occurrence and forms of kranz anatomy in photosynthetic organs

Journal of Experimental Botany, Vol. 59, No. 7, pp. 1755–1765, 2008

doi:10.1093/jxb/ern020 Advance Access publication 24 April, 2008

SPECIAL ISSUE RESEARCH PAPER

Occurrence and forms of Kranz anatomy in photosyntheticorgans and characterization of NAD-ME subtype C4

photosynthesis in Blepharis ciliaris (L.) B. L. Burtt(Acanthaceae)

Hossein Akhani1,*, Maraym Ghasemkhani1, Simon D. X. Chuong2 and Gerald E. Edwards3,*

1 Department of Plant Sciences, School of Biology, College of Science, University of Tehran, PO Box 14155-6455,Tehran, Iran2 Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada3 School of Biological Sciences, Washington State University, Pullman, Washington, WA 99164-4236, USA

Received 17 October 2007; Revised 14 December 2007; Accepted 16 January 2008

Abstract

Blepharis (Acanthaceae) is an Afroasiatic genus com-

prising 129 species which occur in arid and semi-arid

habitats. This is the only genus in the family which is

reported to have some C4 species. Blepharis ciliaris

(L.) B. L. Burtt. is a semi-desert species with distribu-

tion in Iran, Oman, and Pakistan. Its form of photosyn-

thesis was investigated by studying different organs.

C4-type carbon isotope composition, the presence of

atriplicoid type Kranz anatomy, and compartmentation

of starch all indicate performance of C4 photosynthe-

sis in cotyledons, leaves, and the lamina part of bracts.

A continuous layer of distinctive bundle sheath cells

(Kranz cells) encircle the vascular bundles in cotyle-

dons and the lateral vascular bundles in leaves. In

older leaves, there is extensive development of ground

tissue in the midrib and the Kranz tissue becomes

interrupted on the abaxial side, and then becomes

completely absent in the mature leaf base. Cotyledons

have 5–6 layers, and leaves 2–3 layers, of spongy

chlorenchyma beneath the veins near the adaxial side

of the leaf, indicating bifacial organization of chloren-

chyma. As the plant matures, bracts and spines

develop and contribute to carbon assimilation through

an unusual arrangement of Kranz anatomy which

depends on morphology and exposure to light. Stems

do not contribute to carbon assimilation, as they lack

chlorenchyma tissue and Kranz anatomy. Analysis of

C4 acid decarboxylases by western blot indicates B.

ciliaris is an NAD-malic enzyme type C4 species, which

is consistent with the Kranz cells having chloroplasts

with well-developed grana and abundant mitochondria.

Key words: Acanthaceae, Blepharis, C4 plants, desert

adaptation, Kranz anatomy, NAD-ME type, photosynthetic

enzymes.

Introduction

Globally, 79% of all C4 plants belong to the monocots andonly 21% belong to the dicots. C4 dicots have been foundin seven orders, and the largest number of species hasbeen discovered in the family Chenopodiaceae (Akhaniet al., 1997; Sage et al., 1999; Jacobs, 2001; Kadereitet al., 2003; Sage, 2004). The most evolutionarilyadvanced C4 dicot lineages are found in three orders ofEuasterids: Lamiales (Scrophulariaceae, Anticharis, Acan-thaceae: Blepharis), Solanales (Boraginaceae, Helio-tropium), and Asterales (Asteraceae: Flaveria) (Sageet al., 1999; Sage, 2004; McKown et al., 2005; Akhani,2007; Muhaidat et al., 2007).Middle Eastern and South African deserts are rich,

biodiverse centres for the evolution of Old World desert

* To whom correspondence should be addressed. E-mail: [email protected]; [email protected]: d13C values, carbon isotope composition; Kranz cells, distinctive bundle sheath cells; L, length; M, mesophyll; NAD-ME, NAD-malic enzyme;NADP-ME, NADP-malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate,Pi dikinase; PPFD, photosynthetic photon flux density; SEM,scanning electron microscopy; TEM, transmission electron microscopy; VB, vascular bundle(s); W, width.

ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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flora. Many C4 species and C4-dominated communitiesare known in these areas (Vogel and Seely, 1977; Wergerand Ellis, 1981; Winter, 1981; Ziegler et al., 1981;Batanouny et al., 1988; Schulze et al., 1996; Akhaniet al., 1997; Akhani and Ziegler, 2002; Sage, 2004). Sofar, in these geographical areas C4 plants have beendiscovered in 18 out of the 19 families which are known tocontain C4 species. In the family Acanthaceae, C4 specieshave only been identified in the genus Blepharis. This is anintriguing genus which is composed of 129 species amongthree subgenera (Vollesen, 2000, 2004); with C4 speciesoccurring in the subgenus Acanthodium (Sage, 2004, 2005;R Sage, personal communication). This is an Afroasiaticgenus, which is widely distributed in tropical and southernAfrica, southern parts of the Middle East and central Asia,India, southern China, with the occurrence of one speciesin Indonesia (Vollesen, 2000).

Blepharis ciliaris (L.) B. L. Burtt, is distributed in southand southeastern Iran, Pakistan, and Oman (see Map 21,p. 99 in Vollesen 2000). In this study, the anatomy andform of photosynthesis in various organs of this specieswas analysed to evaluate the adaptive value of thesetraits in hot deserts. Blepharis ciliaris is shown to bea C4 species; the occurrence and form of Kranz anatomyin cotyledons, leaves, and bracts was characterized andthe biochemical subtype of C4 photosynthesis wasdetermined.

Materials and methods

Plant material and growth conditions

Seeds of B. ciliaris (L.) B. L. Burtt were collected from a populationin Hormozgan Province, near Hajiabad in southern Iran on 13 June2004. A herbarium voucher from the natural habitat (Akhani et al.,17837), and two vouchers from the greenhouse and growth chamber(Akhani 18174, 18211) plants, have been collected and preserved atthe Botanical Biodiversity Research Laboratory, School of Biology,University of Tehran. The seeds were initially germinated inFebruary 2005, in Petri dishes at room temperature. After 2–3 d,the young seedlings were transplanted to 10 cm pots containing 10parts commercial potting soil, one part clay, one part sand, and 100grams gypsum, and grown for 3 weeks at room temperature under;50 lmol m�2 s�1 photosynthetic photon flux density (PPFD).After 3 weeks, the plants were transferred to a greenhouse andgrown under natural sunlight plus supplemental light (sodiumvapour lamps providing a PPFD of 400 lmol m�2 s�1) witha maximum midday PPFD on clear days of 1750 lmol m�2 s�1,a 14/10 h light/dark photoperiod and a 2561 �C day and 1860.5�C night.

Light microscopy

Samples from cotyledon leaves, leaves (tip, middle, and base ofvery young and mature leaves), bracts spine and bracts lamina, andyoung stems were fixed in 2% (v/v) paraformaldehyde and 2% (v/v)glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), dehydrated witha graded ethanol series and embedded in London Resin White (LRWhite, Electron Microscopy Sciences, Fort Washington, PA, USA).Semi-thin cross-sections (about 0.8–1 lm thick), made on a Reichert

Ultracut R ultramicrotome (Reichert-Jung GmbH, Heidelberg,Germany) were dried onto gelatin-coated slides and stained with1% (w/v) Toluidine blue O in 1% (w/v) Na2B4O7 for generalanatomy. The periodic acid–Schiff’s procedure (PAS) was used forstaining starch by incubating the sections in periodic acid (1% w/v),washing and then incubating with Schiff’s reagent (Sigma, St Louis,MO, USA).

Transmission electron and scanning electron microscopy

Samples for transmission electron microscopy (TEM) were fixed in2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 Mphosphate buffer (pH 7.2) at 4 �C overnight, dehydrated in anacetone series and then embedded in Spurr’s resin. Ultra-thin cross-sections were made on a Reichert Ultracut R ultramicrotome(Reichert-Jung GmbH, Heidelberg, Germany), stained with 2%(w/v) uranyl acetate followed by 2% (w/v) lead citrate andexamined using a JEOL JEM-1200 EX (JEOL USA, Inc.,Massachusetts, USA). Samples for scanning electron microscopywere fixed as described for light and transmission microscopy, post-fixed in 2% (w/v) OsO4, and then dehydrated in an ethanol series,cryofractured in liquid nitrogen, critical point dried, sputter coatedwith gold, and observed on a Hitachi S570 scanning electronmicroscope (Hitachi Scientific Instruments, Mountain ViewCalifornia, USA).

Western blot analysis

Total proteins were extracted from cotyledons or leaves as de-scribed in Voznesenskaya et al. (2005a). Protein samples (10 lg)were separated by 12% SDS-PAGE, transferred to nitrocellulose,and probed with antisera raised against Spinacea oleracea Rubisco(LSU; 1:10 000; courtesy of B McFadden), Zea mays PEPC (1:40000; Chemicon, Temecula, CA, USA), Zea mays pyruvate,Pidikinase (PPDK) (1:10 000; courtesy of T Sugiyama), Amaranthushypochondriacus NAD-malic enzyme (NAD-ME) (1:5000; courtesyof J Berry), or Zea mays NADP-malic enzyme (NADP-ME)(1:5000; courtesy of C Andreo). Goat anti-rabbit IgG-alkalinephosphatase conjugate (dilution of 1:50 000) was used as thesecondary antibody for detection of the enzymes. Blots weredeveloped with 350 lg ml�1 nitroblue tetrazolium and 175lg ml�1 5-bromo-4-chloro-3-indolyl phosphate in detection buffer(100 mM TRIS-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2).

In situ immunolocalization

Leaf samples were fixed at 4 �C in 2% (v/v) paraformaldehyde and1.25% (v/v) glutaraldehyde in 50 mM PIPES buffer, pH 7.2,dehydrated with a graded ethanol series and embedded in LondonResin White (LR White, Electron Microscopy Sciences, FortWashington, PA, USA) acrylic resin. Antibodies used were anti-Spinacea oleracea L. Rubisco (LSU) and commercially availableanti-Zea mays L. PEPC IgG. Non-immune serum was used forcontrols. Cross-sections, 0.8–1 lm thick, were dried in a drop ofwater on gelatin-coated slides and blocked for 1 h with TBST+BSA[10 mM TRIS-HCl, 150 mM NaCl, 0.1% (v/v) Tween 20, 1% w/vBSA, pH 7.2]. They were then incubated for 3 h with either thenon-immune serum diluted in TBST+BSA (1:100), anti-Rubisco(1:500 dilution) or anti-PEPC (1:200 dilution), treated for 1 h withprotein A-gold (10 nm particles diluted 1:100 with TBST+BSA)and exposed to a silver enhancement reagent for 20 min accordingto the manufacturer’s directions (Amersham, Arlington Heights, IL,USA). The sections were stained with 0.5% (w/v) Safranin O andimaged in a reflected/transmitted mode using a Bio-Rad 1024confocal system (Bio-Rad, Hercules, CA, USA). The backgroundlabelling with non-immune serum was very low, as demonstratedpreviously with these antibodies. Images were processed using

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Adobe Photoshop image processing software (Version 6.0, AdobeSystem Inc., San Jose, CA, USA).

Carbon isotope composition

Measures of the carbon isotope composition (d13C values), weredetermined at Washington State University on leaf, bracts and stemsamples taken from plants grown under a controlled environment,and from leaves of plants collected in a natural habitat fromSouthern Iran, using a standard procedure relative to PDB (Pee DeeBelemnite) limestone as the standard (Bender, 1971).

Results

Habitat and general morphology

A living form of the species in its natural habitat insouthern Iran (Fig. 1A), and its form in cultivation isillustrated in Fig. 1B. The seeds, 9–11 mm in length(L)34–5 mm in width (W), germinate within a few hoursafter moisture is provided. After 3–4 d, the ovatecotyledon leaves (W3L of 5–6 mm) become green andglabrous on the adaxial surface (Fig. 1C). As the

cotyledons enlarge, the anchoring hairs on the abaxialsurface shed and, eventually, the two reniform cotyledonleaves reach their maximum size (up to 11 mm L316 mmW), with a well-developed petiole up to 15 mm L (Fig. 1D).By the time the cotyledon leaves are mature, the lanceolateseedling leaves have developed. The plants become matureand produce flowers after 3.5 months (Fig. 1B).The mature leaves are lanceolate, sub-sessile to shortly

petiolate (5–8.5 cm L31–1.5 cm W), and bi-coloured witha shiny dark-green upper surface and a whitish-greenlower surface. The trichomes on the adaxial leaf side aresparser and shorter than those on the abaxial side(Fig. 1F). A few sub-sessile, 4-celled glands are inter-mixed with trichomes (Fig. 1G, H). Such glands have beenreported in many genera of Acanthaceae (Ahmad, 1978).The main difference between glandular hairs and longtrichomes is that glands are located in depressed parts ofthe leaf, while the trichomes are located on elevatedepidermal cells (Fig. 1G, H). The diacytic stomata on theabaxial leaf surface are very dense, and 3.6 times moreprevalent than on the adaxial surface. The overlapping

Fig. 1. Blepharis ciliaris. (A) Natural habit in southern Iran. (B) Habit in greenhouse conditions (Pullman, USA). (C) Young cotyledon leaves, notethe multicellular hairs attached to the abaxial surface and, laterally, the remaining parts of the capsule. (D) Seedling, showing reniform petiolatecotyledon leaves and lanceolate seedling leaves. (E) Light microscopy from a cross-section of cotyledon leaves showing Atriplicoid Kranz typeanatomy. (F) SEM of a cross-section of a leaf blade showing one lateral VB encircled by a continuous bundle sheath; note the denser and longer hairson the abaxial surface. (G) Epidermal surface of the adaxial side of mature leaves showing trichomes and glandular hairs. (H) Cross-section of theadaxial surface of leaves showing one glandular trichome consisting of four cells. (I) Close-up of trichomes on bracts. Labels: PM, palisademesophyll; KC, Kranz cell. Scale bars: (B) 5 cm; (C) 5 mm; (D) 10 mm; (E) 50 lm; (F) 136 lm; (G) 38 lm; (H) 25 lm; (I) 13.6 lm.

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bracts (18–20 mm L36–9 mm W) have a terminal spineup to 30 mm L and 2–3 pairs of lateral spines up to 5 mmL (Fig. 1A). The trichomes, on the spines, which are up to1.25 mm L, are denser than on the midrib of the leaf.Figure 1I shows a close-up view of trichomes on bracts.

Carbon isotope composition

The d13C values of different organs of greenhouse-grownplants and of leaves of plant grown in a natural environ-ment in southern Iran were analysed. Under the green-house growth conditions, cotyledons had the mostnegative d13C value, –15.7�/oo, while mature leaves hada value of –14.1�/oo, and bracts and stems had values of–13.1�/oo and –13.0�/oo, respectively. Mature leavescollected from plants grown in the natural habitat hada value of –13.4�/oo.

Anatomy

Cotyledon leaves: The cotyledon leaves are bifacial,having a layer of palisade cells beneath the adaxialepidermis and 5–6 layers of chloroplast containingspongy, rounded to oval, mesophyll (M) cells towards theabaxial side of the leaf. There is considerable air spacearound the spongy M tissue which occupies a largevolume of the cotyledon section. The cotyledons leaveshave atriplicoid type Kranz anatomy. The vascularbundles (VB) are surrounded by a continuous layer ofKranz cells with centripetal chloroplasts (Fig. 1E), withanother layer of palisade-like chlorenchyma external tothe Kranz cells. Between veins, there are a few rounded orvariously shaped chlorenchymatous cells.

Young leaves: The anatomy of young leaves (7–10 mmL31 mm W) was studied in three sequential parts, fromthe tip, middle, and at the base of the leaf (Fig. 2A–F).The cross-sectional shape of the young leaf tip is crescent-like with VB located towards the adaxial surface, in-cluding a central VB and 10 less prominent lateral VB.The general anatomy is similar to cotyledon leaves, beingbifacial in cross-section and having atriplicoid-type Kranzanatomy. There is generally only one layer of palisadecells on the adaxial side except near the mid-vein. TheKranz cells are surrounded by palisade cells on the adaxialside, and by small chlorenchyma cells on the abaxial sideand between VB (Fig. 2A, D). On the abaxial side thereare 2–3 layers of spongy parenchyma with few chloro-plasts, and considerable intercellular air space. In the tipof the leaf, all VB are completely surrounded by a layer ofKranz cells. PAS staining of polysaccharides showeddense starch grains in the Kranz cells and sparser grainsin the palisade and spongy chlorenchyma cells.The middle section of the leaves is slightly semicircular

in shape, with a prominent midrib on the abaxial surface(Fig. 2B). The leaf has a bifacial structure similar to that

of the leaf tip. While all lateral VB are surrounded byKranz cells, the Kranz layer around the midrib isinterrupted on the abaxial side of the leaves (Fig. 2B, E).PAS staining of polysaccharides showed a denser starchaccumulation in Kranz cells than in the surroundingchlorenchyma cells (Fig. 2E).The basal part of the leaf has a very prominent midrib,

with only small, strongly arcuate lateral blades (Fig. 2C).The epidermal layer is densely covered with elongate andglandulose trichomes (Fig. 2C, F). In the midrib, theKranz cells appear only on the adaxial side and occupyonly half of the VB. The Kranz cells in lateral veins areless prominent than in the middle and tip sections of theleaf and show a lower accumulation of starch. Somestarch grains appear in Kranz cells around the mid-vein(Fig. 2F), but they are not as dense as in the Kranz cells ofthe lateral veins. Starch grains are evenly distributedthroughout the chlorenchyma tissue.

Mature leaves: Mature leaves (55 mm L312 mm W) werestudied in three parts, including the tip, middle, and basalsections (Fig. 2G–O). The general structure of matureleaves is similar to young leaves, with respect to havingatriplicoid-type Kranz anatomy and being bifacial.Other than in the midrib, the Kranz cells of the leaf lamina

form a continuous layer around the VB (Fig. 2M–O). Onthe adaxial side, there is a single layer of palisade cells,many of which are in direct contact with Kranz cells.Between the VB and beneath the VB there is a layer ofsmall chlorenchyma cells with a more oblate shapeadjacent to the Kranz cells, and two or three layers oflarge spongy parenchyma cells, with large intercellularairspaces, next to the lower epidermis.The VB in the leaf tips are closer to each other and the

Kranz cells are more densely packed with chloroplaststhan in the middle and basal parts. The Kranz cells of leaftips are smaller in size and are largely occupied bychloroplasts, while those in the middle and base areconcentrated centripetally and occupy only about one-third of the volume of rather larger cells. PAS staining ofpolysaccharides shows a rather strong accumulation ofstarch in Kranz cells and surrounding chlorenchyma in theleaf tip, moderate accumulation in the middle, and muchlower levels in the basal part of the leaf (Fig. 2M–O).The VB of the midrib of the mature leaf tip is

surrounded by an incomplete Kranz layer which isinterrupted by parenchymatous cells on the abaxial halfof the midrib volume (Fig. 2G, J). In the middle of theleaf there is a further reduction in the development ofKranz cells around the mid-vein with only a few Kranzcells on the adaxial side (Fig. 2H, K). Then, in the basalsection, Kranz cells are completely absent around the mid-vein; rather, there is a layer of very small bundle sheathcells, containing very few chloroplasts, around thevascular tissue which, in turn, is surrounded by ground

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tissue (Fig. 2I, L). Also, around the leaf mid-vein, theappearance of starch decreases from the tip towards thebasal part of the leaf, which parallels the decrease indevelopment of Kranz cells around the vein (Fig. 2J–L).

Bracts: Under natural conditions, bracts appear soon aftersenescence and demarcation of leaves, and at thebeginning of periods of reproductive development (Fig.

1A, B). The bracts produce a very long terminal spine (upto 3 cm), and two pairs of lateral spines, of which theupper pair is longer than the lower pair (Fig. 1A). Bracts,which are much smaller than leaves, have a well-de-veloped central midrib, with two parallel lateral veins thatterminate in the upper pair of lateral spines, and twomarginal veins which terminate in the lower spine pair.The most obvious difference between bracts and leaves is

Fig. 2. Comparison of a leaf cross-section at three different parts (tip, middle, and base) and the developmental stages of young and mature leaves ofB. ciliaris. (A–C) Toluidine blue staining of young leaves. (D–F) PAS staining for starch in young leaves. (G–I) Toluidine blue staining of midrib ofmature leaves. (J–L) PAS staining in midrib of mature leaves. (M–O) PAS staining of mature leaf lamina. Labels: KC, Kranz cells; CVB, centralvascular bundle; PM, palisade mesophyll; VB, vascular bundle. Scale bars: (A, D, E, J, M, N, O) 50 lm; (B, C, F, G, K, L) 100 lm; (H, I) 150 lm.


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the mode of orientation to sunlight (Fig. 1A). While theadaxial surfaces of leaves are exposed to light, the adaxialsides of bracts are shielded from light by their orientationand coverage by flowers while the abaxial side of bracts ismore exposed to direct light. The spines are triangular incross-section, being convex on the dorsal (abaxial) andslightly concave on the ventral (adaxial) side (Fig. 3D, E).A cross-section of the bract lamina shows that the Kranz

layer is almost continuous around the lateral VB (Fig. 3A)and more or less continuous in the marginal veins (Fig.3C); but, it is interrupted on the adaxial and, sometimes,on the abaxial side of the central vein (Fig. 3B). Incontrast to bifacial leaves, in which the palisade paren-chyma on the adaxial side is well-differentiated fromspongy parenchyma on the abaxial side, those of thebracts are more or less uniform. On both sides of thebracts, there are c. three layers of chlorenchymatous cellsaround the Kranz cells, of which the innermost layer onthe abaxial surface is slightly more elongated than the twoouter layers. The association of considerable air space onthe adaxial side of the marginal vein shows that thesecells, though less distinct, are comparable to the spongychlorenchyma of the abaxial side of leaves. PAS stainingfor polysaccharides showed a denser starch accumulationin photosynthetic cells in the abaxial than those in theadaxial sides (Fig. 3C).A cross-section of young spines show distinctive Kranz

anatomy with a layer of chlorenchyma cells surroundingthe Kranz cells on the adaxial side; and there are one or

two additional layers of large parenchyma cells next to theepidermis. The density of chloroplasts (Fig. 3E) and thedense staining of polysaccharides (not shown) in theadaxial edges of the spine suggest that it is morephotosynthetically active than other regions. On theabaxial side of the spine, Kranz anatomy is interruptedat the apex where there is lack of chlorenchyma tissue(Fig. 3D); with several layers of large parenchyma cellsbetween the epidermis and small bundle sheath cells.

Young stems: The young stems, which are circular incross-section, are composed of an epidermal layer whichis underlain by several layers of small, rounded paren-chyma cells, and c. 6 layers of cortical parenchyma cellsand a central vascular cylinder. Toluidine blue staining(Fig. 3F) shows that there are no Kranz cells and noapparent chlorenchyma tissue. The vascular cylinder ofthe stem is associated with a cambial ring.

Biochemical features and ultrastructure of matureleaves

Studies on mature leaves were conducted to characterizethe form of C4 photosynthesis in B. ciliaris, includingimmunolocalization of PEPC and Rubisco, levels ofphotosynthetic enzymes by western blots, and ultrastruc-ture of Kranz anatomy.

Immunolocalization: The middle part of mature leaf blades(50 mm L310 mm W) was used for analysis of

Fig. 3. Bract and stem anatomy of B. ciliaris. (A) Bract lamina, showing central and lateral vascular bundles. (B) Bract lamina, enlarged centralvein stained by PAS. (C) Bract lamina, lateral bundle stained by PAS. (D) Parts of dorsal (abaxial) side of spine tip showing interupted Kranz layer.(E) Enlarged edge of ventral (adaxial) side of spine. (F) Enlarged cortex part of young stem, note the presence of cambial ring. Labels: KC, Kranzcells, C, cortex; CVB, central vascular bundle; CA, cambial ring; M, mesophyll cells; VB, vascular bundle. Scale bars: (D) 50 lm; (B, C, E, F) 100lm; (A) 150 lm.

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immunolocalization of Rubisco and PEPC. There wasstrong labelling for Rubisco LSU in chloroplasts in Kranzcells. There is labelling for PEPC in the layer ofchlorenchyma cells immediately surrounding the Kranzcells, but little or no labelling was detected in Kranz cells,or the spongy parenchyma towards the abaxial side of theleaf (Fig. 4).

Kranz ultrastructure: TEM results from samples takenfrom the middle part of mature leaves show the distribu-tion of a few chloroplasts along the peripheral walls of Mcells, and the dense Kranz chloroplasts arranged centri-petally towards the VB. Both M (Fig. 5A, B) and Kranzchloroplasts (Fig. 5C–E) are rather similar in their granalstacking. They usually form small grana consisting of 2–5

Fig. 4. Immunolocalization of Rubisco and PEPC in mature leaf of B. ciliaris. Transmitted/reflected confocal images of immunogold labelling, withlabel appearing as yellow particles. (A) Rubisco is selectively localized in Kranz cells. (B) PEPC is abundant in M cells, with low amount in Kranzcells. Scale bars: 50 lm.

Fig. 5. Electron microscopy of M and Kranz cells. (A) A section of a M cell showing several elongated chloroplasts. (B) Higher magnification ofa M chloroplast. (C, D) Chloroplasts of Kranz cells showing large starch grains and associated mitochondria. (E) magnification of a Kranz cellchloroplast. Labels: KC, Kranz cells; Ch, chloroplast; Mit, mitochondria; M, mesophyll cells; S, starch grains. Scale bars: (A) 1000 nm; (B, C) 500nm; (D) 1000 nm; (E) 200 nm.


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aggregated thylakoids (Fig. 5B–E). Measurements weremade on the average number of thylakoids per granum(n¼50); M chloroplasts had 3.1 thylakoids per granum(83% of the grana had 2–3 thylakoids per granum), whileKranz chloroplasts had 3.4 thylakoids per granum (68%had 2–3 thylakoids per granum). The TEM ultrastructurestudy also reveals that there are more mitochondria in theKranz cells which are associated with chloroplasts than inM cells (Fig. 5C, D). The chloroplasts are dimorphic inthat those in Kranz cells store starch and are larger than Mchloroplasts.

Western blots: SDS-PAGE blots of total proteins extractedfrom leaves were probed immunologically to test for C4

enzymes and Rubisco LSU (Fig. 6). The molecularmasses of the immunoreactive bands corresponded to theexpected mass of the different polypeptides. The resultsshow a strong immunoreactive band for Rubisco LSU at56 kDa in all species. With antibodies to C4 aciddecarboxylases, there was immunolabelling for NAD-ME(65 kDa) in both cotyledons and leaves of B. ciliaris, nodetectable labelling for NADP-ME (62 kDa) (Fig. 6), andno labelling for phosphoenolpyruvate carboxykinase (notshown). As controls, the NAD-ME-type species Bienertiacycloptera shows labelling for NAD-ME and no labellingfor NADP-ME; the NADP-ME species Stipagrostisraddiana shows high labelling for NADP-ME and lowlabelling for NAD-ME. The C3 control Suaeda physo-phora has very low labelling for NAD-ME, no labellingfor NADP-ME, and low labelling of the other C4 cycleenzymes, PEPC and PPDK. Cotyledons and leaves of B.ciliaris had levels of labelling of PEPC and PPDKcorresponding to those of the C4 species B. cyclopteraand S. raddiana.

Discussion

Life form and morphology

Desert plants show great diversity in adaptation to harshenvironmental conditions (Fahn and Cutler, 1992; Gutter-man, 1994). The most important limiting factors in desertareas are deficiency in water and high temperature. The C4

type of photosynthesis is highly advantageous in suchhabitats, as it enables species to grow successfully in poorand dry soils due to their higher light, water and nitrogenuse efficiency, and higher productivity (Long, 1999).Blepharis ciliaris shows strong plasticity in life form,growing normally as an annual plant; but, in some habitatsit may be biennial, perennial, and even a small shrub, asreported in Oman (Vollesen, 2000). In Iran, it is restrictedto very hot and dry conditions, usually on stony desertswith very sparse vegetation. The unique germination inthis species is very effective for establishing itself indesert conditions. The bilocular capsules of the Acantha-ceae, in general, and Blepharis, in particular, explode andgerminate ;50 min after being exposed to moisture(Gutterman, 1972; Witztum and Schulgasser, 1995).The seeds of B. ciliaris are covered by integumentary

hairs. They occur on the abaxial sides of the cotyledons(Fig. 1C), and hydrate rapidly when exposed to a wetsubstrate, stand erect and reflexed, and orient the seed atan angle to the soil surface (Gutterman and Witztum,1977). Upon germination, the large, broad cotyledonleaves (Fig. 1D), which perform C4 photosynthesis,support the rapid growth and successful establishment ofthis species in the fragile desert ecosystem. A comparisonof plants cultivated under greenhouse conditions, withample water and nutrients and moderate temperature, withnaturally-grown plants in southern Iran, shows the strongphenotypic plasticity of this species in forming a highlybranched plant with long internodes under sufficient waterconditions (Fig. 1B), and a short-growing plant, with mostleaves replaced by strongly thorny bracts under dryconditions (Fig. 1A).The bracts are photosynthetically active, as indicated by

the presence of Kranz anatomy in bract lamina and spines(Fig. 3A–E), and the fact that starch is accumulated in Mand Kranz cells. The seeds and capsules are well-protectedfrom seed predation by the overlapping spiny bracts (Naritaand Wada, 1998). The coriaceous bracts hold an aerial poolof seed on dead plants. It flowers almost year long, stillhaving full flowers even in very hot months when mostannuals have already died, (see Fig. 1A, e.g. June and July).It tolerates long periods of drought; in southern Iran whereextensive droughts have occurred many species have diedout, whereas Blepharis successfully remains in many places.

Anatomy and photosynthetic type

The data presented in this paper, including leaf anatomy,d13C values, presence, and ultrastructure of M and Kranz

Fig. 6. Western blot analyses for PEPC, PPDK, NAD-ME, NADP-ME,and Rubisco using total proteins extracted from cotyledons and leavesof B. ciliaris, and leaves of B. cycloptera, S. raddiana, andS. physophora. Numbers to the left indicate molecular mass in kDa.

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cells, western blot, and immunocytochemical studies,collectively show that B. ciliaris is a C4 plant. This agreeswith previous reports of the occurrence of C4 photosyn-thesis in the genus Blepharis based on leaves havingatriplicoid-type Kranz anatomy and d13C values (Sankhlaet al., 1975; Raghavendra and Das, 1978; Muhaidat et al.,2007). In the eudicot C4 lineages, atriplicoid-type anatomyis the most common form; it has arisen independently in21 out of a total of 32 known C4 eudicot lineages,providing a striking example of extensive evolutionaryconvergence. This domination of atriplicoid leaf anatomyin C4 eudicots is suggested to be due to the prevalence oflaminate leaves in C3 ancestral taxa (Muhaidat et al.,2007). The three known C4 species in the genus,B. ciliaris, B. scindica, and B. attenuata, belong to sectionAcanthodium (Vollesen, 2000).In the present study, it is demonstrated that B. ciliaris

cotyledon leaves, normal leaves, and bracts all have Kranzanatomy, C4 photosynthesis, and show accumulation ofstarch (Figs 1E, 2A–O, 3A–E). The cotyledons and leaveshave atriplicoid-type Kranz anatomy and they are bifacial,in that beneath the epidermal layer of the adaxial surfacethere are chlorenchymatous palisade cells, VB with Kranzcells, and spongy parenchyma towards the abaxial surface.The cotyledons have 5–6 layers of spongy parenchymacells (perhaps contributing to storage of seed reservesto support the developing seedling), while the leaves have2–3 layers of spongy parenchyma cells. While thecotyledons have a C4-type carbon isotope value, it isa little more negative than that of leaves and stems whichsuggests cotyledons may be less efficient in capturingCO2 by the C4 cycle and transferring it to Rubisco. Bothbifacial and unifacial forms of atriplicoid-type leafanatomy occurs among C4 dicot species, see summary by(Muhaidat et al., 2007).As the mid-vein develops in leaves and becomes larger

near the base with multiple layers of parenchyma on theabaxial side, there is loss of Kranz anatomy and the abilityto function photosynthetically. Besides the decrease inKranz cells, in the surrounding chlorenchyma there is littleaccumulation of starch. This may be due to the diffusionallimitations for CO2 from the atmosphere to the VB,especially on the abaxial side of the leaf, which alsoappears to lack stomata. Thus, the basal midrib developsfor structural support of the leaf with a diminished role inphotosynthesis.The anatomy of the bracts and spines is more complex.

The lamina of bracts are oriented such that the adaxiallamina is facing the flowers and shielded from light whilethe abaxial side is more exposed to the atmosphere andlight. The anatomy of the bract lamina indicates thatchlorenchyma cells immediately surrounding the Kranzcells are the same on the abaxial or the adaxial sides(oval shaped with considerable intercellular air space,Fig. 3A–C), which is quite different from the anatomy in

cotyledons and leaves. The thin bract spines are orientedat an opposite angle to the bract lamina, so that theadaxial, concave, and lateral sides are exposed to sunlight.The presence of Kranz tissue in young spines (Fig. 3E),except at the lower-most end of the abaxial side (Fig. 3D),shows that they have a role in C4 photosynthesis.The absence of Kranz anatomy in the stem indicates

they lack C4 photosynthesis. The stems are not green andlack chlorenchyma cells (from light microscopy) indicat-ing they are not contributing to carbon assimilation. Inaddition, young stems of B. ciliaris, in natural conditions,are soon covered by the overlapping bracts (Fig. 1A),which practically prevents light being available forphotosynthesis. This is in contrast to many succulent,(semi-) aphyllous and articulated desert plants in whichthe stems are green and contribute to carbon assimilation,i.e. in many genera in Chenopodiaceae, Polygonaceae(Calligonum), Cactaceae, and Apocynaceae.C4 photosynthesis evolved from C3 species having

different types of leaf anatomies resulting in structurallydifferent forms of Kranz. In addition, within C4 species,there is also structural variation in Kranz anatomyoccurring in different photosynthetic organs, which isillustrated in the current study with B. ciliaris. Thisspecies performs C4 photosynthesis throughout its lifecycle through cotyledons, leaves, and bracts. The presenceof well-developed leaves at an earlier stage of the lifecycle, and extensively-developed bracts which subse-quently assume a major role in carbon assimilation duringa large portion of the life cycle, contribute to its success incarbon acquisition, enabling these species to establish andsurvive in deserts.

C4 biochemical subtype

Western blots on C4 acid decarboxylases show thatB. ciliaris is an NAD-ME-type C4 species. In bothcotyledons and leaves (Fig. 6), there is a strong immuno-reactive band with NAD-ME, and no detectable NADP-ME (also observed with a different set of antibodies, cour-tesy MV Lara). The results with B. ciliaris are the same aswith B. cycloptera, a known NAD-ME-type species(Voznesenskaya et al., 2002, 2005b), and unlike S. raddiana,a known NADP-ME-type genus (Voznesenskaya et al.,2005a). In addition, ultrastructural studies are consistentwith B. ciliaris being an NAD-ME-type C4 species, in thatthere are well-developed grana in chloroplasts of Kranzcells, and abundant mitochondria in the Kranz cells,which is characteristic of this subtype (Edwards andWalker, 1983).Earlier controversy about the nomenclature of Blepharis

resulted in later confusion over the correct naming ofB. ciliaris. Blepharis persica Burm., described fromsouthern Iran, is a homotypus synonym of B. ciliaris,which is referenced in Flora Iranica (Rechinger, 1966).Thus, the species used in this study is B. ciliaris.


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However, the name B. ciliaris was used erroneously invarious Middle Eastern literature and Floras (Gutterman,1972; Tackholm, 1974; Feinbrun-Dothan, 1978) for plantsgrowing in Egypt, Israel/Palestine, and Jordan, rather thanthe correct name, B. attenuata Napper. This also led to theerroneous naming in a recent publication by (Muhaidatet al., 2007), in which they characterized the type ofKranz anatomy in a number of eudicot families, includingB. attenuata (from Jordan) from family Acanthaceae,which they described under the name B. ciliaris.B. attenuata in that study, like B. ciliaris in the currentstudy, has atriplicoid Kranz-type anatomy. However, theyreported that B. attenuata is an NADP-ME-type speciesbased on assays of C4 acid decarboxylating enzymes inleaf extracts. Consistent results have been obtained in sub-typing of C4 species by immunodetection versus enzy-matic assay of C4 decarboxylases (Walker et al., 1997;Wingler et al., 1999; Voznesenskaya et al., 2002). Thus,the results with Blepharis suggest that two biochemicalsubtypes, NAD-ME and NADP-ME, may have evolvedamong C4 species in the genus. Since genus Blepharis isdiverse in having both C3 and C4 species, a combinedanalysis of photosynthetic types, anatomy, and biochemi-cal and ultrastructural studies for C4 subtyping in this andrelated genera would be of interest.

Acknowledgements

This research was made possible by support to H Akhani from theResearch Council, University of Tehran, for a sabbatical leave andthrough research project No. 6104037/1/01. It was also supportedin part by the National Science Foundation under Grant NosIBN-0236959 and IBN-0641232. We also thank the FranceschiMicroscopy and Imaging Center of Washington State University foruse of their facilities and staff assistance by Drs V Lynch-Holm andC Davitt, and E Voznesenskaya for her help at an early stage of thework.

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occurrence and forms of kranz anatomy in photosynthetic organs

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