Journal of Experimental Botany, Vol. 50, No. 341, pp. 1779–1795, December 1999
Anatomy, chloroplast structure and compartmentation of
enzymes relative to photosynthetic mechanisms in leaves
and cotyledons of species in the tribe Salsoleae
(Chenopodiaceae)
Elena V. Voznesenskaya1, Vincent R. Franceschi2, Vladimir I. Pyankov3 and
Gerald E. Edwards2,4
1 Department of Anatomy and Morphology, VL Komarov Botanical Institute of Russian Academy of Sciences,
Prof. Popov Street 2, 197376 St Petersburg, Russia
2 Botany Department, Washington State University, Pullman, WA 99164–4238, USA
3 Department of Plant Physiology, Ural State University, Ekaterinburg 620083, Russia
Received 15 March 1999; Accepted 23 July 1999
Abstract
Certain members of the family Chenopodiaceae are
the dominant species of the deserts of Central Asia;
many of them are succulent halophytes which exhibit
C -type CO fixation of the NAD- or NADP-ME (malic
4
2
enzyme) subgroup. In four C species of the tribe
4
Salsoleae, the Salsoloid-type Kranz anatomy in leaves
or stems was studied in relation to the diversity in
anatomy which was found in cotyledons. Halocharis
gossypina, has C NAD-ME Salsoloid-type photosyn4
thesis in leaves and C photosynthesis in dorsoventral
3
non-Kranz cotyledons; Salsola laricina has C NAD-ME
4
Salsoloid-type leaves and C NAD-ME Atriplicoid-type
4
cotyledons; Haloxylon persicum, has C NADP-ME
4
Salsoloid-type green stems and C isopalisade non3
Kranz cotyledons; and S. richteri has C NADP-ME
4
Salsoloid-type leaves and cotyledons. Immunolocalization studies on Rubisco showed strong labelling in bundle sheath cells of leaves and cotyledons of
organs having Kranz anatomy. The C pathway enzyme
4
phosphoenolpyruvate carboxylase was localized in
mesophyll cells, while the malic enzymes were localized in bundle sheath cells of Kranz-type tissue.
Immunolocalization by electron microscopy showed
NAD-ME is in mitochondria while NADP-ME is in
chloroplasts of bundle sheath cells in the respective
C types. In some C organs, it was apparent that
4
4
subepidermal cells and water storage cells also contain some chloroplasts which have Rubisco, store
starch, and thus perform C photosynthesis. In
3
non-Kranz cotyledons of Halocharis gossypina and
Haloxylon persicum, Rubisco was found in chloroplasts
of both palisade and spongy mesophyll cells with the
heaviest labelling in the layers of palisade cells,
whereas C pathway proteins were low or undetect4
able. The pattern of starch accumulation correlated
with the localization of Rubisco, being highest in the
bundle sheath cells and lowest in the mesophyll cells
of organs having Kranz anatomy. In NAD-ME-type
Kranz organs (leaves and cotyledons of S. laricina and
leaves of H. gossypina) the granal index (length of
appressed membranes as a percentage of total length
of all membranes) of bundle sheath chloroplasts is 1.5
to 2.5 times higher than that of mesophyll chloroplasts.
In contrast, in the NADP-ME-type Kranz organs (S.
richteri leaves and cotyledons and H. persicum stems)
the granal index of mesophyll chloroplasts is 1.5 to
2.2 times that of the bundle sheath chloroplasts. The
mechanism of photosynthesis in these species is
discussed in relation to structural differences.
Key words: Immunolocalization, photosynthetic enzymes,
C plants, anatomy, chloroplasts, ultrastructure, Cheno4
podiaceae.
4 To whom correspondence should be addressed. Fax: +001 509 335 3517. E-mail: [email protected]
Abbreviations: NAD-ME, NAD-malic enzyme; NADP-ME, NADP-malic enzyme; PEPC, phosphoenolpyruvate carboxylase; Rubisco, ribulose
1,5-bisphosphate carboxylase oxygenase.
© Oxford University Press 1999
1780 Voznesenskaya et al.
Introduction
Chenopodiaceae is one of the most interesting families
with respect to having species with a large diversity in
the structure of the carbon assimilating organs (Monteil,
1906; Khatib, 1959; Carolin et al., 1975; Voznesenskaya
and Gamaley, 1986) with different types of photosynthesis, namely C , C , and C -C or C -CAM inter3 4
3 4
4
mediates (Hatch et al., 1972; Gutierrez et al., 1974;
Glagoleva et al., 1978; Zalenskii and Glagoleva, 1981;
Pyankov et al., 1997). Among species of the family having
C -type photosynthesis there are differences in biochem4
istry, primary products of photosynthesis and C decarb4
oxylating enzymes, and this occurs even within Salsola,
one genus of the family (Pyankov and Vakhrusheva,
1989; Pyankov et al., 1992a, 1997). These differences in
biochemistry correlate with the variations in ultrastructure of the assimilating tissues ( Voznesenskaya, 1976;
Voznesenskaya and Gamaley, 1986; Glagoleva et al.,
1992). A previous study of the structure of assimilating
organs during the development of the seedlings of some
Chenopodiaceae species showed that there are also differences in the structure of cotyledons. Even if leaves have
the C Salsoloid (according to Carolin et al., 1975)
4
structure, all variants of cotyledon structure are represented in species of the genus Salsola and some nearrelated genera, from the dorsoventral C -type of anatomy,
3
to the C isopalisade and to different types of C anatomy
3
4
(Butnik, 1979; 1984; Butnik et al., 1991; Pyankov et al.,
1999a). The purpose of the present study was to determine
the relationship between the occurrence and localization
of key enzymes of C and C photosynthesis by in situ
4
3
immunolocalization in relation to the anatomy of assimilating organs (both leaves and cotyledons), and the
ultrastructure of chloroplasts in chlorenchyma cells in
selected species of the tribe Salsoleae of the family
Chenopodiaceae.
Materials and methods
Plant material
The plants were grown in a greenhouse from seeds collected in
the deserts of Central Asia, Uzbekistan (Samarkand and
Bukhara region) in the autumn of 1996. Seeds were stored in a
refrigerator (3–5 °C ) for two or more weeks before planting.
After refrigeration seeds were germinated on moist paper under
greenhouse conditions and were then transplanted to soil. Four
species of Chenopodiaceae in the tribe Salsoleae were used
which have C photosynthesis in leaves/stems: Halocharis
4
gossypina Korov. & Kinzikaeva having NAD-ME-type C
4
leaves and C -type cotyledons, Salsola laricina Pall. having
3
NAD-ME-type photosynthesis in leaves and cotyledons, Salsola
richteri (Moq.) Karel. ex Litv. having NADP-ME-type leaves
and cotyledons, and Haloxylon persicum Bunge having NADPME-type stems and C -type cotyledons (Pyankov et al., 1999a;
3
Pyankov et al., unpublished results). Plant names are according
to Czerepanov (Czerepanov, 1995).
Halocharis gossypina is an annual plant, while the three other
species are perennials. Haloxylon persicum is practically aphyllous as it has extremely reduced scale-like leaves (1.5–2 mm
long), with the carbon assimilatory functions being essentially
fulfilled in this species by the photosynthetic primary stem
cortex of young shoots. The other three species have cylindrical
succulent leaves; H. gossypina and S. laricina have small leaves
up to 1.5 cm long and nearly 0.3 mm thick, while S. richteri
has rather long leaves up to 8–9 cm long and 1–2 mm thick.
Mature leaves, or young stems (H. persicum), were sampled
from 1–4-month-old plants growing in a greenhouse under the
same climatic and soil conditions as that for cotyledons.
Cotyledons in all species were well developed, having the
same green colour as leaves. Two species, S. laricina and
H. gossypina, have flattened cotyledons about 1 cm long, while
H. persicum and S. richteri have succulent terete cotyledons
which are up to 1 cm and 5 cm long, respectively. They were
fully expanded after 10–12 d of growth of plants in soil and
they were used simultaneously for the determination of enzyme
activity and anatomical studies.
Light and electron microscopy
All samples for microscopy, immunolocalization and starch
analysis were taken from fully developed leaves and cotyledons
around midday. Samples for ultrastructural characterization
were fixed overnight at 4 °C in 2% (v/v) paraformaldehyde–2%
(v/v) glutaraldehyde solution in 0.1 M phosphate buffer
(pH 7.2), post-fixed in 2% (w/v) OsO , and then after a standard
4
dehydration procedure, embedded in a mixture of Epon and
Araldite. Cross-sections were obtained on an Ultracut ultramicrotome (Austria). For light microscopy, semi-thin sections
were stained with 1% (w/v) Toluidine blue O in 1% (w/v)
Na B O ; ultra-thin sections were stained for electron micro2 4 7
scopy with 2% (w/v) lead citrate or 154 dilution of 1% (w/v)
KMnO 52% (w/v) uranyl acetate. For electron microscopy
4
studies Hitachi H-600 and JEM-1200 EX transmission electron
microscopes were used.
The number of thylakoids per granum was counted on 10–15
micrographs of median chloroplast sections. The lengths of
appressed and non-appressed thylakoid membranes (including
both intergranal and end-granal thylakoid membranes) were
measured with a curvimeter in at least ten chloroplasts. The
chloroplast area in sections was estimated with an Image
Analysis program (Image Tool for Windows, version 128) on
the same micrographs.
In situ immunolocalization
Samples were fixed for 12–24 h at 4 °C in 2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer,
pH 7.2. The samples were dehydrated with a graded ethanol
series and embedded in London Resin White (LR White)
acrylic resin. The four antibodies used (all raised in rabbit)
were anti-spinach Rubisco (LSU ) IgG (courtesy of
B McFadden, Washington State University), anti-maize PEPC
IgG (courtesy of R Chollet, University of Nebraska), antimaize 62 KDa NADP-ME IgG (courtesy of C Andreo, Maurino
et al., 1996), and anti-Amaranthus hypochondriacus mitochondrial NAD-ME IgG which was prepared against the 65 kDa a
subunit (courtesy of J Berry, Long et al., 1994). Analyses of
the malic enzyme and PEPC antibodies by Western blots of
maize (NADP-ME-type species), Amaranthus cruentus (NADME-type species), and H. persicum (NADP-ME species)
indicates specificity for immunoreaction of the antibodies with
the respective enzyme (Pyankov et al., 1999b). For specificity
of the Rubisco antibody from Western blots see Everard et al.
( Everard et al., 1993).
Photosynthetic mechanisms in Salsoleae
TEM: Thin sections on coated nickel grids were incubated for
1 h in TBST plus BSA (10 mM TRIS-HCl, pH 7.2, 150 mM
NaCl, 0.1% (v/v) Tween 20 plus 1% (w/v) BSA) to block nonspecific protein binding on the sections. The sections were
then incubated for 3 h with NAD-ME (15100 dilution), or
NADP-ME (1520 dilution) antibodies. After washing with
TBST plus BSA, the sections were incubated for 1 h with
Protein A-gold (20 nm) (Amersham) diluted 1550 with
TBST/BSA. The sections were washed sequentially with TBST
plus BSA, TBST, and distilled water, and then post-stained
with a 154 dilution of 1% (w/v) potassium permanganate and
2% (w/v) aqueous uranyl acetate. Images were obtained using
a JEOL-1200 EX transmission electron microscope.
Light microscopy: Sections, 0.8–1 mm thick, from the same
samples were placed onto gelatin-coated slides and blocked for
1 h with TBST plus BSA. They were then incubated for 3 h
with the preimmune serum with TBST plus BSA, or with
Rubisco (15400 dilution), PEPC (15300 dilution), NAD-ME
(15100), or NADP-ME (1520) antibodies. The slides were
washed with TBST+BSA and then treated for 1 h with protein
A-gold (20 nm particles diluted 1550 with TBST+ BSA). After
washing, the sections were exposed to a silver enhancement
reagent for 20 min according to the manufacturer’s directions
(Amersham), stained with 0.5% (w/v) Safranin O, and imaged
in a reflected/transmitted mode using a BioRad 1024 confocal
system with Nikon Eclipse TE 300 inverted microscope and
Laser sharp image program 3.10. The background labelling
with preimmune serum was low although some infrequent
labelling occurs in areas where the sections were wrinkled due
to trapping of antibodies/label (results not shown).
Staining for polysaccharides
Sections, 0.8–1 mm thick, were made from the same samples on
gelatin-coated slides, incubated in periodic acid (1% w/v) for
30 min, washed and then stained with Shiff ’s reagent (Sigma)
for 1 h. After rinsing they were ready for analysis by light
microscopy.
1781
cells with weaker labelling of mesophyll and water storage
tissue (Fig. 1C ). A large amount of starch is found in
bundle sheath cells and in water storage tissue, while few
starch grains occur in mesophyll cells ( Fig. 2A). For
convenience in comparing the cell-specific compartmentation of enzymes and starch in different tissues of
the species studied, the results are summarized in Table 1.
Cotyledons: While the leaf has a C Salsoloid structure,
4
the flattened cotyledons have a C Atriplicoid structural
4
type with two main photosynthetic cell layers (palisade
mesophyll and bundle sheath cells) around vascular
bundles (Fig. 1D–F ). There is also a layer of large,
irregular-shaped, subepidermal cells which contain some
chloroplasts. On the lower side of the leaf there are 1–2
layers of spongy parenchyma cells which also contain
some chloroplasts. Both the subepidermal and spongy
parenchyma layer on the abaxial side of the cotyledon
have some appearance of water-storage parenchyma and
may function as such. Immunolabelling reveals that
Rubisco levels are very high in bundle sheath cells,
whereas there is very low labelling in subepidermal and
spongy mesophyll cells, and no labelling in palisade cells
( Fig. 1D). In contrast, PEPC is located specifically in
palisade mesophyll cells without labelling of bundle
sheath, subepidermal or spongy mesophyll cells (Fig. 1E).
There is strong labelling of NAD-ME in bundle sheath
cells, and very light labelling in all other tissue, which
may be background (Fig. 1F ). Starch grains are abundant
in bundle sheath cells, and a few grains also appear in
subepidermal and spongy parenchyma cells (Fig. 2B). See
Table 1 for a summary of enzyme and starch compartmentation.
Results
NAD-ME-type C species—light microscopy
4
(A) Salsola laricina: C Salsoloid NAD-ME-type leaves, C
4
4
Atriplicoid NAD-ME-type cotyledons
Leaves: Leaves of this species are classified as having the
C Salsoloid-type of anatomy (see terminology of Carolin
4
et al., 1975) which is characterized by the presence of
two chlorenchymatous layers (palisade mesophyll and
bundle sheath cells) continuous around their periphery.
There is a layer of round subepidermal cells under the
epidermis, which contains few chloroplasts. A main
bundle occurs immersed in the centre of the water-storage
tissue, that contains rather numerous and well developed
chloroplasts, while smaller veins occur just below the
bundle sheath layer (Fig. 1). There is a very strong
labelling for Rubisco in bundle sheath cells and in water
storage tissue (Fig. 1A) whereas there is a high level
of PEPC labelling in mesophyll cells (Fig. 1B). Most
immunolabelling of NAD-ME occurs in bundle sheath
(B) Halocharis gossypina: C NAD-ME-type Salsoloid leaf
4
anatomy, C dorsoventral cotyledons
3
Leaves: Leaves of this species have a Salsoloid C -type
4
anatomy, but are somewhat flattened. The chloroplastcontaining layers of palisade mesophyll and bundle sheath
cells are located on the periphery of the adaxial side of
the leaf and are disrupted on the abaxial side. The water
storage cells, which are not particularly large, also contain
well-developed chloroplasts. Immunolocalization studies
show a high level of Rubisco protein in the C -type
4
bundle sheath cells, and the occurrence of this protein in
water storage tissue is also apparent while it is absent in
mesophyll cells ( Fig. 3A). PEPC is specifically located
in palisade mesophyll cells (Fig. 3B), whereas NAD-ME
is mostly localized in bundle sheath cells, with only traces
of labelling appearing in other tissues (Fig. 3C ). Starch
grains are abundant in bundle sheath cells and water
storage cells, with lesser amounts in mesophyll cells
( Fig. 2C ). See summary in Table 1.
1782 Voznesenskaya et al.
Fig. 1. Light microscopy of in situ immunolocalization of photosynthetic enzymes in leaves and cotyledons of Salsola laricina. (A) Rubisco in leaf,
×280, bar=50 mm. (B) PEPC in leaf, ×230, bar=50 mm. (C ) NAD-ME in leaf, ×260, bar=50 mm. (D) Rubisco in cotyledon, ×210, bar=50 mm.
(E ) PEPC in cotyledon, ×180, bar=50 mm. ( F ) NAD-ME in cotyledon, ×180, bar=50 mm. Note two layers of chlorenchyma on the periphery of
the leaf: palisade mesophyll (PM ) and bundle sheath (BS) cells. In the centre, the main vascular bundle is surrounded by water storage tissue
( WS ), with small vascular bundles ( VB) on its periphery. The layer of subepidermal cells (SE ) is under the epidermis.
Cotyledons: This species has C -type dorsoventral cotyle3
dons with two to three layers of palisade-like cells on
the adaxial side of the leaf and three layers of spongy
parenchyma cells on the abaxial side. In situ immunolocalization studies using antibodies against Rubisco
show that the protein is located in all chloroplastcontaining cells of the mesophyll including palisade and
spongy parenchyma and the C -type bundle sheath,
3
with the prevalence of the labelling in the palisade
layers ( Fig. 3D). Immunolabelling with antibodies to
the C enzymes is completely lacking with PEPC
4
and NAD-ME ( Fig. 3E, F; Table 1). The localization
of starch grains is diffuse in all chloroplast-containing
cells, but they are more prominent in the outer layers
of palisade mesophyll ( Fig. 2D). See summary in
Table 1.
Photosynthetic mechanisms in Salsoleae
1783
Fig. 2. Starch localization in leaves and cotyledons of Salsola laricina and Halocharis gossypina. (A) Salsola laricina leaf. ×300, bar=50 mm. (B)
Cotyledon of Salsola laricina. ×300, bar=50 mm. (C ) Leaf of Halocharis gossypina. ×440, bar=50 mm. (D) Cotyledon of Halocharis gossypina.
×260, bar=50 mm.
NAD-ME species—electron microscopy
Immunolocalization of NAD-ME enzyme: Transmission
electron microscopy was used to determine the subcellular
localization of the main decarboxylating enzymes, which
is NAD-ME in the two species, S. laricina and H.
gossypina. Under the electron microscope it was obvious
that the bundle sheath cells contain numerous mitochondria having an intensive system of tubular or lamellae
cristae, which are usually located along the inner cell
walls or between chloroplasts in the inner parts of bundle
sheath cells. It is well known that such mitochondria,
with some variations in cristae structure, are characteristic
of all NAD-ME plants (Laetsch, 1968; Osmond et al.,
1969; Voznesenskaya and Gamaley, 1986). Immunolabel
for NAD-ME is concentrated in these mitochondria in
bundle sheath cells in the leaves of both species and the
C -type cotyledons of S. laricina (Fig. 4A, B). There was
4
very little immunolabelling with NAD-ME in the C
3
cotyledons of H. gossypina.
Quantitative analysis of thylakoid structure: In addition
to results on immunolabelling with NAD-ME, electron
microscopy studies further established the classification
of these two species as NAD-ME-type plants on the basis
of the structure of chloroplasts and mitochondria in leaf
bundle sheath cells. They have ultrastructural features
characteristic of this C subgroup: in leaves of both
4
species, bundle sheath chloroplasts have an intensive
granal system, while the chloroplasts of mesophyll have
some degree of grana reduction. Organelles have a centripetal position in leaf bundle sheath cells of these species
( Figs 1A–C; 3A–C ). The chloroplasts and mitochondria
in the bundle sheath cells of the C -type cotyledons of S.
4
laricina have the same characteristics.
Quantitative analysis of grana stacking showed distinct
differences between mesophyll and bundle sheath chloroplasts. In leaves of both S. laricina and H. gossypina there
was a clear trend in the bundle sheath having a greater
degree of grana stacking than mesophyll chloroplasts
( Fig. 5). For example, in H. gossypina about 90% of the
grana in the mesophyll had only 2 or 3 thylakoids per
granum and only 10% with four or more thylakoids per
granum, while in the bundle sheath the corresponding
values were 66% and 34%. A similar trend was observed
in leaves of S. laricina (Fig. 5). The maximum number
of thylakoids in the grana of H. gossypina was six for the
mesophyll and 15 for the bundle sheath chloroplasts; and
the corresponding values in S. laricina were 12 in mesophyll and 20 in a bundle sheath.
1784 Voznesenskaya et al.
Table 1. Immunolocalization of the main photosynthetic enzymes and starch in different tissues of selected species of tribe Salsoleae
The relative intensity of labelling in different cell types with each antibody is indicated by the number of + symbols.
Species, sample,
type of anatomy and
biochemistry
Cell type
Rubisco
PEPC
NAD-ME
NADP-ME
Starch
Salsola laricina
Leaf
C , NAD-ME
4
Salsoloid
S. laricina
Cotyledon,
C , NAD-ME,
4
Atriplicoid
Halocharis gossypina
Leaf
C , NAD-ME
4
Salsoloid
H. gossypina
Cotyledon
C , dorsoventral
3
Salsola richteri
Leaf,
C , NADP-ME
4
Salsoloid
S. richteri
Cotyledon,
C , NADP-ME
4
Salsoloid
Haloxylon persicum
Stem,
C4, NADP-ME
Salsoloid
H. persicum
Cotyledon,
C , isopalisade
3
SE
PM
BS
WST
SE
PM/SM
BS
—
—
+++
++
+
−/+
+++
—
+++
—
—
—
+++/−
—
—
+
+++
+
+a
+a/+a
+++
NDb
ND
ND
ND
ND
ND
ND
—
+
+++
++
+
−/+
+++
PM
BS
WST
—
+++
++
+++
—
—
+a
+++
+a
ND
ND
ND
+
+++
+
PM/SM
BS(C )
3
+++/++
++
—
—
—
—
ND
ND
++/+
+
SE
PM
BS
WST
SE
PM
BS
WST
SE
PM
BS
WST
PM/SM
BS(C )
3
+
+
+++
+
+
+
+++
+
+
—
+++
+
+++/++
++
+a
+++
—
—
—
+
—
—
+
+++
—
—
—
—
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+a
+a
++++
—
—
—
+++
—
—
—
+
—
—
—
—
+
++
—
+
++
++
—
—
+
++
—
++/+
+
aLight labelling which may either be background or low levels of the protein.
bND–not determined.
BS, bundle sheath cells; PM, palisade mesophyll cells; SM, spongy mesophyll cells; SE, subepidermal cells; WST, water-storage tissue.
In the C Atriplicoid-type cotyledons of S. laricina
4
there is an obvious shift to a higher degree of grana
stacking in the bundle sheath chloroplasts, which is similar
to that of the Salsoloid-type leaves. For example, nearly
53% of the thylakoids in mesophyll chloroplast grana are
paired compared to 42% in the bundle sheath; the bundle
sheath chloroplasts have about 16% of the grana with
6–8 or more thylakoids per granum compared to only
4% in the mesophyll chloroplasts. The maximum number
of thylakoids per granum in mesophyll chloroplasts is
eight compared to 18 in the bundle sheath chloroplasts.
Measurements of the granal index ( length of all
appressed thylakoid membranes as a percentage of the
total length of all thylakoid membranes in the chloroplast), and appressed versus non-appressed thylakoid density ( length of a given type of thylakoid per area of stroma)
also showed marked differences. In leaves of H. gossypina
and in leaves and the C -type cotyledons of S. laricina,
4
the granal index is about 1.5–2.5 times higher in the bundle
sheath than in mesophyll chloroplasts. Analysis of thylakoid density shows that, in general, for a given species
the total density of thylakoids (appressed plus non-
appressed ) is similar between mesophyll and bundle sheath
chloroplasts. However, in leaves of H. gossypina and leaves
and cotyledons of S. laricina, the appressed thylakoid
density is higher in the bundle sheath than in the mesophyll
chloroplasts, while the non-appressed thylakoid density is
just the opposite. The differences in thylakoid density
between mesophyll and bundle sheath chloroplasts in
leaves of S. laricina are considerably larger than in the
cotyledons ( Table 2). Some other calculations, for
example, the ratio of appressed to non-appressed thylakoids, also showed a significant difference between the two
kinds of cells. This ratio in bundle sheath chloroplasts/
mesophyll chloroplasts was greater than five in S. laricina
and about 2.5 in H. gossypina.
In comparison to the above data with C -type organs,
4
the flattened dorsoventral C -type cotyledons of H. gossy3
pina, have about 86% of the grana with two to five
thylakoids per granum. These chloroplasts have 59%
membranes in appressed thylakoids. The granal development in these C -type chloroplasts of cotyledons is
3
remarkably similar to that of the bundle sheath chloroplasts in H. gossypina leaves ( Fig. 5; Table 2).
Photosynthetic mechanisms in Salsoleae
1785
Fig. 3. Light microscopy of in situ immunolocalization of photosynthetic enzymes in leaves and cotyledons of Halocharis gossypina. (A) Rubisco
in leaf, ×310, bar=50 mm. (B) PEPC in leaf, ×350, bar=50 mm. (C ) NAD-ME in leaf, ×260, bar=50 mm. (D) Rubisco in cotyledon, ×150,
bar=50 mm. ( E ) PEPC in cotyledon, ×220, bar=50 mm. ( F ) NAD-ME in cotyledon, ×170, bar=50 mm.
NADP-ME species—light microscopy
(A) Salsola richteri: C NADP-ME-type Salsoloid leaves and
4
cotyledons
This species has both C Salsoloid-type leaves and cotyle4
dons with two layers of chlorenchyma (palisade mesophyll
and bundle sheath cells) around their periphery.
Cotyledons are somewhat flattened, whereas leaves are
cylindrical and round in cross-section. Both have a layer
of chloroplast-containing subepidermal cells, which in
cotyledons are rather large and nearly of the same size as
the mesophyll cells, whereas in leaves they are randomly
distributed and often contain calcium oxalate crystals.
Cells of water-storage parenchyma, which surround the
main vascular bundle in the centre of the leaf, also contain
some chloroplasts.
Organelles in cotyledon bundle sheath cells of this
species have a more random distribution around the cell
compared to the centripetal position in leaves (Fig. 6A,
D). Bundle sheath cells of the cotyledons also have
thinner cell walls in comparison to that in the leaf bundle
sheath. These features of the bundle sheath cells in
cotyledons of S. richteri are atypical for C plants of
4
this group.
1786 Voznesenskaya et al.
Fig. 4. Electron microscopy of in situ immunolocalization of NAD-ME in Salsola laricina (A, leaf; B, cotyledon) and NADP-ME in Salsola richteri
(C, leaf; D, cotyledon). Ch, chloroplast; M, mitochondria; P, peroxisome.
Leaves: There is an intense immunolabelling for Rubisco
in bundle sheath cells, but also some labelling in subepidermal, mesophyll and water storage cells ( Fig. 6A).
Labelling for PEPC gave a high level of the protein in
mesophyll cells, but very low labelling in the subepidermal
cells, and no label in bundle sheath and water storage
cells ( Fig. 6B). There is selective immunolabelling of
bundle sheath cells with NADP-ME antibody (Fig. 6C ).
The amount of starch grains was generally relatively low,
but higher in bundle sheath than in mesophyll cells.
Results of immunolabelling and starch compartmentation
are summarized in Table 1.
Cotyledons: This organ showed high labelling for Rubisco
in bundle sheath cells, and very little label for Rubisco in
all other tissues ( Fig. 6D). Immunolabelling for PEPC
was found only in mesophyll cells (Fig. 6E), while
NADP-ME is localized in bundle sheath cells (Fig. 6F ).
Starch grains were equally distributed between mesophyll
Photosynthetic mechanisms in Salsoleae
1787
Fig. 5. Relative distribution of thylakoids per granum (%) in chloroplasts of palisade mesophyll (PM ) and bundle sheath (BS) cells. The standard
error of the mean was usually between 2–10% except when number of thylakoids per granum was greater than 9 (due to low frequency).
and bundle sheath cells, and a few starch grains were
found in subepidermal cells ( Table 1).
(B) Haloxylon persicum: C NADP-ME-type Salsoloid stems,
4
C -type cotyledons
3
Stem: This species belongs to the aphyllous plants with
its leaves reduced to very small scales of 1–2 mm in
length. Carbon assimilation occurs predominantly in the
green stem cortex of young shoots, which have the C
4
Salsoloid-type of anatomy with two chlorenchymatous
layers (palisade mesophyll and bundle sheath cells)
around its periphery. The layer of subepidermal cells in
this species consists of rather evenly distributed small
cells. Some chloroplasts also appear in the subepidermal
and water storage cells. The level of immunolabelling for
Rubisco in bundle sheath cells of this species is very high,
as in C leaves; it is also detected in low levels in
4
subepidermal cells and in water storage parenchyma, but
1788 Voznesenskaya et al.
Table 2. Chloroplast granal index and thylakoid density in palisade mesophyll (PM) and bundle sheath (BS) chloroplasts of selected
species of tribe Salsoleae
The standard error was usually from 5–10%.
Species
Cell type
Granal indexa
(%)
Appressed/non-appressed
Appressed
thylakoid
densityb
Non-appressed
thylakoid densityb
Total
thylakoid densityb
Salsola laricina
Leaf
S. laricina
Cotyledon
Halocharis gossypina
Leaf
H. gossypina
Cotyledon
Salsola richteri
Leaf
S. richteri
Cotyledon
Haloxylon persicum
Stem
H. persicum
Cotyledon
PM
BS
PM
BS
PM
BS
25
64
39
56
36
58
0.33
1.75
0.65
1.31
0.57
1.43
16.9
49.1
17.9
24.1
16.8
24.8
50.7
27.9
27.49
18.8
29.7
17.4
67.6
76.9
45.4
42.8
46.5
42.1
PM
PM
BS
PM
BS
PM
BS
59
46
30
59
27
52
31
1.46
0.92
0.47
1.46
0.38
1.12
0.48
22.5
18.7
15.4
22.4
10.7
21.3
9.4
15.3
19.3
38.2
15.4
28.7
19.3
20.4
37.8
38.0
53.6
37.8
39.3
40.7
29.8
PM
62
1.64
15.0
9.3
24.3
aGranal index, the length of all appressed thylakoid membranes as a percentage of the total length of all thylakoid membranes in the chloroplast.
bThylakoid density in this case means the length of thylakoid membranes (in mm) per 1 mm2 of chloroplast stroma area (analysed for the total
area of the chloroplast excluding starch grains).
not in mesophyll cells ( Fig. 7A). A high level of PEPC
was detected by immunolabelling in mesophyll cells and
only low levels occurred in the subepidermal cells
(Fig. 7B), while NADP-ME was found specifically in
bundle sheath cells ( Fig. 7C ). Starch appears in bundle
sheath cells, with lower levels in mesophyll cells. The
starch in mesophyll cells was preferentially located in
their proximal part in relation to the bundle sheath cells.
See summary of results in Table 1.
Cotyledons: This species has C isopalisade cotyledons,
3
which are oval shaped in cross section, with three or four
layers of palisade cells and two-to four layers of spongy
parenchyma around the central-located vascular bundles.
Rubisco was found in all chloroplast-containing cells,
including the C -type bundle sheath cells ( Fig. 7D),
3
whereas there was no detectable labelling with antibodies
to the C enzymes ( Fig. 7E, F ). Starch grains were found
4
in all photosynthetic cells, but were most abundant in the
upper layers of palisade mesophyll cells ( Table 1).
NADP-ME species—electron microscopy
Immunolocalization of NADP-ME: Light microscopy
studies show, as noted above, that NADP-ME is found
mainly in bundle sheath cells of leaves/stems of S. richteri
and H. persicum and in bundle sheath cells of the C
4
cotyledons of S. richteri. At the electron microscopy level
labelling was observed in bundle sheath chloroplasts in
all cases (Fig. 4; results not shown). Although some label
appeared in subepidermal and mesophyll cells of the leaf
of S. richteri, there was no specific association with
organelles in these cells suggesting this may either be
background, or low levels of another isoform of
NADP-ME.
Quantitative analysis of thylakoid structure: Electron
microscopy of chlorenchyma cells in leaves of S. richteri
and the stem of H. persicum reveals ultrastructural features which are consistent with their classification in the
NADP-ME group of plants: bundle sheath cells contain
chloroplasts with some degree of grana reduction along
with a few small mitochondria. On the other hand,
mesophyll chloroplasts in both species have a welldeveloped granal system. Similar structural characteristics
were found in the C -type cotyledons of S. richteri.
4
Clear quantitative differences in grana formation
between the chloroplast types in S. richteri and H. persicum are shown in Fig. 5 and Table 2. In leaves of S.
richteri, there is a clear shift in bundle sheath chloroplasts
having fewer thylakoids per granum than mesophyll (e.g.
64% of the grana in bundle sheath having only two
thylakoids per granum compared to 37% in the mesophyll
chloroplasts). A similar trend is observed in the C
4
cotyledons of S. richteri and in the C stems of H.
4
persicum (Fig. 5). Both species have organelles in bundle
sheath cells in a centripetal position in leaves/stems,
whereas they are randomly or sometimes even centrifugally arranged in S. richteri cotyledons, but the ultrastructural features of bundle sheath chloroplasts in these
species are very similar in all these assimilatory organs.
However, in H. persicum some rather large grana are
found in stem mesophyll chloroplasts (up to 15 thylakoids
per granum) while in leaves of S. richteri the grana of
mesophyll chloroplasts are not as large (maximum of
eight thylakoids per granum).
Photosynthetic mechanisms in Salsoleae
1789
Fig. 6. Light microscopy of in situ immunolocalization of photosynthetic enzymes in leaves and cotyledons of Salsola richteri (NADP-ME-type C
4
species). (A) Rubisco in leaf, ×150, bar=50 mm. (B) PEPC in leaf, ×230, bar=50 mm. (C ) NAD-ME in leaf, ×200, bar=50 mm. (D) Rubisco in
cotyledon, ×180, bar=50 mm. (E ) PEPC in cotyledon, ×200, bar=50 mm. (F ) NAD-ME in cotyledon, ×220, bar=50 mm.
The granal index is about 1.5–2 times higher in
mesophyll chloroplasts than bundle sheath chloroplasts
in S. richteri leaf and cotyledon, and in stem of H.
persicum. Likewise, the ratio of appressed/non-appressed
thylakoids is clearly higher in the mesophyll chloroplast
than the bundle sheath chloroplasts of these organs
( Table 2).
In C isopalisade cotyledons of H. persicum, mesophyll
3
chloroplasts have abundant, though not very large grana;
more than 98% of the grana consist of only two to five
thylakoids (Fig. 5). All other characteristics of the chloroplasts of cotyledons, such as the ratio of appressed to
non-appressed thylakoid length, the percentage of
appressed thylakoids in chloroplast, and the density of
both appressed or non-appressed thylakoids resemble that
of mesophyll chloroplasts in the stem of H. persicum, but
are closer in all characters to that of mesophyll chloroplasts in C cotyledon of H. gossypina.
3
1790 Voznesenskaya et al.
Fig. 7. Light microscopy of in situ immunolocalization of photosynthetic enzymes in stems and cotyledons of Haloxylon persicum (NADP-ME-type
C species). (A) Rubisco in stem, ×160, bar=50 mm. (B) PEPC in stem, ×190, bar=50 mm. (C ) NADP-ME in stem, ×160, bar=50 mm.
4
(D) Rubisco in cotyledon, ×230, bar=50 mm. ( E) PEPC in cotyledon, ×160, bar=50 mm. (F ) NADP-ME in cotyledon, ×180, bar=50 mm.
Discussion
Kranz anatomy–enzyme compartmentation in four species
in tribe Salsoleae
Among the four C species examined in this study from
4
the tribe Salsoleae, the leaves or green stems of aphyllous
shoots have a Salsoloid-type Kranz anatomy, while the
cotyledons show greater anatomical variation from nonKranz, to Salsoloid or Atriplicoid Kranz-type anatomy.
In all of the Kranz-type organs, immunolocalization
studies show PEPC is localized in mesophyll cells and
absent from bundle sheath cells, while Rubisco is localized
in bundle sheath cells with little or no labelling in mesophyll cells. The malic enzymes, NAD-ME and NADPME, are expressed in bundle sheath cells, while the rather
low labelling in a few cases in other cells may either be
background or labelling of a constitutive isoform
( Table 1). This compartmentation is consistent with the
mechanism of C photosynthesis where the primary fixa4
tion of CO takes place in mesophyll cells via PEPC with
2
the production of C acids, malate or aspartate, which
4
subsequently move to the bundle sheath cells, are decarb-
Photosynthetic mechanisms in Salsoleae
oxylated and CO donated to the C cycle ( Edwards and
2
3
Huber, 1981; Edwards and Walker, 1983; Hatch, 1987).
These results also show that cotyledons, as well as leaves,
having the Kranz-type anatomy are performing C
4
photosynthesis.
With respect to Rubisco, in addition to heavy immunolabelling in bundle sheath cells there is also some labelling
in mesophyll cells of leaves and cotyledons of S. richteri.
This suggests there may be a small amount of atmospheric
CO fixed directly via this carboxylase in mesophyll cells
2
provided there is also at least low levels of the other
enzymes of the C cycle. However, this is likely to
3
contribute little to carbon assimilation since the carbon
isotope composition value for S. richteri is as expected
for a C plant (−12.0 to −13.6‰, Pyankov et al., 1997).
4
The occurrence of some Rubisco in mesophyll cells of
species having Kranz anatomy has also been observed by
immunofluorescent labelling in Salsola kali and some
grasses (Hattersley et al., 1977) and in C Flaveria palmeri,
4
F. trinervia and the C -like F. brownii (Bauwe, 1984; Reed
4
and Chollet, 1985). There is also evidence from immunolocalization studies that some other C dicots such as
4
Amaranthus caudatus, A. dubius, Gomphrena globosa, and
Portulaca oleraceae have some Rubisco in mesophyll cells
(Castrillo et al., 1997).
In most cases, PEPC was confined to the palisade
mesophyll cell layer adjacent to the bundle sheath cells.
In some organs such as S. laricina leaves and cotyledons
the protein is absent from subepidermal cells, and is also
absent from the spongy mesophyll of cotyledons. This is
consistent with the proposal that only mesophyll cells
which have an immediate contact with the bundle sheath
cells express PEPC (Langdale et al., 1988; Dengler et al.,
1995), although this is not the case in H. persicum since
some PEPC protein also is found in subepidermal cells.
Studies of immunolabelling by electron microscopy
showed that NAD-ME in bundle sheath cells of leaves
and cotyledons of S. laricina, and leaves of H. gossypina,
is located in the mitochondria, whereas the NADP-ME
was located in the bundle sheath chloroplasts of leaves
and cotyledons of S. richteri and stem of H. persicum.
This is consistent with previous results which show localization of NAD-ME in bundle sheath mitochondria of
the NAD-ME species Atriplex spongiosa ( Kagawa and
Hatch, 1975; Hatch et al., 1975) and Amaranthus hypochondriacus (Long et al., 1994) and NADP-ME in bundle
sheath chloroplasts of Zea mays (Slack et al., 1969;
Maurino et al., 1997) and C Flaveria species (Drincovich
4
et al., 1998).
Non-Kranz anatomy
In C plants, CO from the air is fixed by Rubisco, and
3
2
this can occur in all chloroplast-containing cells. The
immunolabelling for Rubisco in the non-Kranz cotyle-
1791
dons of H. gossypina and H. persicum show that the
labelling occurs in all chloroplasts, including palisade,
spongy mesophyll and bundle sheath parenchyma cells
whereas immunolabelling for C proteins was absent or
4
low. This is consistent with C photosynthesis. The highest
3
levels of Rubisco and starch grains occurred in the
palisade mesophyll cells which are nearest the adaxial
surface of the leaf, suggesting this is the most active site
of photosynthesis.
Water storage tissue
C species of the tribe Salsoleae have prominent water
4
storage tissue located in the centre of the assimilating
organ which, in leaves of some species, may comprise
nearly 40–45% of leaf volume in comparison with 25–30%
for chlorophyllous tissues (Gamaley, 1985). Water storage
tissue contains chloroplasts which vary in abundance
between species (Shomer-Ilan et al., 1979; Bil’ et al.; 1983;
Atachanov, 1986) In Climacoptera crassa (tribe Salsoleae)
the number of chloroplasts (in thousands) in calculations
for 1 mm of leaf length in water storage cells is nearly
three times higher than in mesophyll or in bundle sheath
(Atachanov, 1986). In the current study the number of
chloroplasts in the water storage tissue ranged from high
in H. gossypina to being practically absent in H. persicum.
Rubisco was detected by immunolabelling in all water
storage tissue with the highest intensity in Kranz-type
leaves and cotyledons of S. laricina and leaves of H.
gossypina ( Table 1). This suggests the water storage tissue
contributes to carbon assimilation and raises a question
about the source of CO . Do these cells assimilate CO
2
2
diffusing from bundle sheath cells (where it is concentrated
by the C cycle) to water storage tissue, perform CAM,
4
or re-fix respired CO from the centrally located tissues
2
(e.g. from respiration associated with phloem loading)?
A degree of CAM has been suggested to occur in water
storage cells of H. persicum ( Zalenskii and Glagoleva,
1981) and in some species of the genus Suaeda and
Climacoptera (Bil’ et al., 1983; Atachanov, 1986).
However, the levels of PEPC, and malic enzymes detected
by immmunolocalization in these water storage cells are
too low to suggest the function of CAM or donation of
CO from C acids ( Table 1). Also, in Climacoptera and
2
4
Suaeda species growing in south Tadjikistan, water storage tissue had little or no PEPC, but similar Rubisco
activity to that of whole leaves on a chlorophyll basis
(Pyankov et al. 1992b). Thus, in water storage cells
Rubisco may assimilate any CO available through res2
piration or diffusion from bundle sheath cells. In any
case, the CO fixed by Rubisco in the water storage tissue
2
may be derived from initial fixation of atmospheric CO
2
via PEPC such that the product of photosynthesis in
water storage tissue has the C -type carbon isotope
4
composition.
1792 Voznesenskaya et al.
Subepidermal cells
Desert plants often have not only internal water-storage
tissue, but also some kind of outer structures with the
same function. It may be special water-storing hairs on
the surface of the leaf, or some layers under the epiderm,
developmentally belonging to the epiderm (many-layered
epiderm) or to the leaf ground meristem (subepiderm or
hypoderm) ( Esau, 1965). It was believed that these tissues
practically lack chloroplasts, nevertheless some of the C
4
species of the tribe Salsoleae have anatomically distinct
subepidermal cells with chloroplasts. Specifically, among
the species of the present study, subepidermal cells occur
in leaves and cotyledons of S. laricina and S. richteri and
in stems of H. persicum. This raises the question of how
chloroplasts in subepidermal cells function in relation to
Kranz anatomy. In all of these cases, with the exception
of leaves of S. laricina, Rubisco is expressed in the
subepidermal cells; and occasionally low levels of PEPC
are detected in these cells (S. richteri leaves, H. persicum
stem). These results suggest some atmospheric CO may
2
be directly fixed in the subepidermal cells by the C
3
pathway, along with a small degree of fixation by PEPC
in some species. However, this may only contribute a
small amount to carbon fixation since the carbon isotope
values of these tissues are C -like (–12 to −15‰, Pyankov
4
et al., 1997, 1999b; CC Black, unpublished results).
Comparison of starch content and Rubisco localization in
Kranz-type organs
The distribution of starch in Kranz-type photosynthetic
organs in the representative species of tribe Salsoleae
shows a strong correlation with the occurrence of
Rubisco. Typically the highest starch content was in
bundle sheath chloroplasts, where Rubisco is high. In C
4
monocots, such as maize, bundle sheath chloroplasts are
the principal site of starch storage ( Esau, 1965; Edwards
and Walker, 1983). Starch was also found in waterstorage tissue of S. laricina leaves and cotyledons where
there was also prominent labelling for Rubisco, whereas
there was little or no starch in palisade mesophyll cells
which lack Rubisco ( Table 1).
In cotyledons of S. richteri there was approximately
equal starch content in mesophyll and bundle sheath cells.
Other results also suggest atypical characteristics of the
cotyledons in this species based on the distribution of
organelles in the cells, and the relative thin bundle sheath
cell walls. Also the ratio of activities of PEPC and
NAD-ME relative to the activity of Rubisco are lower,
and the carbon isotope fractionation values are more
negative in the cotyledons than in the leaves ( V Pyankov,
C Black, A Kuzmin, M Ku, and G Edwards, unpublished
results). These results suggest cotyledons of S. richteri
have some features intermediate to those of C and C
3
4
plants. There is also evidence that cotyledons of Kochia
scoparia and Salsola collina of the family Chenopodiaceae
have an intermediate feature; the CO compensation
2
points were about twice as high in cotyledons of these
species (15.4 and 11.6 ppm) as that in leaves (7.4 and
6.7 ppm) (Pyankov et al. 1999a).
Mesophyll and bundle sheath chloroplast ultrastructure in
NADP-ME-type C organs and proposed functions in
4
photochemistry
In the NADP-ME species S. richteri and H. persicum there
is a clear differentiation of bundle sheath and mesophyll
chloroplasts in Kranz-type organs. In both species the
appressed thylakoid density is about 1.5 to 2.2 times higher
in mesophyll than in bundle sheath chloroplasts and the
average granal index for the mesophyll chloroplasts is 52%
compared to 29% for bundle sheath chloroplasts (calculated from the data of Table 2). For the C organs studied
3
(cotyledons of H. gossypina and H. persicum) the granal
index of the mesophyll chloroplasts is about 60% (i.e.
about 60% of the membranes are appressed and 40% nonappressed). Thus, the mesophyll chloroplasts in the Kranz
organs are more like the mesophyll chloroplasts in the C
3
cotyledons in the degree of grana development. C plant
3
mesophyll chloroplasts function primarily in linear electron
flow through the two photosystems with little cyclic electron flow; thus, the high grana stacking in the C mesophyll
4
chloroplasts suggest they function primarily in PSII- and
PSI-mediated linear electron flow. The reduction in grana
formation in bundle sheath chloroplasts suggests a
decreased capacity for NADPH production and an
increased capacity for cyclic photophosphorylation which
would result in a higher ATP/NADPH ratio. It is known
that some NADP-ME C monocots like sorghum have
4
completely agranal bundle sheath chloroplasts; they lack
PSII activity and only function to generate ATP by PSImediated cyclic electron flow, while species like maize with
rudimentary grana have low PSII activity (Edwards and
Walker, 1983). Since there is a significant degree of grana
formation in bundle sheath chloroplasts of S. richteri and
H. persicum it is important to consider how the two cell
types function photochemically to provide the energy for
C photosynthesis. These species are primarily malate
4
formers, and the shuttle of malate to bundle sheath cells
and its decarboxylation results in a donation of both CO
2
and reductive power. The simplified scheme in Fig. 8A
shows how most of the PGA formed as a product of
RuBP carboxylase could be reduced to triose-P in bundle
sheath chloroplasts through the combined production of
some NADPH photochemically by bundle sheath chloroplasts and by NADP-malic enzyme. In this illustration the
mesophyll chloroplasts would produce twice as much
NADPH as the bundle sheath chloroplasts (consistent with
higher grana stacking and higher PSII activity in the
mesophyll chloroplasts), while the ATP production per
NADPH is about 2-fold higher in bundle sheath
chloroplasts.
Photosynthetic mechanisms in Salsoleae
1793
Fig. 8. Simplified schemes illustrating how the photochemical energy requirements could be met in Kranz-type organs of NADP-ME and NAD-ME
species of tribe Salsoleae in relation to differences in chloroplast ultrastructure and intercellular C acid shuttles. The stochiometry shown is for
4
assimilation of 3 CO to 1 triose-P with an overall requirement of 5 ATP and 2 NADPH per CO fixed. For details on enzymatic conversions in
2
2
the NAD- and NADP-ME C pathway see Kanai and Edwards ( Kanai and Edwards, 1999).
4
Mesophyll and bundle sheath chloroplast ultrastructure in
NAD-ME-type C organs and proposed functions in
4
photochemistry
In the NAD-ME species S. laricina and H. gossypina
there is also a pronounced differentiation of bundle sheath
and mesophyll chloroplasts in Kranz-type anatomy. In
this case, for the two species the granal density is about
1.5–2.5 times higher in bundle sheath than in mesophyll
chloroplasts, and the average granal index in mesophyll
chloroplasts is 33% compared to 59% in bundle sheath
chloroplasts (calculated from the data in Table 2). The
bundle sheath chloroplasts of these NAD-ME species are
very similar to that of the mesophyll chloroplasts of C
3
cotyledons in grana stacking. Previous studies on NADME-type C plants indicate they have granal mesophyll
4
and bundle sheath chloroplasts with PSII activity
( Edwards and Walker, 1983). However, the present analysis clearly shows these NAD-ME species of the tribe
Salsoleae have a much lower level of grana development
in the mesophyll chloroplasts which is characteristic for
many, if not all, species of this group (Gamaley, 1985).
Again, less grana stacking suggests an increased demand
for production of ATP relative to NADPH. In NAD-ME
species of the tribe Salsoleae aspartate is the main product
of the C pathway (Pyankov and Vakhrusheva, 1989);
4
1794 Voznesenskaya et al.
whose synthesis from alanine and CO in mesophyll cells
2
only requires the production of ATP. The scheme in
Fig. 8B illustrates how the formation of aspartate in
mesophyll cells and the reduction of the majority of the
PGA in bundle sheath cells would result in the mesophyll
chloroplasts having a primary role in ATP synthesis and
the bundle sheath chloroplasts being more responsible for
photochemical production of NADPH. In these proposed
schemes, the relative requirements for photochemical
production of ATP and NADPH by mesophyll and
bundle sheath chloroplasts in the NAD-ME species is
just the opposite of that in the NADP-ME-type species,
as is the degree of grana development ( Fig. 8A, B). If
linear electron flow to NADP produces 3 ATP and 2
NADPH per O evolved (Rumberg and Berry, 1995), it
2
is clear that substantial production of additional ATP
would be required in bundle sheath chloroplasts of
NADP-ME species (Fig. 8A) and mesophyll chloroplasts
of NAD-ME species (Fig. 8B) which may be generated
by PSI-dependent cyclic electron flow. Further evaluation
of the proposed functions of mesophyll and bundle sheath
chloroplasts will require studies at the cellular level to
determine the capacity for cyclic and linear electron flow,
and the capacity of bundle sheath cells to utilize malate
and asparate as carbon donors to the C pathway.
3
Summary
In conclusion, there are interesting anatomical variations
in the photosynthetic apparatus of leaves/stems and cotyledons of representative species of the tribe Salsoleae. The
immunological, ultrastructural and cytochemical studies
indicate the relationships of these anatomical arrangements to photosynthetic mechanisms. The specific modifications are likely to be related to environmental factors
associated with the evolution of these plants. Besides
Kranz-type organs having mesophyll and bundle sheath
cells, the circular arrangement in the Salsoloid anatomy
in leaves and stems of these four species includes water
storage and, in some cases, subepidermal cells, which
also have biochemical modifications in photosynthesis as
indicated by immunolocalization. There is not an apparent advantage of one C subtype, NAD-ME versus
4
NADP-ME, over the other. It is conceivable that the
NAD-ME-type plants could function photosynthetically
without any PSII activity in mesophyll chloroplasts, but
not in the NADP-ME-type species where there is an
absolute requirement of NADPH for the C cycle to
4
function. If PSII, which can be inactivated by photoinhibition, is more protected in chloroplasts of bundle
sheath cells under certain environmental stresses then the
NAD-ME-type photosynthesis could be advantageous.
The extensive variation in cotyledon structure and photosynthetic function among these species from Salsoloid
Kranz-type with nearly 50% of water storage tissue, to
Atriplicoid Kranz-type with only 10–15% water storage
tissue (according to Gamaley, 1985), to non-Kranz, may
be important for optimizing photosynthesis and survival
under the conditions existing during early seedling development. There is obviously some interesting differential
developmental expression of the photosynthetic mechanism between the cotyledons and leaves of these species
which could shed some new light on the development of
the photosynthetic apparatus of higher plants. This study
in the tribe Salsoleae validates the analysis of structure–
function as a very powerful approach to help understand
evolution of the photosynthetic carbon reduction mechanisms and the range of variations that exist.
Acknowledgements
This work was partly supported by Civilian Research and
Development Foundation Grant RB1–264 and NSF Grant
IBN-9807916. EV Voznesenskaya would like to thank CIES,
Washington DC for a Fulbright Scholar Research Fellowship.
We also thank the Electron Microscope Center of Washington
State University for use of their facilities and staff assistance.
References
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3 4
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