JXB Advance Access published online on April 24, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern020
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RESEARCH PAPER |
Occurrence and forms of Kranz anatomy in photosynthetic organs and characterization of NAD-ME subtype C4 photosynthesis in Blepharis ciliaris (L.) B. L. Burtt (Acanthaceae)
1Department of Plant Sciences, School of Biology, College of Science, University of Tehran, PO Box 14155-6455, Tehran, Iran
2Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada
3School of Biological Sciences, Washington State University, Pullman, Washington, WA 99164-4236, USA
* To whom correspondence should be addressed. E-mail: akhani{at}khayam.ut.ac.in; edwardsg{at}wsu.edu
Received 17 October 2007; Revised 14 December 2007 Accepted 16 January 2008
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Abstract |
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Blepharis (Acanthaceae) is an Afroasiatic genus comprising 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 distribution in Iran, Oman, and Pakistan. Its form of photosynthesis 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 photosynthesis in cotyledons, leaves, and the lamina part of bracts. A continuous layer of distinctive bundle sheath cells (Kranz cells) encircle the vascular bundles in cotyledons 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 chlorenchyma. 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
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Introduction |
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Globally, 79% of all C4 plants belong to the monocots and only 21% belong to the dicots. C4 dicots have been found in seven orders, and the largest number of species has been discovered in the family Chenopodiaceae (Akhani et al., 1997; Sage et al., 1999; Jacobs, 2001; Kadereit et al., 2003; Sage, 2004). The most evolutionarily advanced C4 dicot lineages are found in three orders of Euasterids: Lamiales (Scrophulariaceae, Anticharis, Acanthaceae: Blepharis), Solanales (Boraginaceae, Heliotropium), and Asterales (Asteraceae: Flaveria) (Sage et 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 flora. Many C4 species and C4-dominated communities are known in these areas (Vogel and Seely, 1977; Werger and Ellis, 1981; Winter, 1981; Ziegler et al., 1981; Batanouny et al., 1988; Schulze et al., 1996; Akhani et al., 1997; Akhani and Ziegler, 2002; Sage, 2004). So far, in these geographical areas C4 plants have been discovered in 18 out of the 19 families which are known to contain C4 species. In the family Acanthaceae, C4 species have only been identified in the genus Blepharis. This is an intriguing genus which is composed of 129 species among three subgenera (Vollesen, 2000, 2004); with C4 species occurring in the subgenus Acanthodium (Sage, 2004, 2005; R Sage, personal communication). This is an Afroasiatic genus, which is widely distributed in tropical and southern Africa, southern parts of the Middle East and central Asia, India, southern China, with the occurrence of one species in Indonesia (Vollesen, 2000).
Blepharis ciliaris (L.) B. L. Burtt, is distributed in south and southeastern Iran, Pakistan, and Oman (see Map 21, p. 99 in Vollesen 2000). In this study, the anatomy and form of photosynthesis in various organs of this species was analysed to evaluate the adaptive value of these traits in hot deserts. Blepharis ciliaris is shown to be a C4 species; the occurrence and form of Kranz anatomy in cotyledons, leaves, and bracts was characterized and the biochemical subtype of C4 photosynthesis was determined.
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Materials and methods |
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Plant material and growth conditions
Seeds of B. ciliaris (L.) B. L. Burtt were collected from a population in Hormozgan Province, near Hajiabad in southern Iran on 13 June 2004. 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 at the Botanical Biodiversity Research Laboratory, School of Biology, University of Tehran. The seeds were initially germinated in February 2005, in Petri dishes at room temperature. After 2–3 d, the young seedlings were transplanted to 10 cm pots containing 10 parts commercial potting soil, one part clay, one part sand, and 100 grams gypsum, and grown for 3 weeks at room temperature under
50 µmol m–2 s–1 photosynthetic photon flux density (PPFD). After 3 weeks, the plants were transferred to a greenhouse and grown under natural sunlight plus supplemental light (sodium vapour lamps providing a PPFD of 400 µmol m–2 s–1) with a maximum midday PPFD on clear days of 1750 µmol m–2 s–1, a 14/10 h light/dark photoperiod and a 25±1 °C day and 18±0.5 °C night.
Light microscopy
Samples from cotyledon leaves, leaves (tip, middle, and base of very young and mature leaves), bracts spine and bracts lamina, and young stems were fixed in 2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), dehydrated with a graded ethanol series and embedded in London Resin White (LR White, Electron Microscopy Sciences, Fort Washington, PA, USA). Semi-thin cross-sections (about 0.8–1 µm thick), made on a Reichert Ultracut R ultramicrotome (Reichert-Jung GmbH, Heidelberg, Germany) were dried onto gelatin-coated slides and stained with 1% (w/v) Toluidine blue O in 1% (w/v) Na2B4O7 for general anatomy. The periodic acid–Schiff's procedure (PAS) was used for staining 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 in 2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C overnight, dehydrated in an acetone 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 and examined using a JEOL JEM-1200 EX (JEOL USA, Inc., Massachusetts, USA). Samples for scanning electron microscopy were 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 coated with gold, and observed on a Hitachi S570 scanning electron microscope (Hitachi Scientific Instruments, Mountain View California, USA).
Western blot analysis
Total proteins were extracted from cotyledons or leaves as described in Voznesenskaya et al. (2005a). Protein samples (10 µg) 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 (1:40 000; Chemicon, Temecula, CA, USA), Zea mays pyruvate,Pi dikinase (PPDK) (1:10 000; courtesy of T Sugiyama), Amaranthus hypochondriacus NAD-malic enzyme (NAD-ME) (1:5000; courtesy of J Berry), or Zea mays NADP-malic enzyme (NADP-ME) (1:5000; courtesy of C Andreo). Goat anti-rabbit IgG-alkaline phosphatase conjugate (dilution of 1:50 000) was used as the secondary antibody for detection of the enzymes. Blots were developed with 350 µg ml–1 nitroblue tetrazolium and 175 µg 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 and 1.25% (v/v) glutaraldehyde in 50 mM PIPES buffer, pH 7.2, dehydrated with a graded ethanol series and embedded in London Resin White (LR White, Electron Microscopy Sciences, Fort Washington, PA, USA) acrylic resin. Antibodies used were anti-Spinacea oleracea L. Rubisco (LSU) and commercially available anti-Zea mays L. PEPC IgG. Non-immune serum was used for controls. Cross-sections, 0.8–1 µm thick, were dried in a drop of water 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/v BSA, pH 7.2]. They were then incubated for 3 h with either the non-immune serum diluted in TBST+BSA (1:100), anti-Rubisco (1:500 dilution) or anti-PEPC (1:200 dilution), treated for 1 h with protein A-gold (10 nm particles diluted 1:100 with TBST+BSA) and exposed to a silver enhancement reagent for 20 min according to the manufacturer's directions (Amersham, Arlington Heights, IL, USA). The sections were stained with 0.5% (w/v) Safranin O and imaged in a reflected/transmitted mode using a Bio-Rad 1024 confocal system (Bio-Rad, Hercules, CA, USA). The background labelling with non-immune serum was very low, as demonstrated previously with these antibodies. Images were processed using Adobe Photoshop image processing software (Version 6.0, Adobe System Inc., San Jose, CA, USA).
Carbon isotope composition
Measures of the carbon isotope composition (
13C values), were determined at Washington State University on leaf, bracts and stem samples taken from plants grown under a controlled environment, and from leaves of plants collected in a natural habitat from Southern Iran, using a standard procedure relative to PDB (Pee Dee Belemnite) limestone as the standard (Bender, 1971).
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Habitat and general morphology
A living form of the species in its natural habitat in southern Iran (Fig. 1A), and its form in cultivation is illustrated in Fig. 1B. The seeds, 9–11 mm in length (L)x4–5 mm in width (W), germinate within a few hours after moisture is provided. After 3–4 d, the ovate cotyledon leaves (WxL of 5–6 mm) become green and glabrous on the adaxial surface (Fig. 1C). As the cotyledons enlarge, the anchoring hairs on the abaxial surface shed and, eventually, the two reniform cotyledon leaves reach their maximum size (up to 11 mm Lx16 mm W), with a well-developed petiole up to 15 mm L (Fig. 1D). By the time the cotyledon leaves are mature, the lanceolate seedling leaves have developed. The plants become mature and produce flowers after 3.5 months (Fig. 1B).
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The mature leaves are lanceolate, sub-sessile to shortly petiolate (5–8.5 cm Lx1–1.5 cm W), and bi-coloured with a shiny dark-green upper surface and a whitish-green lower surface. The trichomes on the adaxial leaf side are sparser and shorter than those on the abaxial side (Fig. 1F). A few sub-sessile, 4-celled glands are intermixed with trichomes (Fig. 1G, H). Such glands have been reported in many genera of Acanthaceae (Ahmad, 1978). The main difference between glandular hairs and long trichomes is that glands are located in depressed parts of the leaf, while the trichomes are located on elevated epidermal cells (Fig. 1G, H). The diacytic stomata on the abaxial leaf surface are very dense, and 3.6 times more prevalent than on the adaxial surface. The overlapping bracts (18–20 mm Lx6–9 mm W) have a terminal spine up to 30 mm L and 2–3 pairs of lateral spines up to 5 mm L (Fig. 1A). The trichomes, on the spines, which are up to 1.25 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 13C values of different organs of greenhouse-grown plants and of leaves of plant grown in a natural environment in southern Iran were analysed. Under the greenhouse growth conditions, cotyledons had the most negative 13C value, –15.7°/oo, while mature leaves had a value of –14.1°/oo, and bracts and stems had values of –13.1°/oo and –13.0°/oo, respectively. Mature leaves collected from plants grown in the natural habitat had a value of –13.4°/oo.
Anatomy
Cotyledon leaves
The cotyledon leaves are bifacial, having a layer of palisade cells beneath the adaxial epidermis and 5–6 layers of chloroplast containing spongy, rounded to oval, mesophyll (M) cells towards the abaxial side of the leaf. There is considerable air space around the spongy M tissue which occupies a large volume of the cotyledon section. The cotyledons leaves have atriplicoid type Kranz anatomy. The vascular bundles (VB) are surrounded by a continuous layer of Kranz cells with centripetal chloroplasts (Fig. 1E), with another layer of palisade-like chlorenchyma external to the Kranz cells. Between veins, there are a few rounded or variously shaped chlorenchymatous cells.
Young leaves
The anatomy of young leaves (7–10 mm Lx1 mm W) was studied in three sequential parts, from the 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, including a central VB and 10 less prominent lateral VB. The general anatomy is similar to cotyledon leaves, being bifacial in cross-section and having atriplicoid-type Kranz anatomy. There is generally only one layer of palisade cells on the adaxial side except near the mid-vein. The Kranz cells are surrounded by palisade cells on the adaxial side, and by small chlorenchyma cells on the abaxial side and between VB (Fig. 2A, D). On the abaxial side there are 2–3 layers of spongy parenchyma with few chloroplasts, and considerable intercellular air space. In the tip of the leaf, all VB are completely surrounded by a layer of Kranz cells. PAS staining of polysaccharides showed dense starch grains in the Kranz cells and sparser grains in the palisade and spongy chlorenchyma cells.
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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 by Kranz cells, the Kranz layer around the midrib is interrupted on the abaxial side of the leaves (Fig. 2B, E). PAS staining of polysaccharides showed a denser starch accumulation in Kranz cells than in the surrounding chlorenchyma 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 and glandulose trichomes (Fig. 2C, F). In the midrib, the Kranz cells appear only on the adaxial side and occupy only half of the VB. The Kranz cells in lateral veins are less prominent than in the middle and tip sections of the leaf and show a lower accumulation of starch. Some starch grains appear in Kranz cells around the mid-vein (Fig. 2F), but they are not as dense as in the Kranz cells of the lateral veins. Starch grains are evenly distributed throughout the chlorenchyma tissue.
Mature leaves
Mature leaves (55 mm Lx12 mm W) were studied in three parts, including the tip, middle, and basal sections (Fig. 2G–O). The general structure of mature leaves is similar to young leaves, with respect to having atriplicoid-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). On the 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 of small chlorenchyma cells with a more oblate shape adjacent to the Kranz cells, and two or three layers of large spongy parenchyma cells, with large intercellular airspaces, 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 chloroplasts than in the middle and basal parts. The Kranz cells of leaf tips are smaller in size and are largely occupied by chloroplasts, while those in the middle and base are concentrated centripetally and occupy only about one-third of the volume of rather larger cells. PAS staining of polysaccharides shows a rather strong accumulation of starch in Kranz cells and surrounding chlorenchyma in the leaf tip, moderate accumulation in the middle, and much lower 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 is interrupted by parenchymatous cells on the abaxial half of the midrib volume (Fig. 2G, J). In the middle of the leaf there is a further reduction in the development of Kranz cells around the mid-vein with only a few Kranz cells on the adaxial side (Fig. 2H, K). Then, in the basal section, Kranz cells are completely absent around the mid-vein; rather, there is a layer of very small bundle sheath cells, containing very few chloroplasts, around the vascular tissue which, in turn, is surrounded by ground tissue (Fig. 2I, L). Also, around the leaf mid-vein, the appearance of starch decreases from the tip towards the basal part of the leaf, which parallels the decrease in development of Kranz cells around the vein (Fig. 2J–L).
Bracts
Under natural conditions, bracts appear soon after senescence and demarcation of leaves, and at the beginning of periods of reproductive development (Fig. 1A, B). The bracts produce a very long terminal spine (up to 3 cm), and two pairs of lateral spines, of which the upper pair is longer than the lower pair (Fig. 1A). Bracts, which are much smaller than leaves, have a well-developed central midrib, with two parallel lateral veins that terminate in the upper pair of lateral spines, and two marginal veins which terminate in the lower spine pair. The most obvious difference between bracts and leaves is the mode of orientation to sunlight (Fig. 1A). While the adaxial surfaces of leaves are exposed to light, the adaxial sides of bracts are shielded from light by their orientation and coverage by flowers while the abaxial side of bracts is more exposed to direct light. The spines are triangular in cross-section, being convex on the dorsal (abaxial) and slightly concave on the ventral (adaxial) side (Fig. 3D, E).
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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). In contrast to bifacial leaves, in which the palisade parenchyma on the adaxial side is well-differentiated from spongy parenchyma on the abaxial side, those of the bracts are more or less uniform. On both sides of the bracts, there are c. three layers of chlorenchymatous cells around the Kranz cells, of which the innermost layer on the abaxial surface is slightly more elongated than the two outer layers. The association of considerable air space on the adaxial side of the marginal vein shows that these cells, though less distinct, are comparable to the spongy chlorenchyma of the abaxial side of leaves. PAS staining for polysaccharides showed a denser starch accumulation in photosynthetic cells in the abaxial than those in the adaxial sides (Fig. 3C).
A cross-section of young spines show distinctive Kranz anatomy with a layer of chlorenchyma cells surrounding the Kranz cells on the adaxial side; and there are one or two additional layers of large parenchyma cells next to the epidermis. The density of chloroplasts (Fig. 3E) and the dense staining of polysaccharides (not shown) in the adaxial edges of the spine suggest that it is more photosynthetically active than other regions. On the abaxial side of the spine, Kranz anatomy is interrupted at the apex where there is lack of chlorenchyma tissue (Fig. 3D); with several layers of large parenchyma cells between the epidermis and small bundle sheath cells.
Young stems
The young stems, which are circular in cross-section, are composed of an epidermal layer which is underlain by several layers of small, rounded parenchyma cells, and c. 6 layers of cortical parenchyma cells and a central vascular cylinder. Toluidine blue staining (Fig. 3F) shows that there are no Kranz cells and no apparent chlorenchyma tissue. The vascular cylinder of the stem is associated with a cambial ring.
Biochemical features and ultrastructure of mature leaves
Studies on mature leaves were conducted to characterize the form of C4 photosynthesis in B. ciliaris, including immunolocalization of PEPC and Rubisco, levels of photosynthetic enzymes by western blots, and ultrastructure of Kranz anatomy.
Immunolocalization
The middle part of mature leaf blades (50 mm Lx10 mm W) was used for analysis of immunolocalization of Rubisco and PEPC. There was strong labelling for Rubisco LSU in chloroplasts in Kranz cells. There is labelling for PEPC in the layer of chlorenchyma cells immediately surrounding the Kranz cells, but little or no labelling was detected in Kranz cells, or the spongy parenchyma towards the abaxial side of the leaf (Fig. 4).
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Kranz ultrastructure
TEM results from samples taken from the middle part of mature leaves show the distribution of a few chloroplasts along the peripheral walls of M cells, and the dense Kranz chloroplasts arranged centripetally towards the VB. Both M (Fig. 5A, B) and Kranz chloroplasts (Fig. 5C–E) are rather similar in their granal stacking. They usually form small grana consisting of 2–5 aggregated thylakoids (Fig. 5B–E). Measurements were made on the average number of thylakoids per granum (n=50); M chloroplasts had 3.1 thylakoids per granum (83% of the grana had 2–3 thylakoids per granum), while Kranz chloroplasts had 3.4 thylakoids per granum (68% had 2–3 thylakoids per granum). The TEM ultrastructure study also reveals that there are more mitochondria in the Kranz cells which are associated with chloroplasts than in M cells (Fig. 5C, D). The chloroplasts are dimorphic in that those in Kranz cells store starch and are larger than M chloroplasts.
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Western blots
SDS-PAGE blots of total proteins extracted from leaves were probed immunologically to test for C4 enzymes and Rubisco LSU (Fig. 6). The molecular masses of the immunoreactive bands corresponded to the expected mass of the different polypeptides. The results show a strong immunoreactive band for Rubisco LSU at 56 kDa in all species. With antibodies to C4 acid decarboxylases, there was immunolabelling for NAD-ME (65 kDa) in both cotyledons and leaves of B. ciliaris, no detectable labelling for NADP-ME (62 kDa) (Fig. 6), and no labelling for phosphoenolpyruvate carboxykinase (not shown). As controls, the NAD-ME-type species Bienertia cycloptera shows labelling for NAD-ME and no labelling for NADP-ME; the NADP-ME species Stipagrostis raddiana shows high labelling for NADP-ME and low labelling for NAD-ME. The C3 control Suaeda physophora has very low labelling for NAD-ME, no labelling for NADP-ME, and low labelling of the other C4 cycle enzymes, PEPC and PPDK. Cotyledons and leaves of B. ciliaris had levels of labelling of PEPC and PPDK corresponding to those of the C4 species B. cycloptera and S. raddiana.
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Life form and morphology
Desert plants show great diversity in adaptation to harsh environmental conditions (Fahn and Cutler, 1992; Gutterman, 1994). The most important limiting factors in desert areas are deficiency in water and high temperature. The C4 type of photosynthesis is highly advantageous in such habitats, as it enables species to grow successfully in poor and dry soils due to their higher light, water and nitrogen use efficiency, and higher productivity (Long, 1999). Blepharis ciliaris shows strong plasticity in life form, growing normally as an annual plant; but, in some habitats it may be biennial, perennial, and even a small shrub, as reported in Oman (Vollesen, 2000). In Iran, it is restricted to very hot and dry conditions, usually on stony deserts with very sparse vegetation. The unique germination in this species is very effective for establishing itself in desert conditions. The bilocular capsules of the Acanthaceae, in general, and Blepharis, in particular, explode and germinate
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 wet substrate, stand erect and reflexed, and orient the seed at an angle to the soil surface (Gutterman and Witztum, 1977). Upon germination, the large, broad cotyledon leaves (Fig. 1D), which perform C4 photosynthesis, support the rapid growth and successful establishment of this species in the fragile desert ecosystem. A comparison of plants cultivated under greenhouse conditions, with ample water and nutrients and moderate temperature, with naturally-grown plants in southern Iran, shows the strong phenotypic plasticity of this species in forming a highly branched plant with long internodes under sufficient water conditions (Fig. 1B), and a short-growing plant, with most leaves replaced by strongly thorny bracts under dry conditions (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 M and Kranz cells. The seeds and capsules are well-protected from seed predation by the overlapping spiny bracts (Narita and Wada, 1998). The coriaceous bracts hold an aerial pool of seed on dead plants. It flowers almost year long, still having full flowers even in very hot months when most annuals have already died, (see Fig. 1A, e.g. June and July). It tolerates long periods of drought; in southern Iran where extensive droughts have occurred many species have died out, whereas Blepharis successfully remains in many places.
Anatomy and photosynthetic type
The data presented in this paper, including leaf anatomy, 13C values, presence, and ultrastructure of M and Kranz cells, western blot, and immunocytochemical studies, collectively show that B. ciliaris is a C4 plant. This agrees with previous reports of the occurrence of C4 photosynthesis in the genus Blepharis based on leaves having atriplicoid-type Kranz anatomy and 13C values (Sankhla et al., 1975; Raghavendra and Das, 1978; Muhaidat et al., 2007). In the eudicot C4 lineages, atriplicoid-type anatomy is the most common form; it has arisen independently in 21 out of a total of 32 known C4 eudicot lineages, providing a striking example of extensive evolutionary convergence. This domination of atriplicoid leaf anatomy in C4 eudicots is suggested to be due to the prevalence of laminate 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 section Acanthodium (Vollesen, 2000).
In the present study, it is demonstrated that B. ciliaris cotyledon leaves, normal leaves, and bracts all have Kranz anatomy, C4 photosynthesis, and show accumulation of starch (Figs 1E, 2A–O, 3A–E). The cotyledons and leaves have atriplicoid-type Kranz anatomy and they are bifacial, in that beneath the epidermal layer of the adaxial surface there are chlorenchymatous palisade cells, VB with Kranz cells, and spongy parenchyma towards the abaxial surface. The cotyledons have 5–6 layers of spongy parenchyma cells (perhaps contributing to storage of seed reserves to support the developing seedling), while the leaves have 2–3 layers of spongy parenchyma cells. While the cotyledons have a C4-type carbon isotope value, it is a little more negative than that of leaves and stems which suggests cotyledons may be less efficient in capturing CO2 by the C4 cycle and transferring it to Rubisco. Both bifacial and unifacial forms of atriplicoid-type leaf anatomy 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 the abaxial side, there is loss of Kranz anatomy and the ability to function photosynthetically. Besides the decrease in Kranz cells, in the surrounding chlorenchyma there is little accumulation of starch. This may be due to the diffusional limitations for CO2 from the atmosphere to the VB, especially on the abaxial side of the leaf, which also appears to lack stomata. Thus, the basal midrib develops for structural support of the leaf with a diminished role in photosynthesis.
The anatomy of the bracts and spines is more complex. The lamina of bracts are oriented such that the adaxial lamina is facing the flowers and shielded from light while the abaxial side is more exposed to the atmosphere and light. The anatomy of the bract lamina indicates that chlorenchyma cells immediately surrounding the Kranz cells 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 oriented at an opposite angle to the bract lamina, so that the adaxial, 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 and lack chlorenchyma cells (from light microscopy) indicating they are not contributing to carbon assimilation. In addition, young stems of B. ciliaris, in natural conditions, are soon covered by the overlapping bracts (Fig. 1A), which practically prevents light being available for photosynthesis. This is in contrast to many succulent, (semi-) aphyllous and articulated desert plants in which the 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 structurally different forms of Kranz. In addition, within C4 species, there is also structural variation in Kranz anatomy occurring in different photosynthetic organs, which is illustrated in the current study with B. ciliaris. This species performs C4 photosynthesis throughout its life cycle through cotyledons, leaves, and bracts. The presence of well-developed leaves at an earlier stage of the life cycle, and extensively-developed bracts which subsequently assume a major role in carbon assimilation during a large portion of the life cycle, contribute to its success in carbon acquisition, enabling these species to establish and survive in deserts.
C4 biochemical subtype
Western blots on C4 acid decarboxylases show that B. ciliaris is an NAD-ME-type C4 species. In both cotyledons and leaves (Fig. 6), there is a strong immunoreactive band with NAD-ME, and no detectable NADP-ME (also observed with a different set of antibodies, courtesy MV Lara). The results with B. ciliaris are the same as with 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 consistent with B. ciliaris being an NAD-ME-type C4 species, in that there are well-developed grana in chloroplasts of Kranz cells, and abundant mitochondria in the Kranz cells, which is characteristic of this subtype (Edwards and Walker, 1983).
Earlier controversy about the nomenclature of Blepharis resulted in later confusion over the correct naming of B. ciliaris. Blepharis persica Burm., described from southern 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. However, the name B. ciliaris was used erroneously in various Middle Eastern literature and Floras (Gutterman, 1972; Täckholm, 1974; Feinbrun-Dothan, 1978) for plants growing in Egypt, Israel/Palestine, and Jordan, rather than the correct name, B. attenuata Napper. This also led to the erroneous naming in a recent publication by (Muhaidat et al., 2007), in which they characterized the type of Kranz anatomy in a number of eudicot families, including B. attenuata (from Jordan) from family Acanthaceae, which they described under the name B. ciliaris. B. attenuata in that study, like B. ciliaris in the current study, has atriplicoid Kranz-type anatomy. However, they reported that B. attenuata is an NADP-ME-type species based on assays of C4 acid decarboxylating enzymes in leaf extracts. Consistent results have been obtained in sub-typing of C4 species by immunodetection versus enzymatic assay of C4 decarboxylases (Walker et al., 1997; Wingler et al., 1999; Voznesenskaya et al., 2002). Thus, the results with Blepharis suggest that two biochemical subtypes, NAD-ME and NADP-ME, may have evolved among C4 species in the genus. Since genus Blepharis is diverse in having both C3 and C4 species, a combined analysis of photosynthetic types, anatomy, and biochemical and ultrastructural studies for C4 subtyping in this and related genera would be of interest.
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Acknowledgements |
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This research was made possible by support to H Akhani from the Research Council, University of Tehran, for a sabbatical leave and through research project No. 6104037/1/01. It was also supported in part by the National Science Foundation under Grant Nos IBN-0236959 and IBN-0641232. We also thank the Franceschi Microscopy and Imaging Center of Washington State University for use of their facilities and staff assistance by Drs V Lynch-Holm and C Davitt, and E Voznesenskaya for her help at an early stage of the work.
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Abbreviations |
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13C 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. |
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