Journal of Experimental Botany, Vol. 62, No. 9, pp. 3197–3212, 2011 doi:10.1093/jxb/err021 Advance Access publication 16 February, 2011

RESEARCH PAPER Development of structural and biochemical characteristics of C4 photosynthesis in two types of Kranz anatomy in genus Suaeda (family Chenopodiaceae)

Nuria K. Koteyeva1, Elena V. Voznesenskaya1, James O. Berry2, Simon D. X. Chuong3,†, Vincent R. Franceschi3 and Gerald E. Edwards3,* 1 Laboratory of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences, Professor Popov Street 2, 197376, St Petersburg, Russia 2

Department of Biological Sciences, State University of New York, Buffalo, NY 14260, USA Downloaded from 3 School of Biological Sciences, Washington State University, Pullman, WA 9164-4236, USA y Present address: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada * To whom correspondence should be addressed. E-mail: [email protected]

Received 15 November 2010; Revised 14 January 2011; Accepted 19 January 2011 http://jxb.oxfordjournals.org/

Abstract Genus Suaeda (family Chenopodiaceae, subfamily Suaedoideae) has two structural types of Kranz anatomy consisting of a single compound Kranz unit enclosing vascular tissue. One, represented by Suaeda taxifolia, has mesophyll (M) and bundle sheath (BS) cells distributed around the leaf periphery. The second, represented by

Suaeda eltonica, has M and BS surrounding vascular bundles in the central plane. In both, structural and at OUP site access on April 22, 2013 biochemical development of C4 occurs basipetally, as observed by analysis of the maturation gradient on longitudinal leaf sections. This progression in development was also observed in mid-sections of young, intermediate, and mature leaves in both species, with three clear stages: (i) monomorphic chloroplasts in the two cell types in younger tissue with immunolocalization and in situ hybridization showing ribulose bisphosphate carboxylase oxygen- ase (Rubisco) preferentially localized in BS chloroplasts, and increasing in parallel with the establishment of Kranz anatomy; (ii) vacuolization and selective organelle positioning in BS cells, with occurrence of phosphoenolpyruvate carboxylase (PEPC) and immunolocalization showing that it is preferentially in M cells; (iii) establishment of chloroplast dimorphism and mitochondrial differentiation in mature tissue and full expression of C4 biochemistry including pyruvate, Pi dikinase (PPDK) and NAD-malic enzyme (NAD-ME). Accumulation of rbcL mRNA preceded its peptide expression, occurring prior to organelle positioning and differentiation. During development there was sequential expression and increase in levels of Rubisco and PEPC followed by NAD-ME and PPDK, and an increase in the 13C/12C isotope composition of leaves to values characteristic of C4 photosynthesis. The findings indicate that these two forms of NAD-ME type C4 photosynthesis evolved in parallel within the subfamily with similar ontogenetic programmes.

Key words: C3 plants, C4 plants, Chenopodiaceae, chloroplast ultrastructure, immunolocalization, in situ mRNA hybridization, NAD-ME type, photosynthetic enzymes, Suaeda.

Introduction

C4 photosynthesis evolved as a means of increasing the would otherwise be limiting for photosynthesis. Limitations supply of CO2 to ribulose bisphosphate carboxylase oxy- can occur by reduced stomatal conductance (e.g. in re- genase (Rubisco) and the C3 cycle under conditions that sponse to a water deficit), and by warm temperatures where

Abbreviations: BS, bundle sheath.d13C values, a measure of the carbon isotope composition; LSU, large subunit; M, mesophyll; NAD-ME, NAD-malic enzyme; NADP- ME, NADP-malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PEP-CK, phosphoenolpyruvate carboxykinase; PPDK, pyruvate, Pi dikinase; PPFD, photosynthetic photon flux density; RT, room temperature; Rubisco, ribulose bisphosphate carboxylase oxygenase; SEM, scanning electron microscope/microscopy; TEM, transmission electron microscope/microscopy. ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 3198 | Koteyeva et al. photorespiration and the Rubisco oxygenase/carboxylase a compound Kranz unit with two layers of chlorenchyma activity ratio is increased. characteristic of species with C4 photosynthesis, BS and M C4 plants evolved independently multiple times, with cells, occurring at the leaf periphery surrounding water convergence in a number of specialized features. These storage tissue in which the vascular bundles are embedded. shared features include fixation of atmospheric CO2 into C4 Schoberia type leaves have a compound Kranz unit acids within mesophyll (M) cells through phosphoenolpyr- characterized by two concentric layers of chlorenchyma uvate carboxylase (PEPC), and decarboxylation of C4 acids cells, BS and M, encircling the vascular bundles, and by and donation of released CO2 to Rubisco and the C3 cycle a layer of enlarged water storage hypodermal cells just within bundle sheath (BS) cells. Common to this process is beneath the epidermis. The chloroplasts in BS cells are the selective expression of the carboxylation phase of C4 located in the centripetal position in the Salsina type, and in enzymes in the M cells, with Rubisco and other C3 cycle the centrifugal position in the Schoberia type (Freitag and enzymes accumulating in the chloroplasts, and glycine Stichler, 2000; Schu¨tze et al., 2003; Voznesenskaya et al., decarboxylase in the mitochondria, of the BS. This pathway 2007). Transmission electron microscopy (TEM) shows that is also correlated with distinctive cellular structural features chlorenchyma cells in species representing the two different that provide diffusive resistance to leakage of CO2, so that C4 anatomical types have features characteristic of NAD- it can be effectively donated from the C4 cycle to Rubisco ME type C4 chenopods: the M cells contain chloroplasts (Edwards and Walker, 1983; Hatch, 1987; Sage, 2004). with reduced grana, while BS cells have chloroplasts with The independent evolution of C4 photosynthesis from C3 well-developed grana and large, specialized mitochondria ancestors across 19 different plant lineages has led to (Fisher et al., 1997; Voznesenskaya et al., 2007). Biochem- Downloaded from considerable diversity in biochemistry, with three types of ical and physiological studies show that the availability and C4 cycle operating through the C4 decarboxylases, NADP- selective immunolocalization of key photosynthetic malic enzyme (NADP-ME), NAD-malic enzyme (NAD- enzymes, CO2 compensation points, photosynthetic re- ME), and phosphoenolpyruvate carboxykinase (PEP-CK), sponse curves to varying CO2 and light were similar for as well as diversity in structural features. There are at least Salsina and Schoberia anatomical types, and these were http://jxb.oxfordjournals.org/ 25 forms of Kranz anatomy, and two forms of single-cell C4 typical for C4 plants (Voznesenskaya et al., 2007; Smith without Kranz (Edwards and Voznesenskaya, 2011). The et al., 2009). forms of Kranz anatomy can be classified into two major The establishment of complex C4 systems requires highly groups: one in which two layers of Kranz chlorenchyma coordinated expression of many genes during development, are formed around each vein (multiple Kranz units per under the control of regulatory factors that must function leaf); the other in which layers of Kranz chlorenchyma concurrently with differentiation of Kranz anatomy surround all the veins, forming a single compound Kranz (Hibberd and Covshoff, 2010; Berry et al., 2011). Studies at OUP site access on April 22, 2013 unit, according to the classification of Peter and Katinas on development of leaf structure relative to enzyme (2003). Current information, though limited, indicates that expression have been performed on a limited number of C4 there is also diversity in the ways different enzymes were species belonging to different structural and biochemical recruited during evolution to function in the C4 cycles, and subtypes. Except for Salsola richteri, which has a structural in the mechanism leading to control of selective expres- form of C4 called Salsoloid type with a compound Kranz sion of C4 in M versus BS cells, e.g. transcriptional, post- unit and NADP-ME biochemistry (Voznesenskaya et al., transcriptional, translation control (Hibberd and Covshoff, 2003b), most studies on C4 development have been on C4 2010; Berry et al., 2011; Gowik and Westhoff, 2011). species that have multiple simple Kranz units with chloren- The family Chenopodiaceae includes many species, with chyma surrounding individual veins. Among the eudicots great diversity in forms of C4 and C3 photosynthesis, studied this includes Atriplex rosea (Dengler et al., 1995) including different anatomical, physiological, and biochem- and Amaranthus hypochondriacus (Ramsperger et al., 1996), ical features. Subfamily Suaedoideae is especially interesting which have atriplicoid type leaf anatomy and the NAD-ME as among ;85 species there are four structural forms of C4 biochemical subtype. In monocots, this includes develop- (two different forms having a compound Kranz unit, and mental studies on Arundinella hirta (Wakayama et al., 2003) two single-cell forms without Kranz anatomy, all NAD-ME and Zea mays (Langdale et al., 1988a; Brutnell et al., 1999; biochemical subtype), as well as C3 species (Kadereit et al., Covshoff et al., 2008; Majeran et al., 2010), which belong to 2003; Schu¨tze et al., 2003; Voznesenskaya et al., 2007). In the NADP-ME type in family Poaceae, and on NADP-ME a classification organized according to sections within the and NAD-ME representatives of the family Cyperaceae genus Suaeda, three different types of anatomy were (Soros and Dengler, 2001). All Kranz type C4 plants studied recognized among C3 species (Brezia, Vera, and Schanginia), are characterized by a gradual pattern of anatomical and two C4 Kranz types named Salsina and Schoberia transition from morphologically similar chlorenchyma cells (Schu¨tze et al., 2003). to structural and biochemical dimorphism between BS and The leaves of the two Kranz type Suaeda have a similar M cells (Liu and Dengler, 1994; Dengler et al., 1996; distribution of the vascular network. However, they differ Voznesenskaya et al., 2003a, b; Wakayama et al., 2003). in the arrangement of chlorenchyma and water storage Structural differentiation of M and BS cells is closely tissue relative to the vascular bundles, as well as in the associated with the C4 gene and enzyme expression, positioning of chloroplasts in BS cells. Salsina type has although some differences have been reported in temporal C4 photosynthesis in the genus Suaeda | 3199 relations between the level of chlorenchyma structural humidity. For microscopy and biochemical analyses, leaf differentiation, the establishment of C4 enzyme expression, samples of different lengths (see below) were taken from and their distribution within the two cell types (Wang et al., vegetative branches on the plants that were ;30–45 d old. 1992; Dengler et al., 1995; Soros and Dengler, 2001). These As described in the Results, three stages of development of processes are often found to be dependent on environmental photosynthetic cells were established from studies on (e.g. light) and/or developmental factors, and it has been longitudinal sections of individual leaves (Figs 1, 5, 6, 8), suggested that positional information during vascular tissue and analyses of leaves of different ages (Table 1, Figs 2–4, differentiation may control expression of Kranz around 7). Voucher specimens are available at the Marion Ownbey veins (Langdale et al., 1988a; Dengler et al., 1995; Hibberd Herbarium, Washington State University: S. eltonica (E. and Covshoff, 2010). Voznesenskaya 38), April 2006, WS 369797 and S. taxifolia The purpose of this study was to characterize and compare (E. Voznesenskaya 39) July 2006, WS 369802. the structural and biochemical development of photosynthe- sis in two C4 representatives of the genus Suaeda, Suaeda Sample preparation for light and electron microscopy taxifolia (section Salsina) and Suaeda eltonica (section Schoberia). The anatomical and ultrastructural features, Study of C4 anatomy during leaf development was carried accumulation of key photosynthetic enzymes, and carbon out on expanding leaves with lengths from 0.1 to 0.7 cm, and isotope discrimination at different stages of leaf development on mature leaves after full expansion. For structural studies, are described. In addition, the cellular compartmentation of three samples of each leaf length were harvested from three Downloaded from the BS and M cell C4 marker enzymes, Rubisco large subunit independent plants of each species (a total of nine samples (LSU) and PEPC respectively, were investigated immunohis- from each plant for each leaf length). Cross-sections were tochemically, together with immunogold labelling TEM, to made in the middle part of leaves that were young (0.2–0.3 evaluate the relative amount of these peptides in different cell cm), intermediate (0.5–0.7 cm), or mature, fully expanded (2– types and at different stages of leaf development. In situ 3 cm). Vegetative shoot apices with several of the youngest mRNA hybridization procedure was used to reveal temporal leaf primordia, and intermediate age leaves (0.5–0.7 cm in http://jxb.oxfordjournals.org/ and spatial patterns of Rubisco LSU gene expression in these length), were also sectioned longitudinally. Samples for two C4 species. Unlike previous studies on C4 development structural studies were fixed at 4 Cin2%(v/v)para- where Kranz anatomy occurs around individual veins, this is formaldehyde and 2% (v/v) glutaraldehyde in 0.1 M phos- the first comprehensive study of C4 development in two phate buffer (pH 7.2), postfixed in 2% (w/v) OsO4, and then, forms of Kranz that have a single compound Kranz unit after a standard acetone dehydration procedure, embedded enclosing all the vascular tissue. It was shown that the in Spurr’s epoxy resin. Reichert Ultracut R ultramicrotome pattern of C4 differentiation was not affected by vascular (Reichert-Jung GmbH, Heidelberg, Germany) was used for at OUP site access on April 22, 2013 tissue positioning relative to BS, or by differences in the sectioning. For light microscopy, semi-thin sections were origin of BS cells. Spatial and temporal patterns of Rubisco stained with 1% (w/v) Toluidine blue O in 1% (w/v) LSU transcript and peptide accumulation indicate that Na2B4O7, and studied under an Olympus BH-2 (Olympus control of transcript levels, by transcription or mRNA Optical Co. Ltd) light microscope equipped with LM Digital stability, has a role in determining very early C4 development Camera and Software (Jenoptik ProgRes Camera, C12plus, (before differentiation and positioning of BS chloroplasts). Jena, Germany). For TEM, ultra-thin cross-sections were stained with 4% (w/v) uranyl acetate followed by 2% (w/v) lead citrate. Hitachi H-600 and JEOL JEM-1200 EX TEMs Material and methods were used for observation and photography. Plant material For scanning electron microscopy (SEM), shoot apices with the youngest leaf primordia were fixed as described Seeds of two species of genus Suaeda, S. eltonica Iljin above for structural studies, postfixed in 2% (w/v) OsO4, (provided by H. Freitag from W. Kazakhstan, 28.237, and then dehydrated in an ethanol series to 100% ethanol, KAS) and S. taxifolia Standley (provided by Dr J. Thorsch, critical-point dried, attached to SEM mounts, sputter- University of California Santa Barbara, CA, USA) were coated with gold, and observed with a Hitachi S570 SEM stored at 3–5 C prior to use. Seeds were germinated on (Hitachi Ltd, Tokyo, Japan). moist paper in Petri dishes at room temperature at To observe formation of the leaf vascular pattern, leaves a photosynthetic photon flux density (PPFD) of 20 lmol of different ages, from youngest primordia to fully ex- photosynthetic quanta m2 s1. The seedlings were then panded leaves, were cleared in 70% ethanol (v/v) until transplanted to 10 cm diameter pots with commercial chlorophyll was removed, bleached with 5% (w/v) NaOH potting soil and grown for 3 d under 30 PPFD and 25/15 C overnight, and then rinsed three times in water. At least five day/night temperature regime. Established plants were then vegetative shoot tips with different-aged leaves from two or transferred to a growth chamber (model GC-16; Enconair three different plants were used. The leaves were mounted Ecological Chambers Inc., Winnipeg, Canada) and were in water and examined under UV light (with DAPI filter) on grown under a PPFD of 400 lmol quanta m2 s1, with a Fluorescence Microscope Leica DMFSA (Leica Micro- a 16-h/8-h light/dark photoperiod and 25/15 C day/night systems Wetzlar GmbH, Germany) using autofluorescence temperature regime, atmospheric CO2, and 50% relative of lignified tracheary elements of the xylem. 3200 | Koteyeva et al.

Immunolocalization using confocal and electron In situ localization of mRNAs encoding Rubisco LSU microscopy protein Leaves of different ages were fixed at 4 C in 2% (v/v) Sense and antisense RNA probes for Rubisco LSU were paraformaldehyde and 1.25% (v/v) glutaraldehyde in generated from pBlsl (which contains a 600-bp HindIII 0.05 M PIPES buffer, pH 7.2. The samples were dehydrated fragment from the central coding region of the amaranth with a graded ethanol series and embedded in London LSU gene) using a modification of procedures described in Resin White (LR White; Electron Microscopy Sciences, Wang et al. (1992). Sense and antisense transcripts were Fort Washington, PA, USA) acrylic resin. Antibodies used synthesized and labelled in vitro with Biotin-11-UTP (all raised in rabbit) were anti-Spinacia oleracea Rubisco (Roche) using T7 or T3 polymerase (Roche). Leaf samples LSU IgG (courtesy of B. McFadden) and commercially were fixed in FAA (50% ethanol, 5% glacial acetic acid, 10% available anti-Z. mays L. PEPC IgG (Chemicon, Temecula, formalin) fixative at room temperature overnight. After CA, USA). Preimmune serum was used in all cases for ethanol and t-butyl alcohol dehydration, samples were controls. embedded in Paraplast Plus. The paraffin-embedded sam- Longitudinal sections, 0.8–1 lm thick, were dried from ples were sectioned (thickness 5–10 lm) using a rotary a drop of water onto gelatin-coated slides and blocked for microtome; sections were mounted to poly-L-lysine-coated 1 h with TBST+BSA (10 mM TRIS–HCl, 150 mM NaCl, slides, dried, and stored at 4 C overnight. After deparaffi- 0.1% v/v Tween 20, 1% w/v BSA, pH 7.2). These were then nization by xylene, the sections were rehydrated through an incubated for 3 h with either preimmune serum diluted in ethanol series, incubated in 0.2 M HCl for 20 min at room Downloaded from TBST+BSA (1:100), anti-Rubisco (1:500 dilution), or anti- temperature (RT), and rinsed in H2O. Slides were then PEPC (1:200 dilution). The slides were washed with incubated in 23SSC (13SSC is 0.15 M NaCl, 0.15 M TBST+BSA and then treated for 1 h with protein A–gold sodium citrate) at 70 C for 30 min and rinsed by H2O. 1 10 nm (diluted 1:100 with TBST+BSA). After washing, Treatment of sections with proteinase K (1 lgml in TE: the sections were exposed to a silver enhancement reagent 100 mM TRIS pH 8.0, 50 mM EDTA) for 15 min at 37C http://jxb.oxfordjournals.org/ for 20 min according to the manufacturer’s directions was followed by a brief rinse in PBS (10 mM phosphate (Amersham, Arlington Heights, IL, USA), stained with buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4), blocking in 1 0.5% (w/v) Safranin O, and imaged in a reflected/ glycine (2 mg ml in PBS) for 2 min at RT, and then transmitted mode using a Zeiss Confocal LSM 510 Meta fixation for 20 min in 4% formaldehyde. Prehybridization Laser Scanning Microscope (Carl Zeiss, Inc. Headquarters, was accomplished in prehybridization medium overnight at Thornwood, NY, USA). The background labelling with 50 C after incubation in 23SSC for 10 min at RT. The preimmune serum was very low, although some infrequent probes were first heated at 75 C for 30 s and mixed with labelling occurred in areas where the sections were prehybridization medium with a final transcript concentra- at OUP site access on April 22, 2013 1 wrinkled due to trapping of antibodies/label (results not tion of 0.5 lgml . Hybridizations with the Biotin-labelled shown). transcripts were performed at 50 C overnight in a moist For TEM immunolabelling, thin cross-sections (;70 nm chamber. The slides were then subjected to the following thick) on Formvar-coated nickel grids were incubated for series of washes: prewarmed at 37 C23SSC, 10 min; 1 h in TBST+BSA to block non-specific protein binding on 23SSC, 1 h, RT; 13SSC, 1 h, RT; 0.53SSC, 30 min, 42 C; the sections. They were then incubated for 3 h with either 0.53SSC, 30 min, RT. Hybridized transcripts were detected the preimmune serum diluted in TBST+BSA, or anti- by streptavidin–alkaline phosphate conjugate (NeutrAvidin; Rubisco (1:50), or anti-PEPC (1:20) antibodies. After Pierce) using blocking buffer (100 mM TRIS–HCl, pH 7.5, washing with TBST+BSA, the sections were incubated for 150 mM NaCl). The final detection step was carried out by 1 h with Protein A–gold (10 nm) diluted 1:100 with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3- TBST+BSA. The sections were washed sequentially with indolyl phosphate (Sigma). Reactions were stopped by TBST+BSA, TBST, and distilled water, and then post- placing slides in 13PBS, and the sections were mounted in stained with a 1:4 dilution of 1% (w/v) potassium perman- 50% glycerol in PBS. Slides were studied under Olympus ganate and 2% (w/v) uranyl acetate. Images were collected BH-2 light microscope equipped with LM Digital Camera using a Hitachi H-600 TEM. and Software. The density of labelling was determined by counting the The colour intensity of chloroplast labelling at base, gold particles on digital electron micrographs using an middle part, and tip of leaves was quantified from images image analysis program (UTHSCSA, Image Tool for collected under the same settings and magnification using Windows, version 3.00) and calculating the number per unit an image analysis program (ImageJ 1.37v; Wayne Rasband, area (lm2). Standard errors were determined and analysis of National Institutes of Health, Bethesda, MD, USA) and variance (ANOVA) was performed using Statistica 7.0 expressed relative to level at the tip, which was set as 100%. software (StatSoft, Inc.). Tukey’s honest significant differ- ence (HSD) test was used to analyse differences between Western blot analysis amounts of gold particles at different stages of leaf de- velopment. All analyses were performed at the 95% Total proteins were extracted from leaves (n¼2 for each significance level. species) by homogenizing 0.5 g of tissue in 1 ml of C4 photosynthesis in the genus Suaeda | 3201 extraction buffer [100 mM TRIS–HCl, pH 7.5, 5 mM MgSO4, 10 mM DTT, 5 mM EDTA, 0.5% (w/v) SDS, 2% (v/v) b-mercaptoethanol, 10% (v/v) glycerol, 1 mM phenyl- methylsulfonyl fluoride, and 25 lgml1 each of aprotinin, leupeptin, and pepstatin]. After centrifugation at high speed for 3 min in a microcentrifuge, the supernatant was collected, and protein concentration was determined using a Bradford protein assay (Bio-Rad), with BSA as a stan- dard. Protein samples (10 lg) were separated by 12% SDS– PAGE, blotted onto nitrocellulose, and probed with anti- A. hypochondriacus NAD-ME IgG against the 65 kDa a subunit (Long and Berry, 1996) (1:5 000), anti-Z. mays PEPC IgG (1:10 000), anti-Z. mays pyruvate, Pi dikinase (PPDK) IgG (courtesy of T. Sugiyama) (1:5 000), or anti-S. oleracea Rubisco LSU IgG (1:10 000) overnight at 4 C. Goat anti-rabbit IgG–alkaline phosphatase-conjugated sec- ondary antibody (Bio-Rad) was used at a dilution of 1:50000 for detection. Bound antibodies were visualized by developing the blots with 20 mM nitroblue tetrazolium and Downloaded from 75 mM 5-bromo-4-chloro-3-indolyl phosphate in detection buffer (100 mM TRIS–HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2). The intensities of bands in Western blots were quantified with an image analysis program (ImageJ 1.37v) and expressed relative to level in the mature stage of http://jxb.oxfordjournals.org/ development, which was set as 100%. d13C values d13C values, a measure of carbon composition, were de- termined on leaf samples of different ages taken from plants using a standard procedure relative to PDB (Pee Dee Belemnite) limestone as the carbon isotope standard at OUP site access on April 22, 2013 (Bender et al., 1973). Plant samples from the growth Fig. 1. Vegetative shoot tip structure and longitudinal sections chamber were dried at 80 C for 24 h, and milled to a fine showing early stages of leaf development in S. taxifolia (A, C, E, G, powder. This preparation (1–2 mg) was placed in a tin I) and S. eltonica (B, D, F, H, J). (A, B) Scanning electron capsule and combusted using a Eurovector elemental microscopy of shoot apices. (C, D) Light microscopy of longitudinal sections of shoot apical meristem showing alternate and approxi- analyser. The resulting N2 and CO2 gases were separated by gas chromatography, and admitted into the inlet of mate initiation of leaf primordia. (E, F) General view of shoot tip a Micromass Isoprime isotope ratio mass spectrometer showing patterns of leaf development. (E) Top view of S. taxifolia (IRMS) for determination of 13C/12C ratio (R). d13C values shoot tip. (F) Side view imaging of S. eltonica shoot tip with tightly 13 packed leaves. (G, H) Longitudinal sections showing the formation of were determined where d C¼[(Rsample/Rstandard) –1]31000. d13C is an expression of the 13C/12C of the plant sample cell lineages at the base of young leaves (with direction of maturation from left to right). Right panels represent schemes of mature leaf relative to the standard (reported in parts per 1000), Rsample 13 12 anatomy to show the origin of leaf tissues from precursor lineages of and Rstandard is the C/ C ratio of the plant sample and the PDB limestone, respectively. cell files. The arrowheads mark the origin of two layers as the result of periclinal divisions of subepidermal cells. (I, J) Cleared leaves under UV light showing the basipetal direction of lateral vascular vein Results development in young leaves. E, epidermis; BS, bundle sheath; H, waterstoragehypoderm;L,leaf;M,mesophyll;VB,vascular Structure of shoot apex and leaf growth bundles; WS, water storage tissue. Scale bars: 100 lmforA,B;50 lm for C, D, G, H; 0.5 mm for E, I; 0.25 mm for J; 2 mm for F. Vegetative apical shoot meristems of both S. taxifolia and S. eltonica at the stage of active organogenesis are dome shaped (Fig. 1 A–D). These have a diameter at the base of ;200 lm, and height of 140 lminS. taxifolia (Fig. 1A, C), Fig. 1D for S. eltonica). Cells of the inner layers (L2 and and 160 and 120 lm, respectively, in S. eltonica (Fig. 1B, L3), which make up the body of the apical meristem, divide D). In both species, the outermost layer (L1, also known as both anticlinally and periclinally, generating the inner the first layer of the tunica) divides only anticlinally and tissues of leaf primordium. Leaf primordia in both species gives rise to the epidermis of the leaf primordium (see are initiated alternately and rather often (Fig. 1A, B), 3202 | Koteyeva et al. resulting in the formation of a compact apical bud with process of Kranz anatomy development. In S. taxifolia close positioning of leaves (Fig. 1E, F). (Fig. 1G), periclinal divisions of subepidermal cells of Figures 1E and F show the organization of the shoot tips ground meristem give rise to M and BS layers, while in S. in the two Suaeda species during active vegetative growth. eltonica, periclinal divisions of subepidermal cells generate In the bud, the emergence of leaf tips from the encircling future water storage hypoderm and M layers (Fig. 1H). superior leaves, and their subsequent greening, occurs Bundle sheath cells in S. eltonica originate from the central earlier in S. taxifolia (length of leaf ;0.2 cm) than in layer of ground meristem around the procambial strands S. eltonica (length of leaf ;0.3 cm), while in general the rate (Figs 1H, 3A). Development of the vascular system in of leaf growth was higher in S. eltonica. The extended leaves Suaeda species also takes place in the basipetal direction. turn aside earlier in S. taxifolia (Fig. 1E) while in S. eltonica Lignin autofluorescence was used to investigate the se- (Fig. 1F) they are still tightly packed in the apical bud and quence of development of xylem tracheary elements in maintain a vertical orientation until they reach ;0.4 cm in young leaves. Clearly the xylem in the midvein is established length. first (developing acropetally, not shown) followed by de- Light and transmission electron microscopy: velopment of lateral veins with their connecting loops, developmental stages beginning from the leaf tip (Fig. 1I, J). Following the initial formation of M and BS cells from Development of Kranz anatomy was studied at the light progenitor cells, three developmental stages were character- microscopy level using longitudinal sections of intermediate ized in the progression towards the formation of the Kranz size leaves, 0.5–0.7 cm (Fig. 1G, H), and cross-sections of syndrome. Structural features of these stages are shown in Downloaded from young (0.2–0.3 cm), intermediate (0.5–0.7 cm), and mature cross-sections of young, intermediate, and mature leaves fully expanded (2–3 cm) leaves (Figs 2, 3). These observa- (designated stages 1, 2, and 3, respectively), as observed by tions indicate that in both species, cellular differentiation light and electron microscopy of S. taxifolia (Fig. 2) and during the growth of young leaves proceeds in a basipetal S. eltonica (Fig. 3). In young leaves (0.2–0.3 cm in length), direction, with the youngest undifferentiated (meristematic) in S. taxifolia two clearly defined layers of chlorenchyma http://jxb.oxfordjournals.org/ cells at the leaf base and the most differentiated cells at the cells are located at the leaf periphery next to the epidermal leaf tip. Bundle sheath and M cell progenitors can be cells (Fig. 2A); in contrast, the two chlorenchyma layers are recognized at the base of the young leaf by their position in located under the hypodermal cells between the veins in comparison with adjacent tissues (Fig. 1G, H). Observa- S. eltonica (Fig. 3A). Precursors of M and BS cells are tions of longitudinal sections of young leaves at their base distinct in shape and size; M cells are more palisade shaped show differences in the origin of BS layers during the due to anticlinal divisions especially in S. eltonica, where for at OUP site access on April 22, 2013

Fig. 2. Structural features of stages of development of Kranz anatomy in S. taxifolia shown by light and electron microscopy of leaf cross-sections in the middle part of young, intermediate, and mature leaves. (A, F, K) Light micrographs showing the development of leaf structure from young to mature stage. Note chlorenchyma layers distributed around the leaf periphery. (B, G, L) The thylakoid system in BS chloroplasts. (C, H, M) BS mitochondria ultrastructure. Note the specific cristae in mitochondria in mature BS cells. (D, I, N) Structure of M chloroplasts. (E, J, O) Cross-sections of the lateral vascular bundles. E, epidermis; BS, bundle sheath; M, mesophyll; Mt, mitochondria; SE, sieve element of phloem; TE, tracheary element of xylem; VB, vascular bundle; WS, water storage tissue. Scale bars: 100 lm for A, F, K; 0.5 lm for B, C, D, G, H, I, N; 1 lm for L, M; 20 lm for E, J, O. C4 photosynthesis in the genus Suaeda | 3203 Downloaded from

Fig. 3. Structural features of stages of development of Kranz anatomy in S. eltonica shown by light and electron microscopy of leaf cross-sections in the middle part of young, intermediate, and mature leaves. (A, F, K) Light micrographs showing the development of leaf structure from young to mature stage. Note chlorenchyma layers distributed around vascular bundles in the middle part of the leaf. http://jxb.oxfordjournals.org/ Arrows in (F) and (K) show BS chloroplasts at centrifugal position. (B, G, L) The thylakoid system in BS chloroplasts. (C, H, M) BS mitochondria ultrastructure. Note the specific cristae in mitochondria in mature BS cells. (D, I, N) Structure of M chloroplasts. (E, J, O) Cross-sections of the lateral vascular bundles. H, water storage hypoderm; for other labels see Fig. 2. Scale bars: 100 lm for A, K; 50 lm for F; 0.5 lm for B, D, G, N; 1 lm for C, L, M; 10 lm for E, J; 20 lm for O. each future BS cell, on average there are two future M cells (Figs 2E, 3E). Mature xylem vessels lack cytoplasm and (Fig. 3A). show secondary wall thickening; mature sieve tubes appear at OUP site access on April 22, 2013 In stage 1, BS cells in both species have a developing to lack nucleus, tonoplast, and ribosomes, and they contain central vacuole, and chloroplasts are distributed around the only a peripheral layer of cytosol, with rare occurrence of cell periphery. In M cells, the nucleus has a central position mitochondria, plastids, and smooth endoplasmic reticulum, in S. taxifolia (Fig. 2A), while in S. eltonica, the nucleus is when viewed at higher magnification (not shown). adjacent to the middle part of the radial cell wall due to Stage 2 was observed in cross-sections from the middle earlier formation of the central vacuole (Fig. 3A). At this part of intermediate leaves (;0.5–0.7 cm in length). For stage of leaf development, the cells of all tissues are tightly both species, at this stage M cells show a well-developed packed in both species; minor intercellular air spaces exist central vacuole with organelles distributed in a thin cyto- between M and epidermal cells in S. taxifolia, while more plasmic layer adjacent to the cell walls (Figs 2F, 3F). The developed airspaces occur between hypodermal cells in characteristic positioning of chloroplasts in BS cells for each S. eltonica (Figs 2A, 3A). The TEM observations confirm species has occurred and is almost complete: plastids are that small chloroplasts are evenly distributed throughout distributed centripetally in BS cells of S. taxifolia (Fig. 2F) the cytosol at the cell periphery, and sometimes around the and centrifugally in BS cells of S. eltonica (Fig. 3F). nucleus. These have a relatively well-developed thylakoid Intercellular air spaces throughout the M cell layer are system with numerous small grana and intergranal thyla- formed in both species (Fig. 2F, 3F). Chloroplasts in koids that are structurally similar in both M and BS cells chlorenchyma cells are bigger and more numerous than in (Figs 2B, D, 3B, D). Mitochondria in chlorenchyma cells the previous stage; their thylakoid systems are more de- are few in number, small, and they have crescent-like cristae veloped, but there is similar grana development in BS and (see Figs 2C, 3C for BS cell). The vascular system is not M chloroplasts (Figs 2G, I, 3G, I). Bundle sheath mito- fully differentiated at this stage, although the midvein is chondria, which are colocalized with chloroplasts, show already clearly morphologically evident with fully developed a tendency to locate next to the inner periclinal cell walls in phloem and xylem elements. The developing peripheral S. taxifolia, and the outer periclinal cell walls in S. eltonica vascular bundles usually have only one differentiated xylem (not shown). At this stage of leaf development, the vessel. They still have periclinal cell divisions at the phloem peripheral vascular bundles have two to four mature xylem end, especially in the most lateral bundles, in accordance vessels and one or two mature sieve elements, depending on with gradient of maturation: the farther from the central the distance from the central vein (Figs 2J, 3J). The pattern bundle, the less degree of minor vein differentiation of maturation of peripheral vascular bundles at this stage 3204 | Koteyeva et al. still maintains a gradient, with less differentiated minor Table 1. Carbon isotope composition of leaves of S. taxifolia and veins situated closer to the leaf margins. S. eltonica at different stages of development Whole leaves were In mature leaves (stage 3), the structural features of sampled at different stages of growth and analysed, n¼2. Salsina and Schoberia types of Kranz anatomy are com- pletely formed. In S. taxifolia (Salsina type), the chloren- Leaf age Leaf length S. taxifolia d13C S. eltonica d13C chyma is composed of two continuous layers at the leaf (cm) (&) (&) periphery, and all vascular bundles are embedded within Young 0.2–0.3 –15.2 –18.4 water storage tissue and arranged in one longitudinal plane; Intermediate 0.4–0.6 –14.0 –16.8 BS chloroplasts are positioned centripetally (Fig. 2K). In Nearly mature 0.7–1.0 –14.1 –16.3 S. eltonica (Schoberia type), the inner vascular bundles are Mature >1 –12.3 –14.2 surrounded by chlorenchyma, and water storage tissue is positioned on the outside; BS chloroplasts are located centrifugally (Fig. 3K). Bundle sheath cells of mature leaves in both species contain chloroplasts having numerous grana with 5–20 thylakoids in stacks (Figs 2L, 3L). Intergranal thylakoids are few and short in S. taxifolia (Fig. 2L), and longer and more numerous in S. eltonica (Fig. 3L). For both species, in comparison with BS chloroplasts, M chloroplasts have poorly developed grana which are inter- Downloaded from connected by rather numerous, long intergranal thylakoids (but they are more numerous in S. eltonica compared with S. taxifolia, Figs 2N, 3N). In mature leaves, the BS cells have abundant, large mitochondria with cristae showing a distinct tubular type structure (Figs 2M, 3M). BS http://jxb.oxfordjournals.org/ organelles maintain characteristic positioning in the cell, as was noted in stage 2. In stage 3, all minor veins in both species are fully differentiated and contain three or four xylem vessels and three or four sieve elements (Figs 2O, 3O).

Carbon isotope composition during leaf development at OUP site access on April 22, 2013 The d13C values for leaves at different stages of develop- ment (young, intermediate, nearly mature, and mature) are presented in Table 1. In both Suaeda species, the isotope values become more positive as development progresses from young to intermediate to mature leaves. The values in young versus mature leaves in S. taxifolia were –15.2& versus –12.3&, and in S. eltonica, –18.4& versus –14.2&, respectively, indicating less expression of C4 in the younger leaves.

Accumulation of C4 pathway enzymes and Rubisco during leaf development Figure 4 shows western blot analysis of PEPC, PPDK, NAD-ME, and Rubisco in the total soluble protein extracts from very young up to mature leaves of S. taxifolia and Fig. 4. Western blot analysis from very young up to mature leaves

S. eltonica. In both Suaeda species, Rubisco and to a lesser showing accumulation of C4 enzymes and Rubisco LSU during extent PEPC were present at the youngest stage of leaf leaf development in two Suaeda species. Total soluble proteins development (0.1 cm leaf length), and their levels steadily were extracted from leaves of different ages of S. taxifolia and increased as leaf age progressed. In contrast, very few S. eltonica. Blots were probed with antibodies raised against PPDK or NAD-ME polypeptides were detected in young PEPC, PPDK, NAD-ME, and Rubisco LSU, respectively. Top: leaves (0.3 cm long, corresponding to stage 1). Increased representative Western blots showing detection of each protein. levels of these C4 enzymes were detected in intermediate size Numbers at the left indicate molecular mass in kDa. Bottom: leaves, 0.5 cm in length, with maximum levels occurring in quantitative representation of Western blot data. 100% on the the mature leaves. y axis refers to the level achieved in mature leaves. C4 photosynthesis in the genus Suaeda | 3205

In situ immunolocalization of Rubisco and PEPC during leaf development Intermediate size leaves (0.5–0.7 cm) of both Suaeda species have a basipetal longitudinal gradient in chlorenchyma cell development, with features of stage 1 located at the leaf base where cell divisions and expansions occur, stage 2 in the mid-section, and stage 3 at the tip. The pattern of Rubisco LSU and PEPC expression, by antibody labelling, along this gradient was studied in longitudinal sections by confocal microscopy (Figs 5A–C, J–L, 6A–C, J–L). For quantitative evaluation of selective enzyme accumulation, immunogold labelling and detection using TEM was applied to cross-sections of young, intermediate, and mature leaves representing similar stages of development (Figs 5D–I, M–R, 6D–I, M–R), with subsequent counting of the number of gold particles detected in the different cell compartments (Fig. 7). In both S. taxifolia (Fig. 5A)and

S. eltonica (Fig. 6A), in stage 1, labelling for Rubisco was Downloaded from detected by confocal imaging after the start of BS cell vacuole extension, but before specific positioning of the BS organelles. At this stage, Rubisco was present mainly in the BS chloroplasts (Figs 5A, 6A), a finding that was confirmed by TEM immunolocalization (Figs 5D, E, 6D, E). Only in http://jxb.oxfordjournals.org/ S. eltonica did M chloroplasts contain a noticeable quantity of labelling for Rubisco at this stage; however, levels were still only half that of BS chloroplasts (Fig. 7A). Subsequently by stage 2, Rubisco LSU labelling was Fig. 5. In situ immunolocalization of Rubisco LSU (A–I) and PEPC strongly associated with BS chloroplasts in both species (J–R) in leaves of S. taxifolia at different stages of development. (Figs 5B, F, G, 6B, F, G,), with the density of labelling in Reflected/transmitted confocal imaging of base, middle, and tip of BS cell chloroplasts increasing >2-fold (Fig. 7A). At this

longitudinal leaf sections show Rubisco LSU (A–C) selectively at OUP site access on April 22, 2013 stage, the density of gold particles in M chloroplasts localized to BS cells beginning from stage 1. Labelling also occurs dropped to half the density of the earlier stage (Fig. 7A). in WS chloroplasts but not in M. Appearance of PEPC (J–L) At stage 3, the pattern of Rubisco LSU labelling preferentially localized to M cells at stages 2–3. Immunogold distribution did not change much qualitatively; however, labelling at the TEM level in cross-sections of young, intermediate, the density of gold particles in the BS chloroplasts reached and mature leaves shows Rubisco localized within BS chloroplasts its highest level, while in M chloroplasts LSU labelling did (D, F, H), and the absence of this protein in M chloroplasts (E, G, I) not differ significantly from background (Figs 5C, H, I, 6C, at stages 1–3. PEPC is selectively localized in the cytosol of M H, I). Quantification of these data in Fig. 7A indicates cells beginning from stage 2 (P) and continuing through stage 3 a very similar pattern of Rubisco accumulation in both (R). The number of gold particles in the cytosol of BS cells (M, O, S. taxifolia and S. eltonica. Q) at all developmental stages and in M cells at stage 1 (N) is In both species, in the earliest stage PEPC was undetect- comparable to the background level in control samples (not able by both immunolocalization methods applied (Figs 5J, shown). Antibody labelling appears as yellow particles in the M, N, 6J, M, N). Nevertheless, at stage 2 (from the mid- confocal images and as black dots on TEM micrographs. BS, section of intermediate leaves), specific labelling of PEPC bundle sheath; Ch, chloroplast; E, epidermis; M, mesophyll; Mt, was observed in M cells (Figs 5K, O, P, 6K, O, P). In stage mitochondria; WS, water storage. Scale bars: 50 lm for A–C, J–L; 3, labelling for PEPC was at its maximum throughout the 0.5 lm for D–I, M–R. cytosol of M cells, with little, if any, labelling observed in the BS cytosol (Figs 5L, Q, R, 6L, Q, R). The graph shown in Fig. 7B indicates a very similar trend in PEPC Temporal and spatial accumulation of rbcL mRNA at accumulation and partitioning at the different developmen- different stages of leaf development tal stages for both Suaeda species. Taken together, the two imaging methods, coupled with Using in situ hybridization, longitudinal sections of young quantification of the number of gold particles in the and intermediate leaves (0.2–0.6 cm long) of S. taxifolia different tissues and stages, have clearly revealed the spatial and S. eltonica were studied to examine the temporal and temporal patterns of Rubisco and PEPC accumulation and spatial distribution of Rubisco LSU mRNA. Specific during leaf development (Fig. 7A), with very similar results hybridization of Biotin-11-UTP-labelled antisense RNA for both Suaeda species. probes transcribed using T7 polymerase from a vector 3206 | Koteyeva et al. Downloaded from http://jxb.oxfordjournals.org/

Fig. 7. Graphical representation of data from Figs 5 and 6 showing the density of immunolabelling for Rubisco LSU (A) in BS Fig. 6. In situ immunolocalization of Rubisco LSU (A–I) and PEPC versus M chloroplasts and PEPC (B) in BS versus M cytosol from (J–R), in leaves of S. eltonica at different stages of development. cross-sections of young, intermediate, and mature leaves of Reflected/transmitted confocal imaging of base, middle, and tip of S. taxifolia (squares) and S. eltonica (triangles). In both graphs the 2 longitudinal leaf sections shows Rubisco LSU (A–C) selectively y axis represents the number of gold particles per lm of at OUP site access on April 22, 2013 localized to BS cells at developmental stages 1–3, and the chloroplast or cytosol area, and the x axis represents the appearance of PEPC (J–L) specifically in M cells at stage 2. TEM developmental stage. For each cell type and stage of micrographs of immunogold labelling in cross-sections of young, development, 10–15 cell areas were used for counting. intermediate, and mature leaves from stages 1, 2, and 3 show Rubisco localized within BS chloroplasts at each stage (D, F, H). Low-level LSU labelling was observed within M chloroplasts at (Fig. 8B, F, respectively). Beginning at stage 1, there was stage 1 (E), but none was detected in M plastids at stage 2 or 3 a clear hybridization signal within the BS chloroplasts (G, I). PEPC was not detected in M cells at stage 1 (N), but was of both species (Fig.8B,C,F,G), with no (in the case observed within the cytosol of M cells at stage 2 (P) and stage 3 of S. taxifolia, Fig. 8C), or very low (in S. eltonica, (R). Gold particles were not present in M cell cytosol at stage 1 (N) Fig. 8G), signal in M cells. At stage 2 of leaf development or in cytosol of BS cells (M, O, Q) at any of the developmental (Fig. 8B, D, F, H) in both species, expression levels of the stages. Antibody labelling appears as yellow particles in the Rubisco LSU gene appeared enhanced, and were confined confocal images and as black dots in TEM micrographs. BS, to BS chloroplasts (Fig. 8B, D for S. taxifolia and Fig. 8F, bundle sheath; Ch, chloroplast; E, epidermis; H, water storage H for S. eltonica). Colour intensity quantification per hypoderm; M, mesophyll; Mt, mitochondria. Scale bars: 50 lm for individual chloroplast area (not shown) revealed no A–C, J–L; 0.5 lm for D–I, M–R. difference for S. taxifolia and a slight (1.1-fold) increase in labelling for S. eltonica at stage 2 comparing with stage 1 containing the central coding region of an LSU gene, are (at P<0.05). The intensity of the colour in the section clearly visualized as a dark purple colour, as seen in Fig. 8. increased from stage 1 to stage 2 due to increase in As a negative control, the sense probes transcribed using chloroplast size and number, and their arrangement T3 polymerase from the same plasmid showed no purple towards one side of BS cells. There were no significant hybridization signal (Fig. 8A). In both S. taxifolia and changes in the pattern of labelling or signal intensity (at S. eltonica, a very low hybridization signal (slightly over P<0.05) in stage 3 at the leaf tip (Fig.8B,E,F,I), background) in pre-BS and M chloroplasts was detected indicating that the mRNA accumulating patterns estab- at the earliest stages of development at the very base of lished at stage 2 were maintained throughout the re- the young leaf, before expansion of vacuoles begins mainder of leaf development. C4 photosynthesis in the genus Suaeda | 3207

et al., 2011). This can make it difficult to assimilate information between studies in order to describe the sequence of events in C4 development. To determine the timing and order of development of the various C4 traits in a certain Kranz/biochemical type of C4, a number of analyses need to be made simultaneously (e.g. as in maize, Li et al., 2010; Majeran et al., 2010). In the present study we examined longitudinal and cross-sections and compared anatomical and ultrastructural features of chlorenchyma cells as well as vascular elements at different developmental stages of two Kranz type species of Suaeda. These data were complemented with quantitative expression analysis of several C4 photosynthetic enzymes, including the in situ localization of the two carboxylases, PEPC (protein) and Rubisco (protein and mRNA).

Sequence of events in development of two forms of

Kranz in genus Suaeda Downloaded from

Basipetal maturation gradient. The two eudicot C4 species S. taxifolia and S. eltonica in subfamily Suaedoideae represent two forms of Kranz anatomy, Salsina and Schoberia, respectively. In both types, the development of lateral veins and cellular differentiation during growth of http://jxb.oxfordjournals.org/ the semi-terete leaves proceeds in a basipetal direction along the base-to-tip maturation gradient, which is useful for studying the progression of differentiation to form C4. A similar progressive developmental gradient has been described for monocot leaves and was exploited for the Fig. 8. In situ hybridization of rbcL mRNA with longitudinal leaf study of photosynthetic tissue differentiation (Miranda sections of S. taxifolia (A–E, from a leaf 0.3 cm long) and et al., 1981; Langdale et al., 1988a; Nelson and Langdale, at OUP site access on April 22, 2013 S. eltonica (F–I, from a leaf 0.5 cm long). The dark purple signal 1989; Soros and Dengler, 2001; Wakayama et al., 2003; Li indicates the specific hybridization to antisense mRNA probes. et al., 2010; Majeran et al., 2010). (A) Sense probe control is presented only for S. taxifolia to show Origins of M and BS. Analysis of cell files near the leaf the low background staining that occurred in these reactions. base on longitudinal and cross-sections revealed that both (B, F) Basipetal gradient of rbcL transcript accumulation from tip BS and M cells of the two Suaeda species originate early in (right) to base (left). Preferential localization of rbcL mRNA in BS leaf development from the ground meristem. There is, cells is shown for stage 1 (C, G), stage 2 (D, H), and stage 3 (E, I) however, different positioning of the chlorenchyma tissue of leaf development. BS, bundle sheath; E, epidermis; M, in the leaf, so that S. taxifolia has Kranz around the mesophyll; WS, water storage. Scale bars: 500 lm for A, B, F; periphery, and S. eltonica has Kranz around the veins in 25 lm for C, G–I; 50 lm for D, E. a central plane. Study of tissue differentiation showed that the origin of the BS layer is restricted to different cell Discussion lineages in the two species: to subepidermal cells in S. taxifolia, and to central cells in S. eltonica. Bundle sheath A common feature of C4 development is the gradual and M cells in S. taxifolia represent sister cells separated by differentiation of BS and M cell anatomy to produce the a single periclinal cell division (followed by anticlinal final functioning Kranz syndrome, starting from structur- divisions of M cells). In S. eltonica, M cells are sister to ally uniform cells and ending with specialized C4 photosyn- hypodermal water storage cells as a result of periclinal thetic cells (Nelson and Langdale, 1989; Liu and Dengler, division of subepidermal cells; hence in this species, BS cells 1994; Dengler et al., 1996; Wakayama et al., 2003). To originate earlier than M cells. According to several studies examine this progression, leaf development in C4 plants has on leaf tissue origin by clonal analysis, in eudicots the been defined in various ways, e.g. according to leaf age (Liu epidermis is derived from the L1 layer of the shoot apical and Dengler, 1994; Voznesenskaya et al., 2003a, b, 2005), meristem; the L2 forms a single layer immediately beneath distance from the leaf base (Wakayama et al., 2003; the epidermis, giving rise to future subepidermal M layers, Majeran et al., 2010), development of leaf anatomy (e.g. and L3 is a progenitor of more central tissues (the vascular vascular system differentiation, Dengler et al., 1986), tissues and inner spongy M cells) (Telfer and Poethig, 1994; chlorophyll content (Kirchanski, 1975), and changes in Leyser and Day, 2003). Thus, the most probable scenario is photosynthetic enzymes (transcripts and proteins) (Berry that from the very beginning, BS cells of the two Suaeda 3208 | Koteyeva et al. species are derived from different meristematic precursors. enzymes suggest that some capacity for biochemistry of C4 In general, BS cells of C4 species can ontogenetically function is being established. The expression of PEPC, originate from the procambium or from the leaf ground along with Rubisco, begins to reach high levels in stage 2 of meristem, while M can be derived only from ground chlorenchyma differentiation and low levels of the C4 meristem (Esau, 1965; Dengler et al., 1985, 1986; Liu and enzymes PPDK and NAD-ME appear. Immunolocalization Dengler, 1994; Dengler et al., 1995; Soros and Dengler, shows that Rubisco is selectively localized in BS cells and 2001). The BS cells in C4 plants are an example of PEPC in M cells. convergent evolution of tissues having different ontogenetic Stage 3 of C4 development. This occurs when the leaf origins, structures, and C4 biochemistry, yet still carrying tissue is nearly fully mature, and the structural features of out the common functions of decarboxylating C4 acids and the two Kranz types are completely formed with M and BS donating the released CO2 to the C3 cycle. cells distributed around the leaf periphery in S. taxifolia, Although BS cells of the two Suaeda Kranz types have and with M and BS cells surrounding vascular bundles in different origins, and originate earlier in ontogeny in the central lateral plane in S. eltonica. In both species, TEM S. eltonica than in S. taxifolia, both species pass through shows that chloroplasts have become structurally differenti- similar anatomical (vacuolization, specific positioning of ated in this final stage; M cells have chloroplasts with organelles, and organelle differentiation) and biochemical reduced grana, while BS cells contain chloroplasts with stages (timing of cell-specific Rubisco expression) during well-developed grana. Also, BS cells have large mitochon- their development, which in both plants coincides with M dria that are specialized to function in C4 acid decarboxyl- Downloaded from cell differentiation. This is consistent with two previous ation, a feature that is characteristic of NAD-ME type C4 studies which show that the differentiation of BS and M chenopods. Western blots show that NAD-ME and PPDK cells in C4 plants is tightly coordinated irrespective of the and the carboxylases reach maximum levels at this stage. anatomical and biochemical types of C4 species or the Following morphological differentiation and final posi- ontogenetic origin of the BS (Dengler et al., 1986, 1996). In tioning of organelles within BS cells during stage 2, there http://jxb.oxfordjournals.org/ particular, in a study of two C4 Panicum species, the origin is ultrastructural differentiation of chloroplasts and mito- of ground meristem-derived BS cells occurs earlier in chondria in stage 3. This order of development has also development in Panicum effusum in comparison with the been observed in some NADP-ME type C4 species. In BS of Panicum bulbosum, which originates from the pro- Arundinella hirta, completely dimorphic chloroplasts cambium; this disparity had little effect on the timing of BS (agranal in M, granal in BS, characteristic of NADP-ME cell differentiation (Dengler et al., 1986). type C4) appear only at the latter stage of development after Stage 1 of C4 development. Anatomical and ultrastruc- specific positioning of organelles in BS cells (from analysis tural criteria were initially used to distinguish between the of figures in Dengler et al., 1996). Other NADP-ME species at OUP site access on April 22, 2013 main stages of leaf development. Stage 1 was defined as that have been studied also show specific differentiation of lacking structural and ultrastructural differences between the thylakoid system relatively late in development (Laetsch BS and M cells. Leaf vasculature at this stage consisted of and Price, 1969; Brangeon, 1973; Leech et al., 1973; a differentiated central vein, while minor veins had only one Kirchanski, 1975; Dengler et al., 1986; Voznesenskaya or two xylem vessels and no differentiated phloem elements. et al., 2003b; Majeran et al.,2010). Also, in the two single- C4 was not established biochemically, as there was sub- cell NAD-ME type C4 plants in subfamily Suaedoideae, stantial Rubisco, low PEPC, and lack of expression of the Bienertia cycloptera (Voznesenskaya et al., 2005) and other C4 enzymes PPDK and NAD-ME (western blots on Suaeda aralocaspica (formerly Borszczowia)(Voznesenskaya extracts from young leaves). From the earliest detection of et al., 2003a), completion of chloroplast differentiation carboxylases by immunolocalization using TEM on leaf occurs after positioning of organelles to two different cross-sections there was preferential expression of Rubisco compartments that are analogues of BS and M in Kranz in BS cells, and PEPC was not detected in stage 1 (Fig. 7). types. Stage 2 of C4 development. At this stage, the two The structural differentiation that occurs in mitochondria chlorenchyma cell layers were clearly defined. Palisade M in the Suaeda spp. in stage 3 coincides with increasing levels cells showed a well-developed central vacuole; BS cells were of mitochondrial NAD-ME. Similar findings have been mainly characterized by organelles becoming positioned to reported for the single-cell C4 species S. aralocaspica one side of the cell (centripetal in S. taxifolia and (Voznesenskaya et al., 2003a), and for B. cycloptera centrifugal in S. eltonica). The ultrastructure of chloroplasts (Voznesenskaya et al., 2005). In C4 plants NAD-ME is with respect to grana development does not differ between localized in mitochondria of BS cells (Hatch and Kagawa, BS and M cells. In stage 2, BS mitochondria appeared more 1974; Kagawa and Hatch, 1975; Edwards and Walker, numerous and bigger than in stage 1; however, the structure 1983; Long et al., 1994) as is glycine decarboxylase, a photo- of the cristae was similar to M cell mitochondria. This respiratory enzyme (Rawsthorne, 1992) that is also associ- shows that the positioning of organelles in BS cells in both ated with the completion of BS mitochondria differentiation species occurs prior to the structural differentiation of (Voznesenskaya et al., 2003a). The findings that develop- chloroplasts and mitochondria which is observed in stage 3. ment of C4 biochemistry in BS mitochondria occurs as This stage is also characterized by the first appearance of leaves reach maturity indicates that complete structural differentiated sieve elements in the lateral veins. Analyses of differentiation of these organelles, in coordination with C4 photosynthesis in the genus Suaeda | 3209 other C4 developmental processes, is essential for full C4 both cytoplasmic domains, indicative of an initial C3-like function. stage (Voznesenskaya et al., 2003a, 2005). Another repre- Analysis of carbon isotope composition of Suaeda species sentative of the Chenopodiaceae family, Salsola richteri,an leaves shows that C4 type values are only obtained in NADP-ME species with Salsoloid type of anatomy, showed mature leaves, which coincides with structural and bio- a similar localization pattern during early leaf development, chemical evidence of full development of C4 in mature with low levels of Rubisco expression in plastids of all 13 leaves. The d C values for C4 plants are typically between tissues of very young leaves, where cell divisions leading o o –10 /oo and –15 /oo; whereas values for C3 species are to the formation of pre-BS and M cells are evident o o typically between –24 /oo and –30 /oo (Cerling, 1999). (Voznesenskaya et al., 2003b). In cases where a C3-default Young leaves of the Suaeda spp. have a carbon isotope stage of Rubisco localization occurs, timing of the transi- o composition that is 3–4 /oo more negative than mature tion to BS cell specificity for Rubisco is species dependent. leaves (a shift of 20–25% towards C3 type values), which is This transition may occur early, in parallel with the consistent with incomplete development of the C4 system. initiation of anatomical differentiation, as in S. richteri The degree of shift towards C3 type carbon isotope values in (Voznesenskaya et al., 2003b), or later after differences the biomass of young leaves may be limited by their import between the two Kranz layers are clearly evident (in of some carbon from C4 photosynthesis in mature leaves. A. hypochondriacus)(Wang et al., 1992). The observation The late development of C4 biochemistry in the Suaeda spp. that a late transition similar to amaranth also occurs for indicates that leaves will only function in full C4 mode as the two intracellular compartments in single-cell C4 species they mature. (Voznesenskaya et al., 2003a, 2005) during the structural Downloaded from differentiation of chloroplasts, indicates that similar de- Immunohisto- and cytochemistry of compartmentation velopmental processes may work to establish C4-like of carboxylases in Suaeda versus other forms of C localization in these plants as well. It is clear from this and 4 previous findings that the timing of Rubisco expression http://jxb.oxfordjournals.org/ Immunohistochemistry using light microscopy on longitudi- during development of C4 photosynthesis does not follow nal sections, as well as analysis of gold particle density by a fixed pattern across the many species that utilize this TEM, revealed that the intercellular partitioning of Rubisco pathway, suggesting the evolution of alternative regulatory to BS cells, and PEPC to M cells, began to occur as soon as mechanisms to control these convergent processes. their expression became evident. There was no stage where In the Kranz type Suaeda leaves expression of PEPC is either Rubisco or PEPC were equally labelled in BS and M initiated later in development than Rubisco, and is cell cells. After their first appearance, both of the carboxylases type specific at its first occurrence. This pattern is in underwent dramatic increases in abundance in coordination agreement with most previous studies, regardless of C4 at OUP site access on April 22, 2013 with leaf maturation. Preferential expression of Rubisco in anatomical or biochemical type (Wang et al.,1992; BS chloroplasts at the earliest stages of leaf development Dengler et al.,1995; Ramsperger et al.,1996; Soros and has also been observed in the NAD-ME type eudicot Dengler, 2001; Voznesenskaya et al.,2003b; Wakayama Atriplex rosea, which has atriplicoid type anatomy (Dengler et al.,2003). However, three different anatomical and et al., 1995), as well as in NADP-ME type monocots Zea biochemical types of C4 species in the family Cyperaceae mays (Martineau and Taylor, 1985; Langdale et al., 1988a) accumulatelowlevelsofPEPCinbothMandBScells and A. hirta (Wakayama et al., 2003). In these cases, as in within the extension zone of young leaves; and, surpris- the present study, regulatory processes responsible for cell ingly, PEPC remains non-specific in Rhynchospora rubra type specificity for Rubisco are active well before the final (NADP-ME, rhynchosporoid anatomical type) even in the anatomical differentiation of the two photosynthetic cell mature state (Soros and Dengler, 2001). In the single-celled types. C4 chenopods, PEPC is not strictly compartmentalized to However, in other species there is evidence for expression one cytoplasmic domain, which is expected, since this is of Rubisco in both chloroplasts before selective localization a cytosolic enzyme (Voznesenskaya et al.,2003a, 2005). in Rubisco with leaf maturation. A different and more Moreover, in S. aralocaspica there is a detectable amount of complex pattern of Rubisco accumulation in leaf develop- PEPC (immunolocalization by light microscopy) from the ment was shown for Amaranthus hypochondriacus, another earliest stages of development (1-mm leaf length), simulta- NAD-ME type plant with atriplicoid type anatomy (Wang neously with Rubisco (Voznesenskaya et al.,2003a). et al., 1992; Ramsperger et al., 1996; Patel and Berry, 2008). There are also differences between species as to when In the youngest leaves (2 mm in length), Rubisco peptide PEPC expression is first initiated relative to development of accumulation showed a C4 pattern confined to precursors of Kranz anatomy. In the two Kranz type Suaeda species, this BS cells (Ramsperger et al., 1996); but, later (5-mm-long occurs only after completion of specific BS organelle leaves), Rubisco protein was present in both M and BS cells positioning in stage 2; however, it precedes chloroplast in a C3-like pattern, and became completely BS specific in ultrastructural differentiation. For a C4 representative of the C4 pattern only at a later stage of development (Wang the family Cyperaceae, Pycreus polystachyos (NADP-ME, et al., 1992). Also, in the single-cell C4 species in subfamily chlorocyperoid anatomical type) the accumulation of PEPC Suaedoideae, S. aralocaspica and B. cycloptera (NAD-ME was suggested also to occur only after cell structural type), Rubisco occurs initially within chloroplasts present in differentiation (Soros and Dengler, 2001). However, this 3210 | Koteyeva et al. conclusion was made based only on light microscopy level of Rubisco enzyme increased towards the tip of the observations; no data were available on organelle position- leaf, while levels of the corresponding mRNAs peaked near ing during development, or on their ultrastructure. In the base of the blade and decreased towards the tip contrast, in Z. mays and A. rosea, it was shown that PEPC (Langdale et al., 1988a; Nelson and Dengler, 1992). In accumulation, which is M specific, occurs before morpho- contrast, in developing amaranth leaf primordium, Rubisco logical differentiation of BS and M cells (Langdale et al., transcripts and proteins are detected simultaneously from 1988a; Dengler et al., 1995). Finally in amaranth, abundant the earliest stages (Ramsperger et al., 1996). levels of PEPC were detected in M cells at the base of the youngest leaves (2 mm long), which have structurally similar BS and M cells (Ramsperger et al., 1996). Relationships between development of vascular tissue and C4 traits Cellular localization of rbcL mRNA and RbcL protein in In this current study we have observed correlations between Suaeda species vascular tissue development, stages of chlorenchyma differ- At all stages of leaf development in both Suaeda species, entiation, and expression of C4 enzymes in the two Kranz Rubisco rbcL mRNA was preferentially localized to BS type Suaeda species. The transitions to the C4 features that cells, followed by corresponding expression of the LSU appear in stage 2, which include specific organelle position- peptide. Accumulation of transcripts for Rubisco LSU in ing in BS cells, significant increase in the level of Rubisco in Downloaded from BS cells increased basipetally in parallel with the formation BS chloroplasts, and initiation of selective expression of C4 of Kranz anatomy in young leaves. The partitioning of enzymes, coincide with phloem sieve element differentiation. mRNA and LSU peptide to BS cells did not correlate with Additional research will be required to determine the chloroplast structural differentiation, since rbcL mRNA relationship between vascular tissue differentiation and accumulation was observed in both stages 1 and 2 prior to development of C4 anatomy and biochemistry in leaves of http://jxb.oxfordjournals.org/ chloroplast differentiation. The spatial and temporal pat- C4 Suaeda. terns and co-localizing of Rubisco rbcL transcript and It is interesting to note that during leaf development in protein to BS cells during chlorenchyma development amaranth, changes in patterns of C4 photosynthetic gene indicates that rbcL mRNA accumulation (either transcrip- expression are coordinated with the maturation of the tional control or stability of transcripts) has a role in smaller veins and sink-to-source transition; but do not targeting to the BS and in the very early development of C4 correlate with the morphological development of Kranz (before differentiation and positioning of BS chloroplasts). anatomy, which occurs much earlier (Wang et al., 1992; A pattern similar to this for Rubisco gene expression was Wang et al., 1993; Ramsperger et al., 1996). at OUP site access on April 22, 2013 described for light-grown Z. mays, in which Rubisco Analysis of BS and M cells during development in mRNAs are expressed mainly in BS cells from the earliest monocots suggests that the veins have a crucial role in cell- stages of leaf development before structural differentiation specific expression of photosynthetic enzymes, and that the of M and BS cells (Martineau and Taylor, 1985; Langdale leaf vascular tissue provides a positional signal that induces et al., 1988a). A more complex pattern of development was C4 photosynthetic differentiation (Langdale et al., 1988b; shown for A. hypochondriacus leaves (Wang et al., 1992; Nelson and Langdale, 1989; Langdale and Nelson, 1991; Ramsperger et al., 1996). Rubisco rbcL transcripts were Soros and Dengler, 2001). According to this hypothesis, present in both M and BS cells until later stages of each vein would control the differentiation of at least two development (leaves up to 5 and 10 mm in length), while cells in radius beyond the vascular tissue (Langdale et al., Rubisco peptides accumulated in the youngest regions of 2- 1988b; Nelson and Dengler, 1992). mm leaves in a C4 manner (only BS), followed by a C3-like However, in the C4 monocot A. hirta, the leaves have distribution (in BS and M) in 5-mm leaves. At a more a special type of BS cell (so-called distinctive cells) that is advanced developmental stage (leaves 10 mm in length), not associated with vascular bundles. Thus, in these plants Rubisco was expressed exclusively in BS cells (Wang et al., the C4 expression pattern does not depend on direct contact 1992; Ramsperger et al.,1996). This two-step developmental of BS cells with veins (Wakayama et al., 2003). pattern suggests post-transcriptional control of Rubisco The two forms of Kranz in Suaeda have a single expression at the earliest stages of amaranth leaf development compound Kranz unit surrounding the vascular tissue. In (Ramsperger et al.,1996), and additional regulation at the the Schoberia Kranz type (as in S. eltonica), there is contact level of mRNA accumulation (transcription or stability) in the between most, but not all, of the BS cells and the vascular following stages (Wang et al.,1992; PatelandBerry,2008). bundles, whereas in the Salsina type (as in S. taxifolia), In both of the Suaeda species, there was an increase in there is no contact. Yet, in both cases there is a similar Rubisco LSU mRNA accumulation which temporally pre- pattern in the development of C4 features. These observa- ceded the peptide expression, since maximal signal intensity tions support the idea that direct contact with veins is not of mRNA was observed beginning from stage 2, while essential for cellular differentiation and C4 pattern of gene the peptide reached its highest labelling in mature tissue. expression. In this case, there would need to be a different A similar pattern was observed for Z. mays, where Rubisco distribution of vein-derived signals, compared with that LSU transcripts accumulated ahead of the peptides, and the with Kranz tissue surrounding individual veins. How C4 photosynthesis in the genus Suaeda | 3211 positional signals could be transmitted and received in the Dengler NG, Dengler RE, Hattersley PW. 1986. Comparative case of a single compound Kranz unit is not clear. bundle sheath and mesophyll differentiation in the leaves of the C4 Taken together, our findings indicate that these two types grasses Panicum effusum and P. bulbosum. American Journal of of Kranz anatomy, which are suggested to have evolved Botany 73, 1431–1442. independently in subfamily Suaedoideae (Kapralov et al., Dengler NG, Donnelly PM, Dengler RE. 1996. Differentiation of

2006) with NAD-ME type C4 photosynthesis, have similar bundle sheath, mesophyll, and distinctive cells in the C4 grass ontogenetic programmes for development of C4 anatomy Arundinella hirta (Poaceae). American Journal of Botany 83, and biochemistry. Additional studies are needed on repre- 1391–1405. sentatives from different lineages where C has evolved to 4 Edwards GE, Voznesenskaya EV. 2011. C photosynthesis: Kranz determine the pattern in C development and its regulation 4 4 forms and single-cell C in terrestrial plants. In: Raghavendra AS, Sage in relation to structural diversity in Kranz anatomy and 4 RF, eds. Photosynthesis and related CO concentrating mechanisms. evolution. 2 Advances in Photosynthesis Research. Dordrecht: Kluwer Academic Publishers, 29–60.

Acknowledgements Edwards GE, Walker DA. 1983. C3,C4: mechanisms and cellular This material is based upon work supported by the and environmental regulation of photosynthesis. Oxford: Blackwell National Science Foundation under Grants IBN-0236959, Scientific Publications, 542 pp. IBN-0641232, and MCB0544234, NSF Isotope Facility Esau K. 1965. Plant anatomy. New York: Wiley Interscience, 767 pp. Downloaded from Grant DBI-0116203, partly by the Russian Foundation of Fisher DD, Schenk HJ, Thorsch JA, Ferren WR. Jr. 1997. Leaf

Basic Research under Grant 08-04-00936 and a collaborative anatomy and subgeneric affiliation of C3 and C4 species of Suaeda grant between the Russian Foundation of Basic Research, (Chenopodiaceae) in North America. American Journal of Botany 84, 10-04-95512 and the Civilian Research and Development 1198–1210. Foundation, RUB1-2982-ST-10. We are grateful to the

Freitag H, Stichler W. 2000. A remarkable new leaf type with http://jxb.oxfordjournals.org/ Franceschi Microscopy and Imaging Center of Washington unusual photosynthetic tissue in a Central Asiatic genus of State University for use of their facilities and staff Chenopodiaceae. Plant Biology 2, 154–160. assistance, and to C. Cody for plant growth management. Gowik U, Westhoff P. 2011. The path from C3 to C4 photosynthesis. Plant Physiology 155, 56–63.

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