Salsola arbusculiformis, a C3-C4 Intermediate in - Annals of Botany

Annals of Botany 88: 337±348, 2001doi:10.1006/anbo.2001.1457, available online at http://www.idealibrary.com on

Salsola arbusculiformis, a C3±C4 Intermediate in Salsoleae (Chenopodiaceae)

ELENA V. VOZNESENSKAYA{, ELENA G. ARTYUSHEVA{, VINCENT R. FRANCESCHI} ,VLADIMIR I. PYANKOV{, OLAVI KIIRATS} , MAURICE S.B. KU}

and GERALD E. EDWARDS*}

{Laboratory of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences,Prof. Popov Street 2, 197376 Saint-Petersburg, Russia, {Department of Plant Physiology, Urals State University,Lenin Avenue 51, 620083 Ekaterinburg, Russia and }School of Biological Sciences, Washington State University,

Pullman, WA 99164-4236, USA

Received: 23 January 2001 Returned for revision: 13 March 2001 Accepted: 9 April 2001

cells, while

0305-7364/0

* For corrwsu.edu

Salsola arbusculiformis is identi®ed as a C3±C4 intermediate species based on anatomical, biochemical and physio-logical characteristics. This is the ®rst report of a naturally occurring intermediate species in the Chenopodiaceae, thefamily with the largest number of C4 species amongst the dicots. In the genus Salsola, most species have Salsoloidanatomy with Kranz type bundle sheath cells and C4 photosynthesis, while a few species have Sympegmoid anatomyand were found to have non-Kranz type bundle sheath cells and C3 photosynthesis. In the cylindrical leaves of C4Salsola with Salsoloid type anatomy, there is a continuous layer of distinct, chlorenchymatous Kranz type bundlesheath cells surrounded by a single layer of mesophyll cells; whereas species with Sympegmoid type anatomy have anindistinct bundle sheath with few chloroplasts and multiple layers of chlorenchymatous mesophyll cells. However,S. arbusculiformis has intermediate anatomical features. While it has two-to-three layers of mesophyll cells,characteristic of Sympegmoid anatomy, it has distinctive, Kranz-like bundle sheath cells with numerous chloroplastsand mitochondria. Measurements of its CO2 compensation point and CO2 response of photosynthesis showS. arbusculiformis functions as an intermediate species with reduced levels of photorespiration. The primary means ofreducing photorespiration is suggested to be by re®xing photorespired CO2 in bundle sheath cells, since analysis ofphotosynthetic enzymes (activity and immunolocalization) and 14CO2 labelling of initial ®xation productssuggests minimal operation of a C4 cycle. # 2001 Annals of Botany Company

Key words: Immunolocalization, photosynthetic enzymes, C ±C intermediate, C -plants, leaf anatomy,
3 4 4Chenopodiaceae, Salsola arbusculiformis.
INTRODUCTION

Most Salsola species ( family Chenopodiaceae) haveso-called Salsoloid (Carolin et al., 1975) or `crown-centric'(Voznesenskaya and Gamaley, 1986) Kranz leaf anatomy.In leaves of this type, chlorenchyma is represented by twolayers of green cells positioned around the periphery of theleaf: the outer layer is composed of palisade mesophyll cellsand the inner layer of bundle sheath cells. The mainvascular bundle is in the centre of the assimilating organssurrounded by the water-storage tissue, and only small,peripheral bundles have contact with chlorenchyma.

It is known that all species with Salsoloid Kranz anatomyin photosynthetic organs (irrespective of whether these areleaves, stems or cotyledons) have C4 type photosynthesis(Zalenskii and Glagoleva, 1981; Pyankov and Vakhrusheva,1989; Pyankov et al., 1999, 2000). However, it is also knownthat some species in the genus Salsola (tribe Salsoleae) havea di�erent type of leaf anatomy. Rojanovskii (1970)described the leaf anatomy of two closely related specieswith di�erent structural features: one (Salsola arbusculaPall.) has Salsoloid type leaf anatomy with hypodermal

the other species (S. arbusculiformis Drob.) does

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espondence. Fax 001 509 335 3184, e-mail edwardsg@

not have a hypoderm and its chlorenchyma consists oftwo-to-three layers of palisade cells and a third, discontin-uous layer of bundle sheath cells, arranged only adjacent tothe peripheral bundles. This latter type of anatomy,described by Carolin et al. (1975) in the genus Sympegmaand in Salsola webbii, was designated as `Sympegmoid'.These authors de®ned Sympegmoid as having non-Kranztype anatomy when they observed multiple layers ofmesophyll chlorenchyma and indistinct bundle sheath cellswith few chloroplasts. The same leaf structure was alsodescribed in Salsola tamariscifolia (Voznesenskaya, 1976).However, Butnik (1984) examined Salsola montana andSalsola pachyphylla, which have the same leaf anatomy, butshe de®ned it as centric, Kranz-discontinuous type anatomy.Recently, we examined the relationship between structureand biochemistry in Salsola oreophila, another species with asimilar Sympegmoid leaf structure. We showed thatS. oreophila has C3 type d

13C values, high Rubisco activity,but low activity of C4 enzymes (Pyankov et al., 1997). It alsohas two-to-three layers of mesophyll cells and thin-walledbundle sheath cells with few chloroplasts usually distributedin the centrifugal position; thus, all structural features in thisspecies are C3-like. In contrast, in S. arbusculiformis, thebundle sheath cells were found to be Kranz-like with rather
numerous chloroplasts, and the walls of these cells were
# 2001 Annals of Botany Company

thicker than those of the mesophyll (Pyankov et al., 1997).Since S. arbusculiformis has two-to-three layers of meso-phyll cells, but a much more distinctive chlorenchymatoussheath than found in Sympegmoid species like S. oreophilia,it was suggested it might be a C3±C4 intermediate (Pyankovet al., 1997).

The aim of the present study was to determine whetherS. arbusculiformis is a C3 or a C3±C4 intermediate species.Comparative studies were made on the leaf anatomy, andbiochemical and physiological characteristics of photo-synthesis in this species and the C4 Salsoloid NADP-ME

338 Voznesenskaya et al.ÐPhotosynthetic

type species, S. arbuscula.

micrograph).

PAS (periodic acid � Schi�'s) staining.

MATERIALS AND METHODS

Plant material

For light and electron microscopy studies and investigationof the polysaccharide content and enzyme activity, Salsolaarbusculiformis Drob. and S. arbuscula Pall. were grown ina growth chamber (model GC-16; Enconair EcologicalChambers Inc., Winnipeg, Canada) from seeds collected indeserts of the Bukhara region of Uzbekistan in Central Asiaduring autumn 1996. Seeds were stored at 3±5 8C beforegermination. After refrigeration, seeds were germinated onmoist paper at room temperature before being transplantedto the soil. Plants were grown in a growth chamber underapprox. 400 mmol photosynthetic quanta mÿ2 sÿ1 with a14/10 h light/dark photoperiod and 25/18 8C day/nighttemperature regime. Both species have linear or teretesucculent leaves, which are rather long (up to 4±5 cm) inyoung plants and rather small (1.5±2 cm) in older ones.Leaves were sampled from 6-week-old plants. They werefully expanded and samples were taken at the same time fordetermination of enzyme activity and light and electronmicroscopy.

For studies on quantitative anatomy and analysis ofphotosynthetic products, plants were grown under ®eldconditions in the summer (mid June±mid July) at the UralsState University ®eld station, about 50 km from Ekaterin-burg, Russia.

For analysis of CO2 compensation point (G) and rates ofphotosynthesis under various CO2 concentrations, plantswere grown in a greenhouse during the summer at
Washington State University, Pullman WA, USA.
Light and electron microscopy

All samples for microscopy, immunolocalization andstarch analysis were taken from fully developed leaves ofplants grown in a growth chamber. Samples for ultra-structural characterization were ®xed overnight at 4 8C in2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehydein 0.1M phosphate bu�er (pH 7.2), post®xed in 2% (w/v)OsO4, and then, after a standard acetone dehydrationprocedure, embedded in Spurr's resin. Cross-sections weremade on a Reichert Ultracut R ultramicrotome (Reichert-Jung GmbH, Heidelburg, Germany). For light microscopy,semi-thin sections were stained with 1% (w/v) Toluidine
blue O in 1% (w/v) Na2B4O7; ultra-thin sections were
stained for electron microscopy with 2% (w/v) lead citrateor 1 :4 dilution of 1% (w/v) KMnO4 :2% (w/v) uranylacetate. Hitachi H-600 and JEM-1200 EX transmissionelectron microscopes were used for electron microscopystudies. The lengths of appressed and non-appressedthylakoid membranes (including both inter- and end-granalthylakoid membranes) were measured with a curvimeter onat least ten chloroplasts. The fraction of cell volumeoccupied by mitochondria and chloroplasts was estimatedby measuring their areas relative to total cell area in leafcross-sections. All areas were measured with an ImageAnalysis program (Image Tool for Windows, version 128,University of Texas Health Science Center, San Antonio,TX, USA) on at least ten micrographs (one cell per

Intermediate Chenopodiaceae Species

Staining for carbohydrates

Sections 0.8±1 mm thick were made from the samesamples, dried onto gelatin coated slides, incubated inperiodic acid (1% w/v) for 30 min, washed and thenincubated with Shi�'s reagent (Sigma, St. Louis, MO,USA) for 1 h. After rinsing, the sections were analysed bylight microscopy. This procedure is referred to in the text as

Quantitative anatomy

The quantitative anatomy of photosynthetic tissues wasstudied according to the method of Mokronosov (1981) andas described in detail recently (Pyankov et al., 1998). Cellnumbers per leaf area were counted on material ®xed in3.5% glutaraldehyde in 0.05 M phosphate bu�er (pH 7.2).Five-to-ten leaves of known area were macerated in 1 N

HCl. Cell numbers were counted in a haemocytometricchamber under the light microscope independently formesophyll and bundle sheath cells that were clearlydistinguished from each other by shape and size (20replications). These data were used to calculate the cellsurface area per unit leaf area (Acell/AL) (see below).For calculation of cell volume, the width and length of 30

mesophyll and 30 bundle sheath cells were determinedunder the light microscope using an ocular micrometer in apressed preparation. For this, ®xed material (three-®veleaves) was placed in 0.5N HCl, left for 5±30 min at roomtemperature and then macerated. Cell volume and cellsurface area were calculated according to earlier studies(Tcelniker, 1978; Pyankov et al., 1998) where

V � pLd

2

� �2

K if L=d is4 2�6;

V � 4

3pL

2

d

2

� �2

if L=d is5 2�6;

S � p2d�2L � d�

where V and S are the volume and surface area of the cellrespectively, L and d are the length and width of the cell
respectively and K is the cell shape-dependent coe�cient.

The surface area of mesophyll and bundle sheath cells per

Voznesenskaya et al.ÐPhotosynthetic

unit of leaf area was calculated as ®xed was determined.

A =A � NS
cell L
where Acell/AL is the cell surface area per unit of leaf area, Nis the cell number per unit leaf area and S is the cell surface
area.
ron-Messelektronic 20046 model, Dresden, Germany).

trapping of antibodies/label (results not shown).

Enzymes extraction and assays

Enzymes were extracted from illuminated leavesharvested during the day. To assay phosphoenolpyruvate(PEP) carboxylase, NAD-malic enzyme (NAD-ME) andNADP-malic enzyme (NADP-ME), about 0.1 g tissue wasground in a pre-chilled mortar with 1 ml of grindingmedium with the aid of acid-washed sand. The grindingmedium contained 50 mM Tris-HCl (pH 7.0), 10 mM

MgCl2, 2 mM MnCl2, 1 mM EDTA-Na2, 5 mM DTT,10% glycerol, 5% insoluble PVP and 1% soluble PVP(MW 40 000). The crude extract was immediatelycentrifuged at 15 000 g for 10 min at 4 8C and thenassayed at room temperature. Activity of PEPC, NADP-ME and NAD-ME was measured spectrophotometricallyin 1 ml reaction mixture at approx. 30 8C. The assaymedium for PEPC contained 50 mM Tris-HCl (pH 8.0),5 mM MgCl2, 2 mM DTT, 1 mM NaHCO3, 0.5 mM

glucose-6-phosphate, 0.2 mM NADH, 10 mM PEP, 3units MDH and 50 ml extract. The reaction was startedwith PEP. The reaction mixture for the NADP-ME assaycontained 25 mM Tricine-KOH (pH 8.3), 5 mM malate-Na, 0.5 mM NADP, 0.1 mM EDTA-Na2, 20 mM MgCl2and 20 ml extract. The reaction was started by addition ofmalate. All rates were obtained within a range whereenzyme activities were linear with time and extractconcentration. The reaction mixture for the NAD-MEassay contained 25 mM HEPES-KOH (pH 7.2), 2.5 mM

NAD, 25 mM NADH, 5 mM DTT, 0.2 mM EDTA-Na2,5 mM malate, 3 units MDH, 75 mM coenzyme A or 25 mMacetyl coenzyme A, 10 mM MnCl2, and 20 ml extract(Hatch et al., 1982). The reaction was started by additionof MnCl2.

To assay Rubisco, illuminated leaves were frozen inliquid N2 in a small mortar and ground to a ®ne powder atthe temperature of liquid N2. The powder was thentransferred to a Ten-Broeck grinder with cold extractionbu�er (1 ml per 50 mg f. wt) and ground for about 30 s.The extraction bu�er contained 100 mM HEPES-KOH(pH 8.0), 20 mM MgCl2, 5 mM DTT and 2 mM EDTA.The extract was then centrifuged in an Eppendorfmicrocentrifuge (1 min, 14 000 rpm) and assayed immedi-ately. The reaction was run at 25 8C and started by adding50 ml of extract to 450 ml of grinding medium containing1 mM RuBP and 10 mM NaH14CO3. The reaction wasstopped after 30 s by addition of 0.5 ml 1 N HCl � 4 N

formic acid, and the acid-stable 14C counts were deter-mined in a scintillation counter. To calculate the speci®cactivity for CO ®xation (mmol CO incorporated per dpm
2 214CO2 ®xed), a separate reaction was run to completion
with addition of 5 nmol RuBP and the amount of 14C

Intermediate Chenopodiaceae Species 339

Initial products of photosynthesis

Mature leaves that were 30±40 d old (approx. 0.5±1 g f.wt) were placed in a photosynthetic chamber (0.25 l). Gascontaining 14CO2 (about 0

.04%;) was injected into the leafchamber; the speci®c activity was about 5 mCi lÿ1. A plantsample was ®xed after 10 s of 14CO2 assimilation byplunging it into boiling alcohol. The temperature duringexperiments was approx. 25 8C and the light intensity was2000 mmol quanta mÿ2 sÿ1. Labelled products wereextracted and separated using two dimensional paperchromatography, where the ®rst solvent contained buta-nol-formic acid-water (13 :12 :75), and the second solventwas 80% phenol. The positions of labelled compounds wereidenti®ed using x-ray ®lm. After elution from the chroma-tography paper, the radioactivity of individual compoundswas determined with a Geiger-MuÈ ller counter (Veb Robot-

In situ immunolocalization by light microscopy

Leaf samples were ®xed for 12±24 h at 4 8C in 2% (v/v)paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M

phosphate bu�er, pH 7.2. The samples were dehydratedwith a graded ethanol series and embedded in LondonResin White (Electron Microscopy Sciences, FortWashington, PA, USA) acrylic resin. Antibodies usedwere rabbit anti-spinach Rubisco (LSU) IgG (courtesy ofB. McFadden, Washington State University, USA) andcommercially available anti-maize PEPC IgG (Chemicon,Temecula, CA, USA). Cross-sections, 0.8±1 mm thick,were placed onto gelatin coated slides and blocked for1 h with TBST � BSA (10 mM Tris-HCl, 150 mM NaCl,0.1% v/v Tween 20, 1% w/v bovine serum albumin, pH7.2). They were then incubated for 3 h with the preimmuneserum diluted in TBST � BSA (1 :100), anti-Rubisco(1 :500 dilution), and anti-PEPC (1 :100 dilution) anti-bodies. The slides were washed with TBST � BSA andthen treated for 1 h with protein A-gold (20 nm particlesdiluted 1 :100 with TBST � BSA). After washing, thesections were exposed to a silver enhancement reagent for20 min according to the manufacturer's directions (Amer-sham, Arlington Heights, IL, USA), stained with 0.5%(w/v) Safranin O, and imaged in a re¯ected/transmittedmode using a BioRad 1024 confocal system with NikonEclipse TE 300 inverted microscope and Lasergraph imageprogram 3.10. The background labelling with pre-immuneserum was very low, although some infrequent labellingoccurred in areas where the sections were wrinkled due to

CO2 compensation point (G) and photosynthetic CO2response

The value of G was determined at 25 8C and 19.7 kPa O2by measuring CO assimilation in response to low
2atmospheric partial pressures of CO2 (0±7 Pa for C3 and

C3±C4 species and 0±2 Pa for C4 species) at two lightintensities (213 and 1200 mmol quanta mÿ2 sÿ1), and byextrapolating the initial CO2 response curve through the x-axis (see Dai et al., 1996). An Analytical Development Co.(ADC) infrared gas analyzer (IRGA) (225-MK3; PP


Systems, Haverhill, Mass., USA) and a Bingham Interspace

FIG. 1. General anatomy (A-D) and starch localization (E and F) in leaveand B, Representative leaf cross-sections of S. arbusculiformis (A) and Sshowing the position of palisade mesophyll (PM) and bundle sheath (BSS. arbuscula, x200. Bar � 100 mm. E and F, PAS (periodic acidÐSchi�'s)mesophyll and bundle sheath, x190. Bar � 100 mm. F, S. arbuscula sh

Hypodermis; BS, bundle sheath; PM, palisade mesoph

(Hyde Park, Utah, USA) Model BI-2-dp Mini CuvetteManual Controller System were used.

The CO2 response curve for photosynthesis up to CO2

saturation rates was measured at 1.9 vs. 19.7 kPa O2 undervarying intercellular levels of CO2 at 25 8C and a PPFD of


1200 mmol mÿ2 sÿ1.

s of Salsola arbusculiformis (A,C,E) and Salsola arbuscula (B, D, F). A. arbuscula (B) �90. Bars � 100 mm. C and D, Higher magni®cation) cells in both species. C, S. arbusculiformis, x250. Bar � 100 mm. D,staining for carbohydrates. E, S. arbusculiformis showing starch in bothowing starch is restricted to bundle sheath, x220. Bar � 100 mm. H,yll; VB, vascular bundle; WS, water-storage tissue.

TABLE 1. Types of leaf structure, quantitative anatomy, and chloroplast granality in mesophyll (M) and bundle sheath (BS)cells of Salsola arbuscula and Salsola arbusculiformis

Character Cell type S. arbusculiformis S. arbuscula

Leaf anatomy Sympegmoid, but Kranz-likebundle sheath

Kranz Salsoloid

Index of chloroplast granality* M 58.5 53.7BS 59.2 40.4

Chloroplast number per cell M 62.1 13.0BS 46.8 26.1

Fraction of cell volumeChloroplasts BS 0.26 0.67Mitochondria BS 0.13 0.02

Cell volume, 103 mm3 M 41.0 8.1BS 21.3 26.6

Cell surface area per leaf area (A cell/AL) M 38.4 25.8BS 7.4 21.3

N � 10 for granality, N� 30 for chloroplast number per cell and for measurements of cell size, N � 20 for cell surface area per unit leaf area.Standard errors as % of means were 2±4 % for chloroplast granality, 2±3 % for length and diameter measurements for cell volumedetermination, 7±12 % for chloroplast number per cell, and 3±5 % for cell number per unit leaf area which was used to calculate Acell/AL.

*Index of chloroplast granality is the ratio of the length of all appressed thylakoid membranes as a percentage of the total length of all thylakoidmembranes in the chloroplast.

Voznesenskaya et al.ÐPhotosynthetic Intermediate Chenopodiaceae Species 341

RESULTS

Leaf structure

Figure 1 shows the leaf structure of S. arbusculiformis (A, Cand E) and S. arbuscula (B, D and F). The C4 speciesS. arbuscula has Kranz type Salsoloid anatomy. Underlyingthe epidermis is a layer of chloroplast-containing hypo-dermal cells, often with calcium oxalate crystals, followedby a layer of palisade mesophyll, and then by a continuouslayer of chlorenchymatous bundle sheath cells. An innercore consists of large water-storage cells that also containsome chloroplasts. Small, peripheral vascular bundles are incontact with bundle sheath cells and a large, main vein ispresent in the centre of the leaf surrounded by water-storageparenchyma. Bundle sheath cells have thick secondarywalls; however, on the outer, tangential walls, in places ofcontact between bundle sheath and mesophyll cells, thereare pits containing plasmodesmata (seen by electronmicroscopy; not shown). Organelles occupy most of thebundle sheath cell volume, as in many other species of thisgenus (Fig. 1B and D). Small vacuoles and the nucleus arenear the outer, tangential wall and organelles are in acentripetal position as indicated by electron microscopy(not shown). Bundle sheath chloroplasts have reducedgrana, whereas mesophyll chloroplasts have well developedgrana (Table 1). Thus, this species has the chloroplastdimorphism characteristic of NADP-ME species.

In contrast, the anatomy of S. arbusculiformis leaves ischaracteristic of Sympegmoid type. S. arbusculiformis haswell developed chlorenchyma of two-to-three palisade layersdirectly beneath the epidermis, a discontinuous layer ofbundle sheath cells and large water-storage cells in the centreof the leaf (Fig. 1A, C and E). There is no hypoderm.
Small vascular bundles are arranged under the palisade
parenchyma between the bundle sheath cells and water-storage cells. As in S. arbuscula, a large, main vein is locatedin the centre of the water-storage tissue. Bundle sheath cellwalls in this species have some thickening (Fig. 1A, C; alsosee Pyankov et al., 1997) but, unlike S. arbuscula, they lackpits with plasmodesmata between mesophyll and bundlesheath cells. In S. arbusculiformis, sparse, single plasmo-desmata are distributed throughout the tangential cell wallsbetween mesophyll and bundle sheath cells, as well asthrough radial cell walls between adjacent bundle sheathcells; this is uncharacteristic of C4 plants. However, inS. arbusculiformis, the bundle sheath cells are Kranz-likewith numerous chloroplasts (46.8 per cell, Table 1), incontrast to those in S. oreophila (18 per cell) (Pyankov et al,1997) and S. webbii (unpubl. res.), two known C3 specieswith Sympegmoid anatomy. There is no di�erence in thegranality of chloroplasts in bundle sheath or mesophyll cellsin S. arbusculiformis, while there is obvious di�erentiation inthe chloroplast structure of C4 S. arbuscula, whose bundlesheath chloroplasts have fewer grana (Table 1). Cell volumeswere calculated from measurements made on isolated cells.The volume of bundle sheath cells in S. arbuscula was three-times higher than that of mesophyll cells, whereas inS. arbusculiformis, the volume of mesophyll cells was twicethat of bundle sheath cells (Table 1). Note the volume ofbundle sheath cells relative to that of mesophyll cells isgreater than might appear in leaf cross-sections due to thegreater length of bundle sheath cells in the longitudinaldirection. The main di�erence is that the mesophyll cells ofS. arbusculiformis are larger than those of S. arbuscula. InS. arbusculiformis, the number of chloroplasts per cell ishigher in mesophyll than in bundle sheath cells, while thereverse is true in S. arbuscula. Also, the mesophyll cell
surface area to leaf surface area ratio was higher in

TABLE 2. Activities of photosynthetic enzymes in leaves ofSalsola arbuscula and Salsola arbusculiformis

Activity (mmol mgÿ1 Chl hÿ1)

Enzymes S. arbuscula S. arbusculiformis

RuBP carboxylase 173.4 612.3PEP carboxylase 767.1 66.4NADP-ME 194.1 18.9NAD-ME ND 15.2

ND, Not detected.Results are the average of two separate experiments with di�erent


S. arbusculiformis, while the bundle sheath cell surface areato leaf surface area ratio was higher in S. arbuscula. Thefraction of the bundle sheath cell volume occupied bychloroplasts is very similar in the two species, while formitochondria, the value is much higher in S. arbusculiformis

leaves.

(Table 1).

in S. arbusculiformis than in S. arbuscula (not shown).

Starch localization

PAS staining for carbohydrates demonstrates that starchis preferentially localized in bundle sheath cells of the C4species S. arbuscula, with only a few, small grains inmesophyll and water-storage tissue (Fig. 1F). In contrast,PAS staining for carbohydrates in S. arbusculiformisreveals that numerous starch grains occur in bothmesophyll and bundle sheath cells, while only some smallsingle grains occur in water-storage tissue (Fig. 1E). Also,the density of starch labelling is higher in the bundle sheathcells around the vasculature than in the mesophyll cells
between bundles.
(Table 3).

Enzyme activity

The activities of the photosynthetic enzymes PEPC andNADP-ME were about an order of magnitude higher inS. arbuscula than in S. arbusculiformis (Table 2). PEPC andNADP-ME activity in S. arbuscula were of the magnitudeexpected for NADP-ME type C4 species of Salsoleae(Pyankov et al., 1999, 2000). Activity of NAD-ME, adecarboxylase found in some C4 Salsola species, was alsolow in S. arbusculiformis and undetected in S. arbuscula.In contrast, the activity of Rubisco was higher inS. arbusculiformis than in S. arbuscula. The activities aresimilar between the two species when expressed on the basisof soluble protein (12 and 14 mg soluble protein mg Chlÿ1
for S. arbusculiformis and S. arbuscula, respectively).
high light, respectively).
Immunolocalization of Rubisco and PEPC
Immunolocalization studies on Rubisco in S. arbusculashowed intense labelling in bundle sheath cells, and slightlabelling in mesophyll and water-storage cells, as would beexpected for C4 plants (Fig. 2A). Observations at highmagni®cation indicated that the label was associated with
chloroplasts. The occasional, intense labelling in the vacuole
of hypodermal cells represents an artifact caused by non-speci®c binding of the antibodies to the calcium oxalatecrystals. In contrast to Rubisco distribution, labelling forPEPC was found almost exclusively in the mesophyll cellcytoplasm, with some slight labelling in the hypodermalcells (Fig. 2B).

In contrast, immunolabelling for Rubisco inS. arbusculiformis showed that this enzyme is present inthe chloroplasts of both mesophyll and bundle sheath cells(Fig. 2C). Rubisco was also detected in low levels in somewater-storage cells. Immunolabelling for PEPC gave a veryhigh signal in all leaf cells (Fig. 2D and E), but highermagni®cation revealed this label was associated withvacuolar contents (Fig. 2E). Very little labelling forPEPC was seen in the cell cytoplasm of either type ofchlorenchyma cell. We suggest that the PEPC antibody isassociating with phenolic compounds in the vacuole of thisspecies. This material can be seen with certain stainingprocedures and also shows up as a very strong, greenauto¯uorescence when unstained sections are viewed withepi¯uorescence microscopy using a blue excitation(Fig. 2F). This strong auto¯uorescence was not previouslyrecorded when a number of other species in the tribeSalsoleae were examined (Voznesenskaya et al,, 1999). Thisnon-speci®c labelling by the PEPC antibody inS. arbusculiformis prevented evaluation of the intercellulardistribution of PEPC. However, Western blots of leafprotein extracts indicated there is much less PEPC protein


Initial products of photosynthesis

In S. arbuscula, the initial products of CO2 ®xation aretypical of those for C4 plants with about 80% incorpora-tion into C4 acids after 10 s of 14CO2 labelling. On the otherhand, in S. arbusculiformis, the primary initial products ofphotosynthesis are 3-PGA and P-esters while only about11% appeared in the C4 acids malate and aspartate

CO2 compensation point (G)

As described in the methods, G was measured at two lightintensities, 213 and 1200 PPFD. Figure 3 illustratesrepresentative experiments for determining G from thezero intercepts of CO2 ®xation vs. the atmospheric level ofCO2 at 1200 PPFD. In S. arbuscula G was 5 ml lÿ1, which istypical of C4 plants, and is similar to that of the C4 plantSorghum bicolor (Table 4). However, in S. arbusculiformis,G was intermediate (36.7 ml lÿ1 at 220 PPFD and 22 ml lÿ1at 1200 PPFD) compared to the values obtained for the C3plant Nicotiana tabacum (55 and 56.8 ml lÿ1 for low and

Photosynthetic CO2 response

The rate of photosynthetic CO2 ®xation (A) wasmeasured at varying intercellular concentrations of CO2under atmospheric levels of O (19.7 kPa) and 1.9 kPa O
2 2(Fig. 4). S. arbuscula showed the response typical of C4

FIG. 2. In situ immunolocalization of photosynthetic enzymes in leaves as viewed with re¯ected/transmitted confocal microscopy. Label appearsas red dots. A and B, Salsola arbuscula. A, Rubisco, x170. Bar � 100 mm. B, PEPC, x190. Bar � 100 mm. C-F, Salsola arbusculiformis. C,Rubisco, x210. Bar � 100 mm. D and E, PEPC. D, Mesophyll cells with high non-speci®c labelling in vacuoles, x500. Bar � 100 mm. E, Highermagni®cation of leaf fragment with heavily labelled vacuoles due to binding on phenolics, �300. Bar � 100 mm. F, Leaf of S. arbusculiformis

showing strong green auto¯uorescence in vacuoles of all cells, �70. Bar � 100 mm.


TABLE 3. Incorporation of 14CO2 into primary photosyn-thetic products after 10 s photosynthesis in Salsola arbusculaand Salsola arbusculiformis (% of total 14C assimilated)

Species P-esters � PGA Malate Aspartate Others

S. arbuscula* 7.1 47.4 34.7 10.8S. arbusculiformis 46 6.8 5.3 41.9

*Data adapted from Pyankov and Vakhrusheva (1989).The results are from one experiment; the recovery of counts from the

chromatogram was determined routinely and was 85±95 %.

0 20 40 60 80

3

2

1

0

S. bicolorS. arbuscula

S. arbusculiformis

N. tabacum

CO2 (Ca) (µbar)

A (

µmol

m�2

s�1

)

FIG. 3. Plots of initial slopes for response of CO2 ®xation (A) vs.atmospheric CO2 partial pressure (Ca). The results are representativeexperiments for Salsola arbusculiformis, Salsola arbuscula, Nicotianatabacum and Sorghum bicolor for determination of CO2 compensation

TABLE 4. CO2 compensation points (G) for Salsolaarbusculiformis, Salsola arbuscula, Nicotiana tabacumand Sorghum bicolor determined from zero intercepts of

photosynthetic CO2 response curves (see Fig. 3)

SpeciesPPFD

(mmol mÿ2 sÿ1)G

(mbar)

S. arbusculiformis 213 36.71200 22.0

S. arbuscula 213 51200 5

N. tabacum (C3) 213 55.01200 56.8

S. bicolor (C4) 213 21200 2

Results for the two Salsola species are the average of tworeplications, while the results for the control C3 species, N. tabacum

344 Voznesenskaya et al.ÐPhotosynthetic Intermediate Chenopodiaceae Species

plants to increasing CO2 levels. There was no inhibition ofphotosynthesis by O2, rather 19.7 kPa stimulated photo-synthesis under the higher CO2 levels (observed in twoexperiments). The value of G was low, and photosynthesissaturated at an intercellular CO2 of approx. 100 mbar. Thestomata of S. arbuscula also tended to shut down rapidlywith CO2 above atmospheric levels. In S. arbusculiformis,intercellular CO2 levels up to approx. 600 mbar wererequired to become near saturating for photosynthesis,and the higher O2 level was inhibitory. The maximum rateof photosynthesis in S. arbuscula on a leaf area basis waslower than in S. arbusculiformis when measured at 25 8Cunder a PPFD of 1200 mmol photon mÿ2 sÿ1. However,expressed on a chlorophyll basis, the maximum rate forS. arbuscula was 178 mmol mgÿ1 Chl hÿ1 (calculated basedon 112 mg Chl mÿ2) compared to 249 mmol mgÿ1 Chl hÿ1in S. arbusculiformis (calculated based on 232 mg Chl mÿ2).In S. arbusculiformis, the predicted response of photo-

synthesis was simulated using a model for C3 photosyn-thesis under saturating RuBP (Fig. 4). The results showthat the predicted rate of photosynthesis in S. arbusculi-formis was lower than the experimental rate whenintercellular CO was less than 60 mbar, and higher than

points at 1200 PPFD (see Table 4).

2the experimental rate at CO2 levels above 60 mbar.

DISCUSSION

The two Chenopodiaceae species used in the present study,S. arbusculiformis and S. arbuscula, belong to sectionCoccosalsola, in the subsection Arbusculae, of the genusSalsola (Botschantzev, 1976). They have some very similarmorphological features including size, degree of branching,length and form of leaves, and degree of occurrence ofshort, rigid hairs on young shoots and leaves. They usuallyoccupy similar ecological niches in foothill areas. Thesection Coccosalsola includes Salsola species known to be
NADP-ME C4 type with Salsoloid type Kranz anatomy,
and a few C3 species which have Sympegmoid type non-Kranz anatomy (see scheme in Pyankov et al., 1997).

S. arbuscula is classi®ed as an NADP-ME type C4 specieswith Salsoloid type anatomy (Pyankov and Vakhrusheva,1989), which very closely resembles that of Salsola richteri(Voznesenskaya et al., 1999). In contrast, S. arbusculiformisis classi®ed as a C3±C4 intermediate species based oncharacteristics of leaf anatomy, physiology and bio-chemistry.

In S. arbusculiformis, the arrangement of leaf tissue withmultiple layers of mesophyll cells and a discontinuousbundle sheath adjacent to peripheral bundles is Sympeg-moid type; but the bundle sheath cells are distinctive andKranz-like, having some wall thickening and rathernumerous chloroplasts. The anatomical features ofS. arbusculiformis are characteristic of C3±C4 intermediates,which have a large investment in multiple layers ofmesophyll chlorenchyma and signi®cant investment inchlorenchymatous bundle sheath. In contrast, C3 plantsnormally have many chlorenchymatous mesophyll cells andlittle or no chlorenchyma development in bundle sheathcells, while C4 plants have a shared investment inchlorenchyma tissue between single layers of mesophylland bundle sheath tissue (Edwards and Ku, 1987). C3 plantsand C3±C4 intermediates have a high mesophyll surface areaper leaf area, which facilitates di�usion of CO2 to Rubisco in

and C4 species S. bicolor, are from one experiment.

mesophyll cells, while C4 plants, which possess the CO2

0 200 400 600

Intercellular CO2 (µbar)

CO

2 as

sim

ilat

ion

(µm

ol m

�2 s

�1)

8

6

4

2

0

15

0

5

10

S. arbusculiformis

S. arbuscula

1.9 kPa O2

19.7 kPa O2

19.7 kPa O2

1.9 kPa O2

FIG. 4. Plots of CO2 response curves for rates of CO2 assimilation inSalsola arbuscula and Salsola arbusculiformis at 1.9 and 19.7 kPa O2,leaf temperature of 25 8C, and light intensity of 1200 PPFD.Photosynthesis in S. arbusculiformis was simulated (broken lines inthe ®gure) using a model for C3 phototosynthesis under saturatingRuBP based on Rubisco kinetic properties at 25 8C having Km CO2 of9 mM, Km O2 of 535 mM, speci®city factor for reacting with CO2 vs. O2of 88 (Woodrow and Berry, 1988), and with Vmax of RuBP carboxylaseand oxygenase on leaf area basis of 40 and 27 mmol mÿ2 sÿ1,respectively. The results shown are from one experiment; replication on

Voznesenskaya et al.ÐPhotosynthetic

concentrating mechanism, have a lower internal mesophyllsurface area (Longstreth et al., 1980).

In the C3±C4 intermediate S. arbusculiformis, theinvestment in mesophyll chlorenchyma is clearly higherthan that in the C4 S. arbuscula when chloroplast numbersare compared on a cell, cell volume, or cell surface area perunit leaf area basis (Table 1). On the other hand, inS. arbuscula, the development of bundle sheath is clearlyhigher than in S. arbusculiformis with respect to cell volumeand cell surface area per unit leaf area. However,S. arbusculiformis has Kranz-like bundle sheath cells dueto the large number of chloroplasts per cell (Table 1).

A common feature of all identi®ed intermediates is anobvious chlorenchymatous bundle sheath. Among these, afew intermediates have Kranz type bundle sheath with

a separate day gave similar results.

thickened walls and a high density of chloroplasts like

S. arbusculiformis (i.e. Neurache minor, Flaveria brownii),while others have a more poorly developed Kranz anatomy(Moricandia arvensis) (Edwards and Ku, 1987; Monson,1999).

The combination of Kranz-like bundle sheath cells inS. arbusculiformis, with multiple layers rather than a singlelayer of mesophyll cells, is suggestive of C3±C4 intermediatephotosynthesis. However, more evidence is needed todetermine whether S. arbusculiformis functions as anintermediate. A few species with a clearly chlorenchyma-tous bundle sheath have been classi®ed as C3 species basedon biochemical (i.e. initial products of CO2 ®xation) andphysiological properties (G and O2 sensitivity of photosyn-thesis) (Crookston and Moss, 1970; Edwards et al., 1990).

Gas exchange analysis shows that S. arbuscula ischaracteristic of C4 plants in having a low G, little or nosensitivity to O2, and saturation of photosynthesis atrelatively low levels of CO2. The most important functionaltest for a C3±C4 intermediate is measurement of G(Edwards and Ku, 1987). S. arbusculiformis has anintermediate G, indicating a reduction in apparent photo-respiration compared to C3 plants. The value of G decreaseswith increasing light intensity, which has been observed insome intermediate Flaveria species (Dai et al, 1996). Asnoted in that study, a decrease in G with increasing lightintensity in intermediates may occur by increasing thedegree of re®xation of photorespired CO2 in bundle sheathcells. In terms of the CO2 response of photosynthesis andthe e�ect of O2, S. arbusculiformis has intermediatefeatures. The value of G at 19.7 kPa O2 and 1200 PPFDis 22 mbar. At 19.7 kPa O2, there is an increasingdiscrepancy between the predicted rate of photosynthesisby the C3 model and the experimental rate inS. arbusculiformis as CO2 decreases below 60 mbar. Theresults indicate this species has a lower apparent level ofphotorespiration and O2 inhibition of photosynthesis underlimiting CO2 which is characteristic of C3±C4 intermediates.

When the activities of Rubisco, reported on a chlorophyllbasis in Table 2, are expressed on a leaf area basis, thecorresponding rates are 5.4 and 39.4 mmol mÿ2 sÿ1 forS. arbuscula and S. arbusculiformis, respectively. Thus, themeasured Rubisco activity in S. arbuscula is just su�cient toaccommodate the maximum rate of photosynthesis, while inS. arbusculiformis, the capacity is in excess of the maximumrate, as shown by the simulation, suggesting some otherlimitation on photosynthesis under high CO2 (Fig. 3).

Common features of intermediates are a lower G than inC3 plants (which results in higher rates of CO2 ®xation thanin C3 species under limiting CO2) and numerous chloro-plasts in bundle sheath cells. Also, in intermediates, thebundle sheath chloroplasts ®x CO2 by the C3 cycle.Reduced photorespiration may occur in intermediates bytwo mechanisms (Edwards and Ku, 1987; Rawsthorne andBauwe, 1998; Monson, 1999). One is via re®xing photo-respired CO2 by compartmentation of glycine decarboxy-lase in bundle sheath mitochondria (which are moreprominent than in C3 species), such that any photorespira-tion which occurs as a consequence of RuBP oxygenase inmesophyll cells will result in CO being generated in bundle

Intermediate Chenopodiaceae Species 345

2sheath cells, as in Moricandia arvensis. Obviously, if all

some light on their evolutionary relationships.

photorespired CO2 could be re®xed in bundle sheath cells, Gwould approach zero. The other mechanism which maycontribute to reduced photorespiration in some inter-mediates is a partially functional C4 cycle, e.g. as inFlaveria ramosissima (Edwards and Ku, 1987; Rawsthorneand Bauwe, 1998; Monson, 1999). Without cell speci®ccompartmentation of photorespiration or a partially func-tional C4 cycle, bundle sheath chloroplasts may function asin the mesophyll under normal C3 photosynthesis.Interestingly, in S. arbusculiformis, the fraction of bundle

sheath cell volume occupied by mitochondria is 50% ofthat of chloroplasts and is much higher than that in S.arbuscula (Table 1). This is as expected if S. arbusculiformisfunctions as an intermediate as described above forMoricandia arvensis, whereas in NADP-ME type specieslike S. arbuscula, there is little role for mitochondria in C4

photosynthesis. The results of the present study withS. arbusculiformis indicate that it has little capacity for C4

photosynthesis. Only 10% of the initial products ofphotosynthesis appeared in C4 acids. Furthermore, theactivities of PEPC and NADP-ME enzymes in S. arbuscu-liformis are much lower than in the C4 S. arbuscula.However, the activity of PEPC in S. arbusculiformis(66 mmol mgÿ1 Chl hÿ1, Table 2) is higher than inS. oreophila (12 mmol mgÿ1 Chl hÿ1) (Pyankov et al.,1997), a C3 Salsola species with Sympegmoid anatomyhaving few chloroplasts in bundle sheath cells, whereas theactivities of NADP-ME are low and similar in the twospecies [data of Table 2 and Pyankov et al. (1997)]. At leastpart of this activity in S. arbusculiformis may be constitu-tive, as in C3 plants, and not associated with any capacityfor C4 photosynthesis. As previously shown in studies withthe C3±C4 intermediate Panicum milioides, C4 photosyn-thesis may not occur even if PEPC is somewhat higher thanin C3 species (Edwards et al., 1982).

In S. arbusculiformis, Rubisco occurs in both layers ofmesophyll cells as well as in bundle sheath cells, asdemonstrated by immunolabelling. This can be comparedwith C4 species and some other intermediates. In C4 plants,Rubisco is speci®cally located in bundle sheath cells, andnot in the adjacent mesophyll cells. However, in somestudies with some C4 plants, distal mesophyll cellsÐ notadjacent to the bundle sheathÐ also have Rubisco andexhibit the C3 pathway of photosynthesis, as observed incotyledons of Salsola laricina and the stem of Haloxylonpersicum (Voznesenskaya et al., 1999). A C4-like species,Flaveria brownii, shows a gradient of decreasing activity ofRubisco from bundle sheath to palisade mesophyll to moredistal chlorenchyma cells (Cheng et al., 1988), as does theintermediate F. ramosissima from bundle sheath to palisademesophyll to spongy mesophyll (Moore et al., 1988).However, in other Flaveria intermediates, immuno¯uores-cent labelling indicated a similar distribution of Rubisco inmesophyll and bundle sheath cells, e.g. F. pubescens andF. anomala (Bauwe, 1984) and F. ¯oridana, F. linearis andF. chloraefolia (Reed and Chollet, 1985). This is the case inS. arbusculiformis, where Rubisco is clearly located in bothmesophyll and bundle sheath cells, indicating the C cycle is


3

functioning in both cell types.

In C3 plants, Rubisco discriminates against ®xingatmospheric 13CO2 (resulting in more negative carbonisotope values), which is prevented or minimized in C4plants where atmospheric CO2 is delivered to Rubisco inbundle sheath cells via the C4 cycle. Thus, if intermediates®x atmospheric CO2 via Rubisco in mesophyll cells, andreduce G by re®xing photorespired CO2 in bundle sheathcells, their carbon isotope composition will be like that ofC3 plants. However, if they reduce photorespiration via apartially functioning C4 cycle, the isotope composition isexpected to have an intermediate value (Edwards and Ku,1987). The average carbon isotope values for S. arbusculi-formis, S. oreophila and S. arbusculawere ÿ23.1, ÿ27.2 andÿ13.0, respectively (Pyankov et al., 1997). Thus, theaverage value of S. arbusculiformis is closer to C3 than toC4 plant values. This, along with evidence that there is littlecapacity for C4 in S. arbusculiformis, suggests that it, likemost other intermediates (Edwards and Ku, 1987), reducesphotorespiration mainly by re®xing photorespired CO2 inbundle sheath cells. However, the values in S. arbusculi-formis are less negative than those in S. oreophila, indicatingthat a C4 cycle may make a small contribution tophotosynthesis under certain conditions in this species.

S. arbusculiformis is the ®rst naturally occurring C3±C4intermediate to be identi®ed in the family Chenopodiaceae,a family that has been found to contain the most C4 speciesamong the dicots. Prior to this, intermediates had beenfound in seven families (Amaranthaceae, Asteraceae,Brassicaceae, Cyperaceae, Molluginaceae, Hydrocharita-ceae and Poaceae) and 11 genera (Sage et al., 1999).Although S. arbusculiformis is the ®rst naturally occurringintermediate identi®ed in the family Chenopodiaceae,substantial work has been done on hybrids obtained fromcrossing C4 and C3 species of Atriplex (A. rosea, C4 �A. triangularis, C3), a genus in the same family which hasAtriplicoid type anatomy (Osmond et al., 1980). Althoughindividual hybrids were intermediate in some features (e.g.leaf anatomy, PEPC activity) they did not show thecombined features for functional intermediacy (e.g. reducedphotorespiration) which have been observed in all naturallyoccurring intermediate species including S. arbusculiformis(BjoÈ rkman et al., 1970, 1971).

The evolutionary relationships between Salsoloid vs.Sympegmoid type anatomy in the tribe Salsoleae areunclear. Carolin et al. (1975) proposed that Sympegmoidanatomy evolved from Salsoloid, which is consistent withthe suggestion of Pyankov et al. (1997, 2001) that C3Sympegmoid type Salsola species, some of which occur athigher elevations, may be reversions from C4 species. Onthe other hand, Akhani et al. (1997) proposed thatSalsoloid anatomy evolved from the Sympegmoid type, inwhich case S. arbusculiformis might be an intermediate inthe path of evolution from C3 to C4. A more detailedmolecular phylogenetic analysis of these species may shed


ACKNOWLEDGEMENTS

This work was supported partly by Civilian Research and
Development Foundation, grant RB1-264 (project leaders


V.I.P and G.E.E) and NSF Grant IBN-9807916 (G.E.E.).E.V. Voznesenskaya would like to thank CIES, WashingtonD.C., for a Fulbright Scholar Research Fellowship. We alsothank the Electron Microscope Center of Washington State
University for use of their facilities and sta� assistance.
NOTE ADDED IN PROOF

Immunolocalization of glycine decarboxylase (antibody-against the P-protein provided by Dr D. Oliver, Iowa StateUniversity, Ames Iowa, USA) in S. arbusculiformis byelectron microscopy showed that this enzyme is exclusivelylocated in mitochondria of bundle sheath cells (A, above)without label in mesophyll cells (B, above) (gold particlesshown by black dots, x 25 000. Bar � 0.5 mm. Ch,chloroplasts; M, mitochondria). Localization of thisenzyme of the glycolate pathway in bundle sheathmitochondria is a feature which supports the observationof an intermediate CO compensation point in this C -C
2 3 4species.
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