giganteus: Field Trials
cultivation and as for many C4 grasses of the

US prairie. Others may avoid chilling by taking M. × giganteus Greef & Deu. ex. Hodkinson
advantage of warm microsites. Muhlenbergia & Renvoize is a sterile triploid hybrid of the
glomerata has one of the most northerly distri- diploid M. sinensis and the tetraploid M. saccha-
butions of all known C4 species. A study of the riflorus (44). A single clone of this plant col-
sheltered openings in the partially wooded fen lected from Japan is probably the most studied
microsites in which it occurs in Ontario showed chilling-tolerant C4 plant (43, 44, 51, 53). Al-
that although in July night temperatures may though originally selected for landscaping use,
drop to freezing, daytime leaf temperatures it has now become widely adopted as a highly
could be 25◦ C (56). Similarly, C4 Muhlenbergia productive biomass crop for energy produc-
richardsonis at an elevation of approximately tion. In replicated trials in southern England at
3,800 m in California’s White Mountains 52◦ N, it achieved a peak dry matter of 30 t ha−1
forms prostrate mats 2.5 cm in height. Surface and a harvested yield of 20 t ha−1 , the highest
frictional resistance produces a boundary layer recorded annual dry-matter yield of any crop
in which the temperature can rise significantly in the United Kingdom (11). In contrast to
above the bulk air temperature, which is typi- maize grown nearby, M. × giganteus showed
cally measured at 1 m above the ground surface. no loss of CO2,max during the chilling con-
As a result, when the air temperature is 10◦ C, ditions of spring, and its recorded efficiency in
in full midday sun the leaf temperature can conversion of intercepted visible radiation into
rise to 25–35◦ C and favor C4 photosynthesis dry-matter energy was 0.078 (10–12, 109, 129,
(102). 130). This efficiency equals that of the C4 plant
However, M. sinensis, M. sacchariflorus, and E. polystachya in the Amazon and is approxi-
their hybrid M. × giganteus grow to 1–4 m mately two-thirds of the maximum theoreti-
in height. Therefore, the most photosynthet- cal efficiency for C4 photosynthesis (88, 129,
ically active parts of their canopies are closely 130). The similarity suggests that through the
coupled to air temperature and unable to avoid growing season in the cool temperate climate of
chilling temperatures. Yet these are among the southeast England, M. × giganteus is realizing
most productive C4 species in cool temperate the same high efficiency of conversion of inter-
and continental-microthermal climates. This cepted solar radiation as the most productive C4
appears possible only through their capabil- plants in the tropics. M. × giganteus has proved
ity to acclimate their photosynthetic appara- just as successful in North America, averaging
tus to chilling conditions (10, 11, 79, 107, 117). shoot dry-matter yields of up to 30 t ha−1 in
Spartina is a second genus that also includes tall the US Midwest and accumulating 35 t ha−1 of
C4 species, which can acclimate photosynthe- root and rhizome dry matter within three years
sis to chilling conditions (11, 107). The follow- of planting (19, 42).
710 Long · Spence

In a side-by-side comparison with a modern perature there is almost no difference in

maize commercial hybrid in the US Midwest, Asat for M. × giganteus leaves, regardless of
M. × giganteus produced 59% more biomass. whether a plant was grown at 14◦ C or 25◦ C
Both crops showed very similar high efficiencies (79) (Figure 1). The only exception is that
in conversion of intercepted solar radiation into the plants grown at 14◦ C show approximately
dry matter. The increase in yield was attributed twice the Asat of the plants grown at 25◦ C when
to the ability of M. × giganteus to form a closed measured at leaf temperatures of 5◦ C and 10◦ C
canopy of leaves six weeks earlier than maize (79) (Figure 1). Figure 4 compiles results
and to maintain it for four weeks longer in the that may explain this marked difference in
autumn. Most striking was that by mid-May, the effect of chilling temperatures on the two
when maize was just emerging, M. × giganteus species. By contrast to maize, M. × giganteus
had a leaf area index of approximately 3 and maintains Asat capacity for PEP regeneration
was intercepting 80% of the available sunlight (Vpr ) and CO2,max . In the absence of any gain
(21). These leaves had high rates of leaf or loss of enzyme protein and activation state,
photosynthesis throughout, with the exception with a Q10 of 2.2 for rate-limiting enzymes
of the very end of the growing season in late of carbon metabolism, Asat and Vpr should
October (20). The ability of M. × giganteus decline by 59% on reduction of temperature
to form photosynthetically competent leaves from 25◦ C to 14◦ C. This is a simple result of
earlier and maintain them later is therefore key the direct effect of the lowered measurement
to the 59% higher dry-matter productivity of temperature on enzyme kinetics. Whereas
this crop compared with maize (21). maize shows an even larger decline, consis-
tent with the decline in the amount of key
Miscanthus × giganteus: Physiological enzymes, upon acclimation, M. × giganteus
and Molecular Analysis of Acclimation shows a decline much lower than would
to Chilling be predicted simply from Q10 (Figure 4).
Why is M. × giganteus able to acclimate This could be explained by increases in the
to chilling conditions when maize is not? rate-limiting enzymes, RuBisCO and PPDK.
Figure 3 shows the result of growing leaves of The most striking difference is the amount
both species under warm conditions and then of these two enzymes considered to exert
transferring the plants to chilling conditions. metabolic control on Asat , PPDK and Ru-
The light-saturated photosynthetic capacity BisCO, which increase by approximately 100%
of plants grown at 14◦ C (measured as Asat at and 50%, respectively (Figure 4). Earlier
a leaf temperature of 14◦ C) declines in both studies failed to identify a significant increase
crops for the first two days. In M. × giganteus, in RuBisCO during chilling (79). Improved
Asat recovers over the subsequent seven days to techniques for immunological quantification
90% of the prechill level, whereas it continues of this abundant protein have now revealed a
to decline in maize, eventually losing >60% large, statistically significant increase (107).
of its prechill capacity (Figure 3). The most Theoretical analysis suggests that Asat
striking changes over this nine-day period in C4 photosynthesis could be limited by
are a 40% loss of RuBisCO and 75% loss of PEPc, PPDK, or RuBisCO activity or some
PPDK protein contents in maize (123). By combination of these (121). A decrease in the
contrast, M. × giganteus doubles its PPDK amount or activity of all three has been found
content and the number of transcripts coding in C4 species in response to chilling (79, 100,
for this protein (123) (Figure 3). When maize 122). PEPc would exert control only if Ci were
is grown throughout its life at 14◦ C, Asat is less insufficient to saturate photosynthesis (121),
than 10% of the rate for plants grown at 25◦ C which is generally not the case in either species
at all measurement temperatures (Figure 1). (21, 78) and is even less likely under chilling,
By contrast, at any given measurement tem- where Ci either remains constant or is increased

(Figure 4). Transgenic reduction in enzymes case of M. × giganteus, the increase in PPDK
of carbon metabolism has suggested that con- is striking and proportional to the doubling
trol is shared between PPDK and RuBisCO of photosynthetic capacity at ≤5◦ C observed
(33). However, significant research has pointed in the leaves developed under daytime chill-
to RuBisCO having control during chilling. ing conditions (Figure 1). Transgenic intro-
Transgenic reductions in RuBisCO activity in duction of a cold-tolerant PPDK into maize was
F. bidentis correlate closely with the reductions found to allow a significant 23% increase in Asat
in light-saturated photosynthesis at low tem- at chilling temperatures, providing further evi-
peratures (57). In two C4 species occurring in dence of a limiting role for this enzyme (84, 85).
cold climates, Bouteloua gracilis and Muhlenber- Further evidence of a common pattern comes
gia montanum, Asat at and below 20◦ C were very from the C4 plant sugarcane, in which chilling
similar to the maximum extractable activities of conditions resulted in a significant 60% loss of
RuBisCO (90, 91, 101). In the case of PPDK, PPDK activity, whereas activity was maintained
the tetrameric C4 isoform of the enzyme is cold or increased in two more cold-tolerant Saccha-
labile in vitro, with the subunits dissociating rum species (23). RuBisCO activity was rela-
below approximately 10◦ C (15), and activities tively little affected, and its activity remained
have been found minimally sufficient to sup- higher than PPDK and observed photosyn-
port in vivo rates of photosynthesis (reviewed thetic rates.
in 100). Within the genus Flaveria, different Both M. × giganteus and maize show
C4 species show a range of cold sensitivities an almost 2.5-fold increase in the leaf con-
in the PPDK that they express (15). F. brownii tent of zeaxanthin when grown at 14◦ C
has a more cold-stable form of PPDK, and this (Figure 4). Zeaxanthin formation is key to
has been associated with specific amino acid nonphotochemical quenching of light ab-
substitutions conferring this cold tolerance. By sorbed by the photosynthetic membrane. It
contrast, sequence analysis revealed few differ- accumulates by de-epoxidation of violaxanthin
ences in either PPDK or RuBisCO between in response to excess absorbed light energy
M. × giganteus and maize (79, 122). M. × relative to photosynthetic capacity. It serves to
giganteus and maize PPDK recombinantly dissipate the excess energy as heat and so mini-
expressed in E. coli showed no detectable mizes the potential for production of oxidizing
difference in the decline in in vitro activity radicals on the photosynthetic membrane. On
with decrease in temperature. However, the return to low light, zeaxanthin is epoxidated
concentration of PPDK in vitro at the levels to violaxanthin, removing this route for dis-
indicated for cold-acclimated M. × giganteus sipating absorbed light energy and allowing
did provide some protection against activity the reaction centers to operate at maximum
loss at low temperature (122). This suggests efficiency under light-limiting conditions (66).
two different evolutionary strategies to over- However, maize under chilling conditions re-
coming PPDK limitation: increased content tains zeaxanthin even in the dark, which appears
of the protein in M. × giganteus and increased to be a further factor causing its low CO2,max
cold stability of the protein in F. brownii. under chilling conditions (31) (Figure 4). In
It cannot be determined from the studies M. × giganteus, by contrast, there is no decline
summarized in Figure 4 whether increased in CO2,max (10), indicating that the large
PPDK, increased RuBisCO, or both are key to increase in zeaxanthin serves to dissipate excess
photosynthetic tolerance to low temperature in energy under light-saturating conditions but
M. × giganteus, because both increase with ac- does not impair light-limited photosynthesis.
climation to chilling temperatures. Previously,
it has been strongly argued from comparative Spartina
data with other C4 species that RuBisCO must Spartina, a genus of C4 grasses, is in many
be the limitation (57, 100). However, in the respects a New World parallel to Miscanthus,
712 Long · Spence

extending in distribution from the tropics to though (as in M. × giganteus) to a lesser extent
61◦ N. Particular attention has been given to (a) (107). This common large increase in PPDK in
the highly productive coastal salt-marsh grasses chilling-tolerant C4 species from very separate
S. anglica and S. alterniflora, which extend in evolutionary lines provides further evidence for
distribution northward along the Atlantic the apparent importance of this protein in chill-
coasts to southern Norway and southern ing tolerance of C4 photosynthesis. The differ-
Canada, respectively (69), and (b) the prairie ence in S. pectinata and S. anglica in apparent
and brackish water-marsh grasses S. cynosuroides PPDK content could reflect either the fact that
and S. pectinata, which are considered potential these species are within different subgenera of
bioenergy grasses (14, 92, 120). S. pectinata Spartina (76) or the difficulty of extracting an
extends in distribution as far north as 59◦ N in active PPDK from these grasses (105).
northern Alberta (76). Although somewhat less
productive than M. × giganteus, in side-by-side PROSPECTS FOR ENGINEERING

trials in eastern England at 52◦ N, S. cynosuroides COLD TOLERANCE INTO

and S. pectinata achieved dry-matter yields of C4 CROPS
up to 18 t ha−1 , among the highest of any crop
at this latitude (92). S. anglica growing on the
With C3 Crops Already Available,
Why Attempt to Adapt C4 Crops to
northeast coast of England maintained rela-
the Cold?
tively high photosynthetic rates into November
(67), and metabolite fluxes remained consistent As noted above, maize in the US Midwest
with C4 photosynthesis at low temperatures achieves almost the highest yields of any grain
(114). or seed crop worldwide, yet it is unable to
Whereas Miscanthus and maize are C4 use much of the available solar radiation.
NADP-ME types, Spartina species are C4 PEP- Because of its inability to form and maintain
CK types (73, 105). Despite the ability of S. an- photosynthetically competent leaves at ≤14◦ C,
glica to maintain photosynthetic rates equal to it is planted relatively late and completes
those of the arctic-alpine C3 grass Sesleria albi- its growth cycle early, with the result that
cans at leaf temperatures of ≤10◦ C (Figure 1), much of the available solar radiation is unused
PPDK was almost undetectable in in vitro leaf relative to native C3 plants and trees. As
extracts (105). This led to the suggestion that S. discussed above, if maize had an ability to form
anglica may avoid the problem of PPDK limita- and maintain leaves at chilling temperatures
tion at chilling temperatures by using the PEP comparable to that of M. × giganteus, then
synthesized by PEP-CK in the bundle sheath a 59% yield increase could be achieved in the
for carboxylation in the mesophyll (105). A sub- major grain-producing areas of the globe.
sequent study similarly detected only trace ac- In the past three decades, maize has emerged
tivities of PPDK in S. anglica grown at either as a major silage crop north of regions where it
27◦ C or 10◦ C. In contrast to the cold-intolerant can be viably grown for grain (6). For example,
PEP-CK species Zoysia japonica, S. anglica main- in England and the Netherlands, where it has
tains its RuBisCO activity with growth at 10◦ C been widely adopted, it fails to cover the ground
at a level more than sufficient to support the until well into July, thereby missing the annual
observed relatively high rate of photosynthe- radiation peak (29, 109). However, the effi-
sis (73). However, a recent immunological- ciency of C4 photosynthesis, even in this short
detection study of S. pectinata photosynthetic growing season, makes silage maize competitive
acclimation to growth at 14◦ C relative to with C3 alternatives, yielding approximately
growth at 25◦ C showed a PPDK level similar 10 t ha−1 dry matter and up to 16 t ha−1 at
to that of M. × giganteus (105). This study also the best sites and in the best years (18, 115).
showed a large increase in response to the chill- Again, by comparison with the yields of M. ×
ing treatment. RuBisCO was also increased, al- giganteus grown in the same regions, a similarly

cold-tolerant maize could yield 60–100% more How Might We Make C4 Crops
than current silage maize (11, 12). Because the Chilling Tolerant?
cattle and dairy operations served by maize M. × giganteus and its close relatives M. sinen-
silage will use grain once forage sources are sis, M. sacchariflorus, and M. lutarioriparius have
exhausted, this would also relieve pressure on already emerged as significant chilling-tolerant
global grain markets. C4 crops providing renewable feedstocks for
Sugarcane has emerged as arguably the bioproducts, for bioenergy, and potentially
world’s best biofuel crop. In 2009, Brazil for cellulosic biofuels (43, 53). These species
consumed 14.1 Mt of fossil fuel oil for trans- are capable of high productivities across large
portation, compared with 19.0 Mt of sugarcane areas with cool temperate or continental-
ethanol (50). This has not only almost halved microthermal climates (40, 59, 75). In addition
net greenhouse gas emissions from Brazilian to their chilling tolerance, their high yields and
automobiles but has also enabled the produc- low water and nitrogen requirements (in part
tion of green electricity (106). The fiber or attributable to C4 photosynthesis) minimize

bagasse that remains after crushing the cane both environmental impacts and land require-
is combusted to power the ethanol production ments. Moreover, these perennial grasses
facility, with the excess power added to the increase soil carbon and provide renewable
national grid. It is estimated that Brazil could biomass that, when converted into bioenergy
generate 880 Gl of ethanol, or approximately that replaces fossil fuels, makes the crop system
14% of the current world transportation fuel potentially greenhouse-gas negative (17, 19, 43,
demand, using land resources that are far 131). A single clone of M. × giganteus has been
south of the Amazon and that would have little the subject of most yield trials, physiological
impact on food crop production (106). Sugar- and molecular analyses of cold tolerance, and
cane is attractive because of its ability to grow commercial plantings (35, 108). Its sterility as a
on relatively poor soils and its low fertilizer triploid allopolyploid and interspecific hybrid is
requirements compared with those needed for valued in avoiding the risk of invasiveness that
grain crops while providing very high yields. could be attached to fertile relatives. However,
However, its production is limited to the its ability to form and maintain photosyn-
tropics and wet subtropics, and it is unable to thetically competent leaves earlier than maize
form and maintain functional leaves at ≤18◦ C and other C4 crops leads to a longer growing
(113). In the United States, for example, its season, which allows it to produce high biomass
cultivation is limited to Hawaii, the southern- yields (21). This clone of M. × giganteus is
most areas of Florida and Louisiana, and the considered to have arisen in the southern part
southernmost wet coastal areas of Texas (118). of Honshu, Japan (108). The distribution of the
A small improvement in its cold tolerance parent species extends much further north, to
could allow production on a substantial portion areas including Siberia and high-altitude sites
of the southeast United States, where poor and of Hokkaido, the northernmost large island of
sometimes erodible soils have proved unsuited Japan. This suggests the presence of consider-
to competitive production of annual grain crops ably greater cold tolerance within the potential
and where more than 25% of cropland has been germplasm pool (35, 108). Completion of the
abandoned since 1940 (16, 46). Sorghum is gene space sequence and structural genomics
another C4 crop of interest owing to its greater for one parent, together with provision of
drought tolerance compared with maize, which high-density molecular markers, now provides
makes it attractive for more drought-prone a means to potentially accelerate transfer of
regions (97). However, it is also limited by this increased cold tolerance (35, 72, 111,
chilling conditions, being even less tolerant of 112). Beyond Miscanthus, what prospects
chilling conditions than maize (26, 58). are there for improved cold tolerance of
714 Long · Spence

C4 photosynthesis in our current major C4 of C4 photosynthesis, in common with the re-

crops? cruitment of other C4 genes, has involved the
The comparison of maize and M. × incorporation of new cis-acting elements, in-
giganteus provides some of the best evidence cluding enhancer elements, into the promoter
of how to achieve successful acclimation sequence, which then interact with expres-
allowing efficient C4 photosynthesis in chilling sion modulators. This, for example, is key to
conditions. As outlined above, increased the tissue- and light-dependent expression and
contents of PPDK and to a lesser extent greatly increased level of the plastidic form of
RuBisCO appear to be a common feature PPDK in C4 species (reviewed in 104, 124). It is
of the few C4 grasses so far shown to ac- tempting to speculate that in Miscanthus, signals
climate to chilling temperatures, whereas that might cause decreased expression through
both enzymes decline in chilling-sensitive these cis-acting elements in maize at low tem-
maize. There are clear functional reasons to perature have a reversed response, or that the
expect both to play a key part in allowing C4 modulator is increased rather than decreased.
photosynthesis at chilling temperatures. Both RNA-seq now provides a relatively cheap
enzymes have associated regulatory proteins, method that could be applied to understand
and although less is known about these, it the expression network that underlies the differ-
must be assumed that both are maintained ence in acclimation to chilling between Miscant-
at adequate levels under chilling conditions hus and maize. This could lead back to a small
in M. × giganteus, given its high photosyn- number of changes, at the level of transcrip-
thetic rates. Activation of RuBisCO was only tion control, needed to achieve chilling toler-
slightly decreased in leaves of M. × giganteus ance. These changes may possibly parallel or
developed under chilling conditions (122). In be part of the network that induces the changes
M. × giganteus, the increase in PPDK is closely involved in general plant acclimation to chilling
correlated with an upregulation of transcripts conditions established for the chilling-tolerant
for the single gene that codes for this protein, C3 plant Arabidopsis thaliana (98). Alternatively,
whereas downregulation occurs in maize (123). chilling tolerance may be achieved posttransla-
One strategy to confer cold tolerance to tionally by ubiquitination of PPDK and other
maize might therefore be to transgenically key proteins, slowing protein turnover and al-
upregulate the expression of PPDK. An lowing accumulation of these proteins (124).
alternative strategy would be to evolve a more Miscanthus spp. are assumed to have arisen in
cold-tolerant form of the enzyme that could the tropics and have evolved increasingly cold-
maintain a higher kcat at chilling temperatures. tolerant forms in a relatively short evolution-
Such a change appears to have occurred in ary time frame, allowing the species to colonize
the chilling-tolerant C4 dicot F. brownii but grasslands as far north as Siberia. This suggests
not in M. × giganteus (15, 79, 86, 123). Engi- that the chilling-tolerance syndrome may re-
neering the key amino acid changes conferring quire relatively few genetic changes.
improved PPDK cold tolerance into maize Achieving the above in maize and other ma-
did result in a significant improvement of jor C4 crops is likely to require transgenic modi-
leaf photosynthesis at low temperatures (84, fication, site-directed mutagenesis, or both (84,
85). However, the full advantage of increased 85). Are there also prospects for achieving this
PPDK levels could not be realized because the through “conventional” breeding?
PPDK regulatory protein was not elevated (85).
Another approach would be to discover and
manipulate the regulatory elements that result Interspecific Breeding for
in increases in PPDK, RuBisCO, and associ- Chilling Tolerance
ated proteins in Miscanthus. The recruitment of Although variability in chilling tolerance
the C3 PPDK-encoding gene in the evolution has been identified in maize, sorghum, and

sugarcane germplasm and that of their im- same tribe, maize is significantly more distant
mediate relatives, it is small compared with from Miscanthus in terms of genome similarity
the differences between M. × giganteus (87). However, wide crosses of maize to oat,
and these crops (1, 23, 79, 89). Miscanthus, which is genetically far more distant, have
Saccharum, Sorghum, and Zea are all gen- allowed introgression of maize genes into oat,
era of Andropogoneae and thus are closely suggesting that wide crosses with Miscanthus
related. A recent analysis showed that the would not be impossible (8, 116). Alternatively,
basic Miscanthus genome (n = 19) is in fact maize has been successfully and beneficially
a doubled sorghum genome (n = 10) in transformed with genes from a wide range
which a duplication event was followed by of sources, providing an alternative route for
rearrangement of the genome onto one fewer introducing cold tolerance from Miscanthus.
chromosomes (112). The sugarcane genome, in M. × giganteus is proof that the benefits
contrast, is an eightfold multiplication relative of highly efficient light, nitrogen, and water
to the sorghum genome (n = 80) (72, 126). use associated with C4 photosynthesis can be
Despite differences in ploidy, fertile crosses realized in cool temperate and continental-
between M. sinensis, one of the parents of microthermal climates. Importantly, it shows
M. × giganteus, and sugarcane have been that there is no fundamental barrier to extend-
obtained several times in breeding for cane ing the beneficial aspects of C4 photosynthesis
disease resistance (3). Although these crosses into colder climates. Breeding or engineering
have been with tropical ecotypes of M. sinensis, similar changes into maize could have profound
it seems equally feasible that the cold tolerance effects on global food and feed supply at a time
of northern populations of Miscanthus could when this may be much needed. Achieving this
be similarly introgressed into sugarcane. will require a far deeper understanding of the
Although far less routine, a genetic muta- genetic basis of the changes that have occurred
tion has been identified that overcomes the in Miscanthus to allow the acclimation of the
interspecific infertility barrier in sorghum, C4 apparatus to chilling conditions. This goal
potentially allowing crosses with Miscanthus (7) has been made far more feasible by recent and
and similarly suggesting a means to introgress rapid advances in the gene space sequencing and
chilling tolerance. Although they are within the structural genomics of Miscanthus (111, 112).
DISCLOSURE STATEMENT
S.P.L. is supported by funding from the Energy Biosciences Institute (which is supported by the
BP Group) and from the Bill & Melinda Gates Foundation. A.K.S. was supported in her graduate
studies by the Energy Biosciences Institute and is currently an employee of Procter & Gamble.
ACKNOWLEDGMENTS
We thank Rachel Shekar and an anonymous reviewer for their constructive comments on the
manuscript, and the Energy Biosciences Institute for support to both authors.
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PP64-frontmatter ARI 25 March 2013 10:21
Annual Review of
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Contents Volume 64, 2013
Benefits of an Inclusive US Education System

Elisabeth Gantt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Plants, Diet, and Health

Cathie Martin, Yang Zhang, Chiara Tonelli, and Katia Petroni p p p p p p p p p p p p p p p p p p p p p p p p p19
A Bountiful Harvest: Genomic Insights into Crop Domestication
Phenotypes
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Progress Toward Understanding Heterosis in Crop Plants
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Tapping the Promise of Genomics in Species with Complex,
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Understanding Reproductive Isolation Based on the Rice Model
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Classification and Comparison of Small RNAs from Plants
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Plant Protein Interactomes
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Seed-Development Programs: A Systems Biology–Based Comparison
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Fruit Development and Ripening
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Growth Mechanisms in Tip-Growing Plant Cells
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Future Scenarios for Plant Phenotyping
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v
Microgenomics: Genome-Scale, Cell-Specific Monitoring of Multiple

Gene Regulation Tiers
J. Bailey-Serres p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 293
Plant Genome Engineering with Sequence-Specific Nucleases
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Smaller, Faster, Brighter: Advances in Optical Imaging
of Living Plant Cells
Sidney L. Shaw and David W. Ehrhardt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 351
Phytochrome Cytoplasmic Signaling
Jon Hughes p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 377
Photoreceptor Signaling Networks in Plant Responses to Shade

Jorge J. Casal p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 403

ROS-Mediated Lipid Peroxidation and RES-Activated Signaling
Edward E. Farmer and Martin J. Mueller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429
Potassium Transport and Signaling in Higher Plants
Yi Wang and Wei-Hua Wu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 451
Endoplasmic Reticulum Stress Responses in Plants
Stephen H. Howell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 477
Membrane Microdomains, Rafts, and Detergent-Resistant Membranes
in Plants and Fungi
Jan Malinsky, Miroslava Opekarová, Guido Grossmann, and Widmar Tanner p p p p p p p 501
The Endodermis
Niko Geldner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 531
Intracellular Signaling from Plastid to Nucleus
Wei Chi, Xuwu Sun, and Lixin Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 559
The Number, Speed, and Impact of Plastid Endosymbioses in
Eukaryotic Evolution
Patrick J. Keeling p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 583
Photosystem II Assembly: From Cyanobacteria to Plants
Jörg Nickelsen and Birgit Rengstl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 609
Unraveling the Heater: New Insights into the Structure of the
Alternative Oxidase
Anthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita,
and Kikukatsu Ito p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 637
Network Analysis of the MVA and MEP Pathways for Isoprenoid
Synthesis
Eva Vranová, Diana Coman, and Wilhelm Gruissem p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 665
vi Contents
Toward Cool C4 Crops

Stephen P. Long and Ashley K. Spence p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 701
The Spatial Organization of Metabolism Within the Plant Cell
Lee J. Sweetlove and Alisdair R. Fernie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 723
Evolving Views of Pectin Biosynthesis
Melani A. Atmodjo, Zhangying Hao, and Debra Mohnen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 747
Transport and Metabolism in Legume-Rhizobia Symbioses
Michael Udvardi and Philip S. Poole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 781
Structure and Functions of the Bacterial Microbiota of Plants
Davide Bulgarelli, Klaus Schlaeppi, Stijn Spaepen, Emiel Ver Loren van Themaat,
and Paul Schulze-Lefert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 807
Systemic Acquired Resistance: Turning Local Infection

into Global Defense
Zheng Qing Fu and Xinnian Dong p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 839
Indexes
Cumulative Index of Contributing Authors, Volumes 55–64 p p p p p p p p p p p p p p p p p p p p p p p p p p p 865

Cumulative Index of Article Titles, Volumes 55–64 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 871
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://www.annualreviews.org/errata/arplant
Contents vii

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PP64CH28-Long ARI 28 March 2013 16:18

• Our comprehensive search

Annu. Rev. Plant Biol. 2013. 64:701–22 Keywords

energy capture by a leaf to the synthesis of

forage grass Pennisetum purpureum growing in

702 Long · Spence

plants, such as palms and tree ferns, yet the 60

Leaf CO2 uptake (µmol m –2 s –1)

winter temperatures of ≤−20◦ C, yet an average

of this review is the impact of and adaptation to

chilling temperatures, i.e., temperatures above

www.annualreviews.org • Toward Cool C4 Crops 703

months (21). In a side-by-side comparison, current atmospheric level is sufﬁcient to largely

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chloroplasts, where it is decarboxylated This common difference from C3 plants

www.annualreviews.org • Toward Cool C4 Crops 705

uptake rate is independent of the maximum

there is still a substantial beneﬁt in avoiding

photosynthesis, with the leaves at the upper

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dimorphic photosynthetic cells, of which only temperatures. As shown in Figure 1, maize

www.annualreviews.org • Toward Cool C4 Crops 707

dissipation of excess absorbed light energy,

and the light-harvesting complex (LHC), and

has been attributed to decreased expression

depressed, corresponding to 50% and 30%

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dissipate excess energy or upregulating path-

light energy, leading to increased production of θ

proteins (83). Indeed, this might explain the ob-

served decrease in these proteins and their gene

www.annualreviews.org • Toward Cool C4 Crops 709

chilling temperatures are unlikely, as in maize

cultivation and as for many C4 grasses of the

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In a side-by-side comparison with a modern perature there is almost no difference in

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productive than M. × giganteus, in side-by-side PROSPECTS FOR ENGINEERING

trials in eastern England at 52◦ N, S. cynosuroides COLD TOLERANCE INTO

www.annualreviews.org • Toward Cool C4 Crops 713

tion of green electricity (106). The ﬁber or attributable to C4 photosynthesis) minimize

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C4 photosynthesis in our current major C4 of C4 photosynthesis, in common with the re-

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Ref. 96, pp. 129–46

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China. Glob. Change Biol. Bioenergy 4:545–54

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Plant Biol. 57:739–59

and heterotetramer formation in transgenic maize plants. Transgenic Res. 13:475–85

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from China. Bot. J. Linn. Soc. 164:178–220

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Contents Volume 64, 2013