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Kranz Anatomy. A Sine Qua Non for C4
Photosynthesis? |
One of the
most exquisite examples of the marriage of form and function in plant
biology, at least so I tell my undergraduate students, is the necessity
of Kranz anatomy for C4 photosynthesis. The leaves of
C4 plants typically are characterized by an orderly arrangement of mesophyll cells around a layer of large bundle sheath
cells, so that the two together form concentric layers around the
vascular bundle. This wreath-like, two-layered arrangement of the
chlorenchyma is termed Kranz anatomy (Kranz is the German word for
`wreath'). The bundle sheath cells of C4 plants generally contain thicker walls, more chloroplasts and other organelles, and
smaller central vacuoles than do mesophyll cells. The function of the
mesophyll cells in C4 plants is to fix CO2 into
oxaloacetate by means of phosphoenolpyruvate (PEP)
carboxylase. In the most common C4 scheme, this
oxaloacetate is quickly converted to malate, which is then rapidly
transferred to the bundle sheath cells, where it is decarboxylated. The
released CO2 is rapidly fixed by Rubisco in the bundle
sheath cells. The strict localization of Rubisco to the bundle sheath
cells, and the release of high concentrations of CO2 in the
vicinity of Rubisco, help to increase its carboxylase activity and to
lower its wasteful oxygenase activity. Thus, the spatial separation of
PEP carboxylase in the mesophyll from Rubisco in the bundle sheath
greatly improves the efficiency of photosynthesis under many
environmental conditions. Because of the close correspondence between
Kranz anatomy and C4 photosynthesis, it has become almost
dogma that Kranz anatomy is a sine qua non for C4
photosynthesis. Here, I summarize the results of a number of new
studies that challenge this idea.
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C4 Photosynthesis in a Diatom |
Previous studies have shown that Rubisco enzymes from diatoms
have half-saturation constants for CO2 of 30 to 60 µM. As a result, diatoms growing in seawater that
contains about 10 µM CO2 may be
CO2 limited. Kinetic and growth studies have shown that
diatoms can avoid CO2 limitation, but the biochemistry of the underlying mechanisms remains unknown. Reinfelder et
al. (2000)
have presented evidence that C4-type PEP
carboxylase is abundant in the marine diatom Thalassiosira
weissflogii. Moreover, pulse chase experiments indicated that
malate accumulates before the accumulation of 3-phosphoglycerate
(Rubisco's product). These experiments support the idea that
C4 photosynthesis is the mode of C assimilation in this
species of marine diatom, thus providing a biochemical explanation for
CO2-insensitive photosynthesis in marine diatoms. The
authors suggest that if C4 photosynthesis is common among
marine diatoms, it may account for a significant portion of C fixation
and export in the ocean, and would explain the greater enrichment of
13C in diatoms compared with other classes of
phytoplankton. It is also possible that unicellular C4-type
C assimilation may have predated the appearance of multicellular
C4 plants.
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Strange Chenopodiaceae from Central Asia |
The dicot family with the largest number of C4
species is the Chenopodiaceae. Certain members of this family from the
deserts of Central Asia are succulent halophytes that exhibit
C4-type CO2 fixation of the NAD- or NADP-malic
enzyme (ME) type. The paradigm of the spatial separation of PEP
carboxylase and Rubisco in different cells has been challenged by
recent findings concerning Borszczowia aralocaspica.
Freitag and Stichler (2000) demonstrated that B. aralocaspica has the photosynthetic features of C4
plants, yet lacks Kranz anatomy. Unlike the two-layered chlorenchyma of
a typical Kranz-type leaf, the leaves of B. aralocaspica
are characterized by a single-layered chlorenchyma. The determination
that this species has a 13C value of 13.1% (more typical
of C4 plants) suggests that this one-layered photosynthetic
tissue combines all the essential anatomical characters of a
two-layered chlorenchyma of regular C4 plants. Voznesenskaya et al. (2001) have demonstrated that the large, individual chlorenchyma cells of B. aralocaspica contain
differentiated chloroplasts. The separation of two types of
chloroplasts within a single cell apparently allows for the spatial
compartmentation of photosynthetic enzymes within the chlorenchyma cell cytoplasm.
Pyankov et al. (1999) have described another interesting genus of
Central Asian Chenopodiaceae. The two photosynthetic organs of
Haloxylon aphyllum and Haloxylon
persicum, the photoassimilating shoots and leaf-like
cotyledons, were studied to characterize their photosynthetic types.
13C/12C isotope ratios, the cellular anatomy of
assimilating organs, primary photosynthetic products, and activities of
C metabolism enzymes (Rubisco, PEP carboxylase, MEs, and Asp
aminotransferase) indicate that different pathways of CO2
fixation occur in the photosynthetic organs. Assimilating shoots have
all of the attributes of C4 photosynthesis, whereas
cotyledons lack Kranz-anatomy and incorporate CO2 via
C4 photosynthesis. Cotyledons and seeds, however, have
lower 
13C values compared with shoots, consistent with
some contribution of C3-like CO2 assimilation.
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Intermediate Stages in the Transition of an Amphibious Sedge
from C3 to C4 Photosynthesis |
The amphibious leafless sedge Eleocharis vivipara
develops C4-like traits as well as Kranz anatomy under
terrestrial conditions, but it develops C3-like traits
without Kranz anatomy under submerged conditions (Uchino et al., 1998).
The photosynthetic organ (the mature internodal region of
the culm) of the terrestrial form shows typical Kranz anatomy
with well-developed bundle sheath cells, whereas the bundle sheath
cells of the submerged form are not developed. In the mature internodal
region of the terrestrial form, expression of the genes encoding two
carboxylases, the small subunit of Rubisco, and PEP carboxylase
occurred mainly in bundle sheath cells and in mesophyll cells,
respectively, as seen in a typical C4 leaf. In the
submerged form, Rubisco was expressed in both bundle sheath cells and
mesophyll cells, and no expression of PEP carboxylase was observed. The
C4-type expression pattern was established concomitantly
with the development of bundle sheath cells during tissue maturation in
the terrestrial internode. In contrast to the terrestrial form, the
submerged form maintains C3-type gene expression during
tissue maturation. When the terrestrial culm was submerged, a region of
transition from the terrestrial form to the submerged form was
established in newly sprouting culms. In this transitional region,
C4-type expression of the two carboxylase genes was still
maintained even though the development of bundle sheath cells was
repressed. This suggests that the C4-type cell-specific
gene expression pattern does not depend on the formation of Kranz anatomy.
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C4 Photosynthesis without Kranz Anatomy in Aquatic
Flowering Plants |
The submersed monocot Hydrilla verticillata
exhibits an inducible C4-type photosynthetic cycle, but
lacks Kranz anatomy (Magnin et al., 1997). The authors tracked the
changes in a great many photosyntheitic parameters during the 12-d
period necessary for the induction of C4 photosynthesis in
this species. The CO2 compensation point and O2
inhibition of photosynthesis declined linearly. PEP carboxylase
activity increased 16-fold, with the major increase occurring within
3 d. Asn and Ala aminotransferases were also induced rapidly.
Pyruvate orthophosphate dikinase and NADP-ME activity gradually
increased 10-fold over 15 d. Total Rubisco activity did not
increase, and its activation declined from 82% to 50%. Western blots
for PEP carboxylase, pyruvate orthophosphate dikinase, and NADP-ME
indicated that increased protein levels were involved in their
induction. The O2 inhibition of photosynthesis was doubled
when C4-type but not C3-type leaves were
exposed to diethyl oxalacetate, a PEP carboxylase inhibitor. These
results are consistent with the existence under certain conditions of a
CO2-concentrating C4 cycle in H.
verticillata chloroplasts and indicate that Kranz anatomy is
not obligatory for C4-type photosynthesis.
Another interesting class of aquatic plants to consider in the
evolution of C4 photosynthesis are members of the
Orcuttieae tribe of C4 grasses (Keeley, 1998). Cladistic
analysis supports the conclusion that the Orcuttieae tribe of
C4 grasses reflect evolution from a terrestrial ancestry
into seasonal pools. Aquatic leaves of the least derived genus
Neostapfia have few morphological and anatomical
characteristics specialized to the aquatic environment and have
retained full expression of the C4 pathway, including Kranz
anatomy. Orcuttia spp. have many derived characteristics and are more specialized to the aquatic environment. Aquatic
leaves of Orcuttia spp. lack Kranz or bundle sheath
anatomy, yet 14C pulse chase studies indicate that malate
and Asp account for more than 95% of the initial products of
photosynthesis and these products turn over rapidly to phosphorylated
sugars, indicating a tight coupling of the C4 and
C3 cycles. Apparently, as the Orcuttieae became adapted to
the aquatic environment, they lost their Kranz anatomy, but maintained
their capacity for C4 photosynthesis.
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