Journal of Plant Research (2019) 132:839–855 https://doi.org/10.1007/s10265-019-01135-5

REGULAR PAPER

Transition from C­ 3 to proto‑Kranz to ­C3–C4 intermediate type in the genus Chenopodium (Chenopodiaceae)

Yuki Yorimitsu1 · Aya Kadosono2 · Yuto Hatakeyama1 · Takayuki Yabiku1 · Osamu Ueno1,2,3

Received: 11 July 2019 / Accepted: 26 August 2019 / Published online: 31 August 2019 © The Author(s) 2019, corrected publication 2020

Abstract

The Chenopodiaceae is one of the families including ­C4 species among eudicots. In this family, the genus Chenopodium is considered to include only C­ 3 species. However, we report here a transition from ­C3 photosynthesis to proto-Kranz to ­C3–C4 intermediate type in Chenopodium. We investigated leaf anatomical and photosynthetic traits of 15 species, of which 8 species showed non-Kranz anatomy and a ­CO2 compensation point (Γ) typical of ­C3 plants. However, 5 species showed proto-Kranz anatomy and a ­C3-like Γ, whereas C. strictum showed leaf anatomy and a Γ typical of ­C3–C4 intermediates. Chenopodium album accessions examined included both proto-Kranz and C­ 3–C4 intermediate types, depending on local- ity. Glycine decarboxylase, a key photorespiratory enzyme that is involved in the decarboxylation of glycine, was located predominantly in the mesophyll (M) cells of ­C3 species, in both M and bundle-sheath (BS) cells in proto-Kranz species, and exclusively in BS cells in ­C3–C4 intermediate species. The M/BS tissue area ratio, number of chloroplasts and mitochondria per BS cell, distribution of these organelles to the centripetal region of BS cells, the degree of inner positioning (vacuolar side of chloroplasts) of mitochondria in M cells, and the size of BS mitochondria also changed with the change in glycine decarboxylase localization. All Chenopodium species examined were ­C3-like regarding activities and amounts of ­C3 and 13 ­C4 photosynthetic enzymes and δ C values, suggesting that these species perform photosynthesis without contribution of the ­C4 cycle. This study demonstrates that Chenopodium is not a ­C3 genus and is valuable for studying evolution of ­C3–C4 intermediates.

Keywords C3–C4 intermediate plant · Chenopodium · CO2 compensation point · Glycine decarboxylase · Leaf anatomy · Proto-Kranz plant

Introduction air, one-fourth of photosynthetically fxed ­CO2 was lost by photorespiration, resulting in decreased photosynthetic ef- Photorespiration is an inevitable metabolic process in C­ 3 ciency (Bauwe 2011; Sage et al. 2012). On the other hand, plants that use ribulose 1,5-bisphosphate carboxylase/oxy- ­C4 plants have biochemical traits of photosynthesis asso- genase (Rubisco) for primary fxation of CO­ 2. In ordinary ciated with anatomical diferentiation of leaves to reduce photorespiration (Hatch 1987). In general, ­C4 leaves exhibit Electronic supplementary material The online version of this Kranz-type anatomy, in which an external layer of meso- article (https​://doi.org/10.1007/s1026​5-019-01135​-5) contains phyll (M) and an internal layer of bundle sheath (BS) encir- supplementary material, which is available to authorized users. cle vascular bundles (Edwards and Voznesenskaya 2011; Lundgren et al. 2014). In M cells, atmospheric ­CO is frst * Osamu Ueno 2 [email protected]‑u.ac.jp fxed in ­C4 compounds, and they are moved to BS cells, where they are decarboxylated to supply CO­ 2 for Rubisco. 1 Graduate School of Bioresource and Bioenvironmental Increased CO­ 2 concentration within BS cells suppresses Sciences, Kyushu University, Motooka 744, Nishi‑ku, photorespiration (Hatch 1987). Therefore, C­ plants have Fukuoka 819‑0395, Japan 4 higher photosynthetic efciency than ­C plants in environ- 2 3 School of Agriculture, Kyushu University, Motooka 744, ments that promote high rates of photorespiration. The C­ Nishi‑ku, Fukuoka 819‑0395, Japan 4 trait has evolved independently at least 66 times in fowering 3 Present Address: Faculty of Agriculture, Kyushu University, plants in response to multiple ecological drivers including Motooka 744, Nishi‑ku, Fukuoka 819‑0395, Japan

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decreasing atmospheric ­CO2 concentration (Sage et al. 2011; Sage et al. 2012, 2014). Type I intermediates would 2012). Much efort has recently focused on elucidating the evolve from the proto-Kranz type with predominant accu- evolution from ­C3 to ­C4 plants (reviewed in Christin and mulation of GDC in BS mitochondria and its decrease in M Osborne 2014; Sage et al. 2012, 2014). These works may mitochondria. Complementation of the glycine shuttle by the provide a clue for engineering of C­ 4 elements into C­ 3 crops increasing activity of the ­C4 cycle would then lead to type II (Schlüter and Weber 2016). intermediates. Finally, the ­C4 type would evolve from type An early study reported the existence of plants with traits II intermediates through the C­ 4–like plants (Edwards and Ku intermediate between those of ­C3 and ­C4 plants (Kennedy 1987; Sage et al. 2012, 2014). and Laetsch 1974). These are called C­ 3–C4 intermediate Among eudicots, the goosefoot family Chenopodiaceae plants (Edwards and Ku 1987; Monson and Rawsthorne (Caryophyllales) includes the greatest number of ­C4 species 2000). Leaves of most ­C3–C4 intermediate plants show (about 40% of 1400 species; Sage et al. 1999). ­C4 species have Kranz-like anatomy, in which BS cells contain numerous been detected in four Chenopodiaceae subfamilies: Cheno- chloroplasts and mitochondria (Edwards and Ku 1987; Sage podioideae, Salicornioideae, Salsoloideae, and Suaedoideae et al. 2014). In these plants, the values of the ­CO2 compensa- (Carolin et al. 1975; Jacobs 2001; Kadereit et al. 2003; tion point (Г) and ­O2 inhibition of photosynthesis are inter- Pyankov et al. 2001; Voznesenskaya et al. 2001b, 2002, 2007; mediate between the values of ­C3 and ­C4 plants (Edwards Wen and Zhang 2011). In the Chenopodioideae, ­C4 species and Ku 1987; Monson and Rawsthorne 2000). An appar- occur in the tribes Atripliceae (e.g., Atriplex and Axyris) and ent reduction in photorespiration in ­C3–C4 intermediates is Camphorosmeae (e.g., Bassia and Kochia), but they have not accomplished by a particular biochemical system operat- been found in the tribe Chenopodieae (Freitag and Kadereit ing between the M and BS cells. This system is called the 2014; Kadereit et al. 2010; Sage 2004). In this family, ­C3–C4 glycine shuttle (Monson and Rawsthorne 2000; Rawsthorne intermediate species have been recorded in the genera Salsola, 1992). In ­C3–C4 intermediates, at least the P-protein, one of Rhaphidophyton, and Sedobassia (Freitag and Kadereit 2014; the 4 subunits constituting the glycine decarboxylase (GDC) Schüssler et al. 2017; Voznesenskaya et al. 2001a, 2013; Wen multi-enzyme system, is absent in the M mitochondria, and Zhang 2015). which renders GDC non-functional, and glycine generated in The genus Chenopodium (Chenopodieae) is considered to the M cells must be transported into the BS mitochondria to include only C­ 3 species (Jacobs 2001; Kadereit et al. 2010). be decarboxylated by GDC (Rawsthorne 1992; Rawsthorne This genus is cosmopolitan and includes about 150 species, et al. 1988). In the BS cells of most C­ 3–C4 intermediates, most of which are annual herbs growing in arid and semi-arid mitochondria are located between the centripetally located regions and also on salt-rich soils (Fuentes-Bazan et al. 2012) chloroplasts and the inner tangential walls (Brown and Hat- and weeds of disturbed habitats and cultivated felds (Judd tersley 1989; Muhaidat et al. 2011; Rawsthorne 1992; Sage and Ferguson 1999). The seeds of some Chenopodium spe- et al. 2013; Ueno et al. 2003; Ueno 2011). As a result, a large cies such as C. quinoa, C. berlandieri, and C. formosanum are part of ­CO2 released from mitochondria by decarboxylation used as cereals, and the leaves and young shoots of C. album of glycine is captured by chloroplasts, resulting in suppres- are eaten as vegetables (Judd and Ferguson 1999). Chenopo- sion of ­CO2 loss from BS cells. Many ­C3–C4 intermediate dium album has been often used as a model plant to study the species reduce photorespiratory CO­ 2 loss only by using the physiology of ­C3 photosynthesis (e.g., Haraguchi et al. 2009). glycine shuttle (type I intermediates), but in some interme- In our preliminary study on leaf anatomy of eudicot spe- diates a ­C4 cycle complements the glycine shuttle (type II cies in Japan, we have recently found that C. album has leaf intermediates; Edwards and Ku 1987). structural traits of the proto-Kranz type, which clearly difer Until now, 56 species with C­ 3–C4 intermediate traits have from those of the typical non-Kranz (C­ 3) type. This fnding been found in 2 monocot and 11 eudicot families (Lundgren motivated us to re-examine leaf anatomy and photosynthetic and Christin 2017). Some genera, such as Flaveria (Ku et al. traits of Chenopodium species from various regions of the 1991; Sage et al. 2013), Heliotropium (Muhaidat et al. 2011; world. This study reports, for the frst time, that Chenopodium Vogan et al. 2007), Salsola (Voznesenskaya et al. 2013), Eleo- includes proto-Kranz and C­ 3–C4 intermediate types as well as charis (Roalson et al. 2010; Ueno et al. 1989), Alloteropsis ­C3 types and is therefore a valuable eudicot group to study the (Bianconi et al. 2018), and Neurachne (Christin et al. 2012), evolutionary and genetic transition from ­C3 to proto-Kranz to include ­C3, ­C3–C4 intermediate, and ­C4 types and provide a ­C3–C4 intermediate plants. unique opportunity to trace the evolution from ­C3 to ­C4 plants. Many of these studies suggest that the initial event in the evo- lution of ­C3–C4 and then ­C4 plants is the appearance of chlo- roplasts and mitochondria along the centripetal region of BS cells, with GDC activity present in both M and BS mitochon- dria. This phase is called the proto-Kranz type (Muhaidat et al.

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Materials and methods seedlings were then transplanted to 5-L pots (one plant per pot) with sandy loam soil containing nitrogen (ammonium Plant materials and growth conditions nitrate), phosphorus (calcium superphosphate), and potas- sium (potassium chloride) fertilizers (1.0 g each). Plants The species and accessions of Chenopodium examined in were grown in a greenhouse [natural sunlight, wherein this study are listed in Table 1. Seeds of two accessions of photosynthetic photon flux density (PPFD) at midday −2 −1 C. album were collected in upland felds of the National exceeded 1500 μmol m­ ­s ; 30–34 °C during the day and Institute of Agro-Environmental Sciences, Tsukuba, 24–27 °C during the night] for 1.5–2 months. Plants were Ibaraki, Japan and along the roadside of Fukuoka City, watered daily. Fully expanded upper mature leaves taken Fukuoka, Japan. Seeds of C. fcifolium were also collected from 3 plants per species (per accession for C. album) in upland felds of the National Institute of Agro-Envi- were used for analysis. ronmental Sciences. Seeds of C. quinoa were provided by the NARO Genebank, Tsukuba, Japan. Seeds of fve other Anatomical and ultrastructural studies accessions of C. album and 12 other species of Chenopo- dium were provided by the USDA Germplasm Resources, Samples taken from the midsections of leaves (one leaf USA (Table 1). The seeds of ­C4 species of Amaranthus (A. per plant) were fxed and embedded in Quetol resin (Nis- cruentus, A. dubius, and A. hybridus), which were used shin EM, Shinjuku, Tokyo, Japan) as reported previously as controls, were also a gift from the USDA Germplasm (Tsutsumi et al. 2017). Semithin sections (1 µm thickness) Resources (Table 1). All seeds were germinated in perfo- were cut with glass knives on an ultramicrotome (Reichert rated multiwell nursery boxes flled with loam soil gran- Ultracut S, Leica, Wien, Austria), mounted on glass slides, ules. Seedlings were grown for 3 weeks in a greenhouse at stained with 1% toluidine blue O, and observed under a light the experimental feld of Kyushu University in July. The microscope (Eclipse Ci-L, Nikon Instech Co. Ltd., Tokyo, Japan). The profile areas of M and BS tissues between

Table 1 List of Chenopodium species examined and control C­ 4 species of Amaranthus Species Locality Germplasm source Accession No.

Chenopodium album L. Arizona, USA USDA Germplasm Resources PI 666270 Finland USDA Germplasm Resources PI 658748 France USDA Germplasm Resources PI 262168 Fukuoka, Japan In the feld India USDA Germplasm Resources PI 658735 Poznan, Poland USDA Germplasm Resources PI 658746 Tsukuba, Japan In the feld C. atrovirens Rybd. Arizona, USA USDA Germplasm Resources PI 666273 C. berlandieri ssp. nuttalliae (Staf.) H. D. Puebla, Mexico USDA Germplasm Resources PI 433231 Wilson and Heiser C. fcifolium Sm. Tsukuba, Japan In the feld C. formosanum Koidz. Taiwan USDA Germplasm Resources PI 433378 C. giganteum D. Don Oklahoma, USA USDA Germplasm Resources PI 596371 C. hians Standl. New Mexico, USA USDA Germplasm Resources PI 666310 C. incanum (S. Watson) A. Heller New Mexico, USA USDA Germplasm Resources PI 666313 C. leptophyllum (Moq.) Nutt. Ex S. Watson Nevada, USA USDA Germplasm Resources Ames 29780 C. nevadense Standl. Nevada, USA USDA Germplasm Resources PI 666321 C. pallidicaule Aellen La Paz, Bolivia USDA Germplasm Resources PI 478406 C. quinoa Willd. Tsukuba, Japan NARO Genebank JP No. 53591 C. standleyanum Aellen Iowa, USA USDA Germplasm Resources PI 666323 C. strictum Roth Saxony, Germany USDA Germplasm Resources PI 665284 C. vulvaria L Santarem, Portugal USDA Germplasm Resources PI 614896 Amaranthus cruentus L. California, USA USDA Germplasm Resources PI 647848 A. dubius Mart. Ex Thell. Nepal USDA Germplasm Resources PI 619238 A. hybridus L. Pennsylvania, USA USDA Germplasm Resources Ames 5580

1 3 842 Journal of Plant Research (2019) 132:839–855 adjacent small vascular bundles were measured using the (University of Idaho, Moscow, ID, USA) and the late Dr. Image J software (National Institutes of Health, Bethesda, S. Muto (Nagoya University, Nagoya, Japan), respectively. MD, USA), and the area ratio of M and BS tissues (M/BS tissue area ratio) was calculated. Simultaneously, the sizes Protein A—immunogold electron microscopy (profle areas) of 8 M cells and 8 BS cells per plant were measured, and the size ratio of M and BS cells (M/BS cell To evaluate exactly the accumulation level of GDC-P in size ratio) was calculated. mitochondria of M and BS cells, a quantitative immuno- Ultrathin sections were cut with a diamond knife on the gold labeling study was made under an electron micro- same ultramicrotome, picked up on Formvar-coated grids, scope. Small leaf segments (one leaf per plant) were fxed stained with lead citrate, and viewed under a transmission and embedded in Lowicryl K4 M resin (Chemische Werke electron microscope (JEM-100CX II K, JEOL Ltd., Tokyo, Lowi GmbH, Waldkraiburg, Germany) as described by Japan) at 75 kV. The numbers and intracellular positions Ueno (1992). Ultrathin sections on Formvar-coated grids of chloroplasts and mitochondria were recorded for 5 M were immunolabeled with the antiserum against GDC-P and cells and 5 BS cells per plant as described by Hatakeyama InnovaCoat Gold − 20 nm protein A nanoparticle conju- and Ueno (2016). In BS cells, we counted chloroplasts and gate (Innova Biosciences, Cambridge, England, UK), stained mitochondria in the inner halves of the cells (i.e., along the with lead citrate, and viewed under a transmission electron inner tangential wall and the inner half of the radial wall) microscope as described by Ueno (1992). As a negative and in the outer halves (i.e., along the outer tangential wall control, the antiserum was replaced by non-immune serum. and the outer half of the radial wall). In M cells, we counted The density of GDC-P labeling was determined for mito- mitochondria on the vacuolar side of chloroplasts (inner chondria and other intracellular locations by counting the position) and on the cell-wall side of chloroplasts, includ- gold particles on electron micrographs at 20,000× magnif- ing isolated mitochondria not associated with chloroplasts cation and calculating the number of particles per unit area but adjacent to the cell wall (outer position), as described (μm−2) with Image J software. We examined 13–18 mito- in Hatakeyama and Ueno (2016). In M cells, chloroplasts chondria of palisade M cells and 20 mitochondria of BS in the intracellular position were not counted because all cells in several sections per leaf. The density of labeling was chloroplasts were adjacent to the cell wall. The numbers of calculated as the mean of 3 plants. chloroplasts and mitochondria per unit area were calculated in each of the 5 M and 5 BS cells using Image J software. Western blots On some sections, the sizes (profle areas) of 10 chloroplasts and 10 mitochondria per plant were measured. Leaves were frozen in liquid nitrogen and stored in a deep To measure vein density, samples taken from the midsec- freezer (− 80 °C). Extraction of soluble proteins, SDS- tions of leaves (one leaf per plant) were fxed in a forma- PAGE, and Western blotting were performed as described lin–acetic acid–alcohol mixture and cleared in 80% lactic by Ueno (1992) with antisera against phosphoenolpyruvate acid and chloral hydrate-saturated ethanol as described by carboxylase (PEPC) and pyruvate, Pi dikinase (PPDK) from Tsutsumi et al. (2017). The vein density (vein length per unit maize leaves (provided by Dr. T. Sugiyama, RIKEN, Yoko- leaf area) was measured using Image J software. hama, Japan). For GDC-P and Rubisco LSU, we used the same antisera for immunohistochemistry.

Immunohistochemistry Enzyme assays

Intercellular immunolocalization of photorespiratory and Parts of frozen leaves were used to measure the activities photosynthetic enzymes in M and BS cells was investigated of Rubisco, PEPC, NADP-malic enzyme (NADP-ME), under a light microscope. Small leaf segments (one leaf per and NAD-malic enzyme (NAD-ME) as described by Ueno plant) were fxed and embedded in parafn, as described by (1992), except that all enzymes were assayed at 30 °C. Hatakeyama and Ueno (2016). Sections (10 µm thick) were cut on a rotary microtome (PR-50, Yamato Kohki Industrial Gas exchange measurements Co. Ltd., Saitama, Japan), mounted on slides coated with poly-l-lysine (Sigma-Aldrich Inc., St Louis, MO, USA), Net CO­ 2 assimilation rate (A) was measured using an and dried overnight. Immunostaining for the P-protein of LI-6400 portable photosynthesis system (Li-Cor Inc., Lin- GDC (GDC-P) and the large subunit of Rubisco (Rubisco coln, NE, USA) at a PPFD of 1000 μmol ­m−2 ­s−1, a leaf LSU) was performed as described by Hatakeyama and Ueno temperature of 30 °C, a relative humidity of 60%, and a −1 (2016) with antisera against GDC-P and Rubisco LSU from ­CO2 concentration of 380 μL ­L , as described in Ueno pea leaves. The antisera were provided by Dr. D. J. Oliver et al. (2003). Light within the chamber was provided by a

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6400-02 LED Light Source (Li-Cor Inc.). The Γ value was C. album, C. berlandieri, C. fcifolium, C. formosanum, C. determined by extrapolating the initial slope of A versus the giganteum, and C. nevadense showed proto-Kranz anatomy intercellular CO­ 2 concentration through the x-axis, where (Table 2). In Kranz-like anatomy (Fig. 1e, f), BS cells con- A equals zero. tained many more chloroplasts in the centripetal region than did proto-Kranz-type BS cells, but fewer than did BS cells Carbon isotope ratio of a ­C4 species of Amaranthus (Fig. S3d). Chenopodium strictum and a C. album accession from Arizona showed One leaf from three plants was air-dried at 80 °C and ground Kranz-like anatomy (Table 2). In all 3 anatomical types, M in a mortar with a pestle. Two mg of leaf powder was used was diferentiated into palisade tissue on the adaxial side and to measure 12C and 13C contents. Carbon isotope ratios were spongy tissue on the abaxial side (Fig. 1, S1–S3). measured at SI Science, Kita-katsushika, Saitama, Japan by using the elemental analyzer–isotope ratio mass spectrome- Immunohistochemical localization of GDC‑P ter (EA-IRMS) system (Thermo Fisher Scientifc, Waltham, and Rubisco LSU MA), as described by Sato and Suzuki (2010). The isotope ratio was expressed in δ notation as parts per million (‰) In the non-Kranz type, GDC-P was detected in M cells and with respect to the Pee Dee belemnite standard. to a lesser extent in BS cells (Fig. 2a, b, S4; Table 2). In the proto-Kranz type, GDC-P was detected in both M and BS Statistical analysis cells (Fig. 2c, d, S5, S6a–c), but the degree of staining var- ied among species and C. album accessions (Table 2). In C. Data were presented as mean ± SD (n = 3 plants), except car- giganteum, C. nevadense, and four accessions of C. album bon isotope ratios. These data were analyzed using Statcel4 (from Finland, France, India and Poland), the staining was software (OMS Publisher, Tokorozawa, Saitama, Japan). stronger in BS cells than in M cells, and a distinct brown We tested the signifcance (P < 0.05) of the diferences in ring surrounded the vascular bundle; this ring represented GDC-P labeling density between M and BS mitochondria a dense accumulation of GDC-P in mitochondria, as shown using Student’s t test and that of the diferences in struc- later by ultrastructural observation. In the C. album acces- tural, biochemical, and physiological traits among spe- sion from Arizona and C. strictum, which had Kranz-like cies and among photosynthetic types of Chenopodium by anatomy, GDC-P staining was detected exclusively in BS the Tukey–Kramer test as a post hoc test, associated with cells (Fig. 2e, f; Table 2). In a ­C4 species of Amaranthus ANOVA. For enzyme activities, data of Amaranthus ­C4 spe- also, GDC-P staining occurred exclusively in BS cells (Fig. cies were added to statistical analysis. Pearson’s correlation S6d), as known in many ­C4 species (Yoshimura et al. 2004). coefcients between Γ values and quantitative parameters of In Chenopodium species, regardless of the anatomical type, cells and organelles were calculated. Rubisco LSU was detected in chloroplasts of both M and BS cells (Fig. S7).

Results CO2 gas exchange and carbon isotope ratio

Leaf anatomy As expected from leaf anatomy and GDC-P localization, Chenopodium species showed a large variation in Г (20–66 Light microscopy revealed a large variation in chloroplast µL ­L−1; Table 2). These Г values were higher than that in numbers and arrangement in BS cells among Chenopodium a control C­ 4 species, Amaranthus dubius (Table 2). In the species examined (Fig. 1, S1–S3). We classifed leaf anat- Arizona accession of C. album and C. strictum, which have omy of Chenopodium into 3 types: non-Kranz, proto-Kranz, Kranz-like anatomy, the Г values were 20 and 26 µL L­ −1, and Kranz-like types (Fig. 1; Table 2). In non-Kranz anat- respectively, which are typical for ­C3–C4 intermediates omy (Fig. 1a, b, S1), BS cells contained few chloroplasts in (Edwards and Ku 1987; Monson and Rawsthorne 2000). The the inner half (centripetal region) along the vascular bundle, Г values of non-Kranz species ranged from 45 to 66 µL ­L−1, but many chloroplasts occurred in the outer half adjacent to whereas those of proto-Kranz species ranged from 35 to 51 intercellular spaces. Chenopodium atrovirens, C. hians, C. µL ­L−1 (Table 2). The average Г values of the 3 anatomical incanum, C. leptophyllum, C. pallidicaule, C. quinoa, C. types in Chenopodium signifcantly difered from each other. standleyanum, and C. vulvaria showed non-Kranz anatomy Relative to the average Г value of the non-Kranz type, the (Table 2). In proto-Kranz anatomy (Fig. 1c, d, S2, S3a–c), average Г value of the proto-Kranz type was 23% lower, and BS cells contained more chloroplasts in the centripetal that of the Kranz-like type was 57% lower. The A values of region than did non-Kranz-type BS cells, and those chlo- Chenopodium species ranged from 10 to 25 µmol m−2 ­s−1, roplasts surrounded the vascular bundle. Six accessions of and that of A. dubius was 17.6 µmol m−2 ­s−1 (Table 2). The

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Fig. 1 Leaf anatomy of Chenopodium species. a C. atrovirens; b C. vulvaria; c C. album (India); d C. nevadense; e C. album (Arizona, USA); f C. strictum. BSC bundle-sheath cell, MC mesophyll cell, V vascular bundle. Bars = 50 µm

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Table 2 Leaf anatomy, immunolocalization of the P protein of glycine decarboxylase (GDC-P), carbon isotope ratios, net ­CO2 assimilation rate (A), and ­CO2 compensation point (Γ) in Chenopodium species and control ­C4 species of Amaranthus Species (locality) Leaf anatomy GDC-P localization δ13C A Γ M cells BS cells (‰) (μmol m−2 s−1) (μmol mol−1)

C. atrovirens Non-Kranz +++ − − 32.6 19.8 ± 1.5a 65.5 ± 3.1h C. hians Non-Kranz +++ − − 31.3 22.0 ± 1.7a 49.0 ± 2.7efg C. incanum Non-Kranz + − − 31.1 24.6 ± 4.6a 45.2 ± 3.0cdef C. leptophyllum Non-Kranz +++ − ND ND ND C. pallidicaule Non-Kranz +++ + − 29.4 15.3 ± 1.9a 56.5 ± 3.7g C. quinoa Non-Kranz +++ + ND ND ND C. standleyanum Non-Kranz +++ + − 27.5 19.9 ± 0.4a 49.9 ± 1.2efg C. vulvaria Non-Kranz +++ − − 29.3 20.5 ± 1.5a 55.6 ± 2.9g Non-Kranz type average − 30.2 ± 1.8x 20.4 ± 3.1x 53.6 ± 7.2z C. album (Finland) Proto-Kranz ++ +++ − 30.1 ND ND C. album (France) Proto-Kranz ++ +++ − 30.6 15.0 ± 6.9a 48.3 ± 1.6defg C. album (Fukuoka, Japan) Proto-Kranz +++ +++ ND 10.1 ± 3.0a 39.7 ± 1.4bcd C. album (India) Proto-Kranz ++ +++ − 29.0 18.3 ± 0.9a 38.1 ± 3.6bc C. album (Poland) Proto-Kranz ++ +++ − 29.2 23.9 ± 2.6a 42.9 ± 1.9bcdef C. album (Tsukuba, Japan) Proto-Kranz +++ ++ ND 12.7 ± 1.5a 38.1 ± 2.6bc C. berlandieri Proto-Kranz +++ ++ − 28.4 16.5 ± 2.0a 51.0 ± 3.2fg C. fcifolium Proto-Kranz +++ ++ ND 11.7 ± 2.0a 35.0 ± 0.9b C. formosanum Proto-Kranz +++ ++ − 27.9 18.6 ± 1.9a 42.5 ± 1.6bcdef C. giganteum Proto-Kranz ++ +++ − 26.7 16.0 ± 4.2a 41.7 ± 0.6bcde C. nevadense Proto-Kranz ++ +++ − 27.7 15.5 ± 1.1a 35.9 ± 1.0b Proto-Kranz type average − 28.7 ± 1.3x 15.8 ± 3.9x 41.3 ± 5.2y C. album (Arizona, USA) Kranz-like − +++ − 30.5 16.3 ± 0.7a 20.3 ± 6.2a C. strictum Kranz-like − +++ − 29.9 21.1 ± 5.3a 25.5 ± 4.1a Kranz-like type average − 30.2 ± 0.4x 18.7 ± 3.4x 22.9 ± 3.7x A. cruentus Kranz − +++ ND ND ND A. dubius Kranz ND ND − 13.1 17.6 ± 4.9 Near 0

GDC-P localization: (+) and (−) refer to the relative intensities of staining, with (+++) indicating heavy staining and (−) indicating little or no staining. The values of A and Γ for each species/accession are given as the mean ± SD of three plants. Diferent letters indicate signifcant difer- ence at P < 0.05 M mesophyll, BS bundle sheath, ND not determined

δ13C values of Chenopodium species ranged from − 32.6‰ species, whereas there were no signifcant diferences among to − 26.7‰, whereas that of A. dubius was − 13.1‰ the three anatomical types of Chenopodium. In contrast, activi- (Table 2). These values were within ­C3 and ­C4 range, respec- ties of PEPC were much lower in Chenopodium species than in tively (Ehleringer and Osmond 1991). The average A and the ­C4 species; there were no signifcant diferences among the δ13C values did not difer signifcantly among the anatomical three anatomical types. Similar trends were found for NADP- types of Chenopodium. ME and NAD-ME (Table 3). In several species, there were large diferences in the enzyme activities among three plants Activities and amounts of photosynthetic examined, resulting in large standard deviation. It was consid- and photorespiratory enzymes ered that these diferences were probably caused by those in growth rate of plants. Activities of ­C3 and ­C4 photosynthetic enzymes were measured Western blot analyses of photosynthetic and photorespira- for fve non-Kranz species, six proto-Kranz species (including 4 tory enzymes were done for fve species of Chenopodium (three C. album accessions), the Arizona accession of C. album and C. accessions from C. album) representing the three anatomical strictum (both Kranz-like type), and two control ­C4 species of types and a control C­ 4 species, A. dubius (Fig. 3). The levels of Amaranthus (Table 3). The average activities of Rubisco were Rubisco LSU and GDC-P were higher, and those of PEPC and higher in all anatomical types of Chenopodium than in the ­C4 PPDK were much lower, in Chenopodium than in A. dubius.

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Fig. 2 Immunohistochemical staining of GDC-P in mesophyll and USA); f C. strictum. BSC bundle-sheath cell, MC mesophyll cell, V bundle sheath cells of Chenopodium species. a C. atrovirens; b C. vascular bundle. Bars = 50 µm vulvaria; c C. album (India); d C. nevadense; e C. album (Arizona, 1 3 Journal of Plant Research (2019) 132:839–855 847

Table 3 Activities of photosynthetic enzymes in leaves of Chenopodium species and control ­C4 species of Amaranthus Species (locality) Rubisco (μmol mg PEPC (μmol mg ­ NADP-ME (μmol mg NAD-ME (μmol mg ­chl−1 h−1) chl−1 h−1) ­chl−1 h−1) ­chl−1 h−1)

C. atrovirens 479.1 ± 105.7bedef 27.9 ± 24.0a 4.0 ± 0.7a 4.1 ± 0.9a C. pallidicaule 353.6 ± 115.3abcd 73.3 ± 4.0a 5.4 ± 2.3ab 19.8 ± 9.8a C. quinoa 426.7 ± 60.2abcdef 124.5 ± 11.9a ND ND C. standleyanum 308.5 ± 16.6abe 23.9 ± 1.2a 4.3 ± 2.1a 5.8 ± 1.4a C. vulvaria 575.8 ± 120.4cdef 39.2 ± 33.5a 14.7 ± 3.5ab 31.7 ± 11.5a Non-Kranz type average 428.7 ± 105.2y 57.8 ± 42.1x 7.1 ± 5.1x 15.4 ± 13.0x C. album (India) 395.8 ± 151.3abcdef 34.3±2.0a 10.3 ± 6.3ab 11.3 ± 5.0a C. album (Fukuoka, Japan) 239.3 ± 22.8ab 87.2 ± 7.3a ND ND C. album (Poland) 645.0 ± 58.2ef 80.3 ± 10.5a 13.2 ± 7.5ab 10.8 ± 5.3a C. album (Tsukuba, Japan) 411.0 ± 58.9abcdef 74.2 ± 22.2a ND ND C. berlandieri 367.1 ± 24.6abcd 37.9 ± 7.0a 6.8 ± 0.7ab 22.5 ± 2.2a C. fcifolium 474.4 ± 26.9bcdef 82.3 ± 6.9a ND ND C. formosanum 377.8 ± 86.1abcde 33.2 ± 3.2a 6.6 ± 1.1ab 9.6 ± 1.6a C. giganteum 178.8 ± 65.4a 33.7 ± 24.7a 3.7 ± 1.1a 6.8 ± 2.5a C. nevadense 585.3 ± 82.1def 41.1 ± 9.7a 11.7 ± 3.4ab 25.3 ± 11.4a Proto-Kranz type average 408.3 ± 148.2y 56.0 ± 24.0x 8.7 ± 3.6x 14.4 ± 7.6x C. album (Arizona, USA) 382.4 ± 86.7abcdef 37.4 ± 36.9a 7.1 ± 1.1ab 6.0 ± 2.9a C. strictum 654.2 ± 157.9f 71.8 ± 6.1a 5.6 ± 3.3ab 5.9 ± 4.0a Kranz-like type average 518.3 ± 192.2y 54.6 ± 24.3x 6.4 ± 1.1x 6.0 ± 0.1x A. dubius 193.8 ± 60.1a 596.5 ± 105.1b 19.6 ± 1.4b 82.8 ± 19.7ab A. hybridus 231.1 ± 87.9ab 564.4 ± 138.8b 45.0 ± 23.1c 201.3 ± 155.1b

C4 type average 212.5 ± 70.4x 580.5 ± 111.5y 32.3 ± 20.2y 142.0 ± 118.3y

For each species, values are given as the mean ± SD of three plants. Diferent letters indicate signifcant diference at P < 0.05 ND not determined

three anatomical types. The sectional area of M tissue was highest in the non-Kranz type and lowest in the Kranz-like type, whereas that of BS tissue showed a reverse tendency. As a result, the M/BS tissue area ratio was higher in the non-Kranz type than in the proto-Kranz and Kranz-like types (Table 4). The size of M and BS cells varied greatly among Chenopodium species. For example, the size of M cells in C. atrovirens was more than fve times that in the Indian accession of C. album. Nevertheless, the M/BS cell size ratio was almost constant among species of the same anatomical type but was lowest in the proto-Kranz type and highest in the non-Kranz type (Table 4). There was a high positive cor- relation between Г and the M/BS tissue area ratio (Fig. 4a) Fig. 3 Western blots of leaf extracts of Chenopodium species. Total soluble protein (20 µg for GDC-P, PEPC, and PPDK and 2.5 µg for and a weaker positive correlation between Г and the M/BS Rubisco LSU) was subjected to SDS-PAGE, blotting on nitrocellu- cell size ratio (Fig. 4b). lose membranes, and identifcation with antisera against the indicated photorespiratory and photosynthetic enzymes Ultrastructure and quantifcation of organelles Quantifcation of leaf inner structure In the non-Kranz type, only a few chloroplasts and mito- We investigated the leaf inner structure of the representatives chondria were located in the centripetal region of BS cells of the three anatomical types in more detail (Table 4). There (Fig. 5b, c, S8b, c, e, f). In the proto-Kranz type, more chlo- were no signifcant diferences in vein density among the roplasts and mitochondria were located in the centripetal

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Table 4 Structural traits of leaves in Chenopodium species Species (local- Vein density M tissue area BS tissue area M/BS tissue M cell size (μm2) BS cell size M/BS cell size ity) (mm mm−2) (%) (%) area ratio (μm2) ratio

C. atrovirens 6.7 ± 2.0ab 90.4 ± 2.3cd 9.6 ± 2.3ab 9.8 ± 2.3b 1723.7 ± 507.5b 1287.9 ± 248.3c 1.4 ± 0.3d C. pallidicaule ND 91.5 ± 0.6d 8.5 ± 0.6a 10.7 ± 0.7b 710.5 ± 28.4a 579.0 ± 47.5a 1.2 ± 0.1cd C. vulvaria 7.0 ± 2.0ab 91.2 ± 0.5d 8.8 ± 0.5a 10.4 ± 0.7b 732.5 ± 97.1a 615.7 ± 90.8ab 1.2 ± 0.2bcd Non-Kranz type 6.7 ± 0.2x 91.5 ± 0.3x 9.0 ± 0.5x 10.3 ± 0.5y 1055.6 ± 578.7y 827.5 ± 399.1x 1.3 ± 0.1z average C. album (India) 9.4 ± 2.1c 84.9 ± 1.1ab 15.1 ± 1.1cd 5.6 ± 0.5a 293.7 ± 66.7a 510.0 ± 75.9a 0.6 ± 0.1a C. album 6.7 ± 2.0ab 86.8 ± 1.6bc 13.2 ± 1.6bc 6.6 ± 0.9a 681.7 ± 88.5a 1225.4 ± 336.3bc 0.6± 0.1a (Poland) C. nevadense 8.5 ± 2.3c 85.7 ± 1.0ab 14.3 ± 1.0cd 6.0 ± 0.5a 386.9 ± 14.1a 784.1 ± 385.9ab 0.6 ± 0.1a Proto-Kranz 8.2 ± 1.4x 85.8 ± 0.9y 14.2 ± 0.9y 6.1 ± 0.7x 454.1 ± 202.5x 839.8 ± 360.9x 0.6 ± 0.0x type average C. album (Ari- 11.8 ± 2.6d 82.2 ± 1.3a 17.8 ± 1.3d 4.6 ± 0.2a 636.4 ± 156.8a 800.5 ± 217.0ab 0.8 ± 0.0abc zona) C. strictum 5.8 ± 1.4ab 83.1 ± 1.7ab 16.9 ± 1.7cd 5.0 ± 0.4a 1450.2 ± 165.2b 1898.1 ± 359.9c 0.8 ± 0.1ab Kranz-like type 8.8 ± 4.2x 82.7 ± 0.7z 17.3 ± 0.7z 4.8 ± 0.5x 1043.3 ± 575.5y 1349.3 ± 776.1x 0.8 ± 0.0y average

The values for each species/accession are given as the mean ± SD of three plants. Diferent letters indicate signifcant diference at P < 0.05 M mesophyll, BS bundle sheath region, where many mitochondria were located between (Fig. 4f), the number of chloroplasts and mitochondria per BS chloroplasts and vascular tissues (Fig. 5e, f, S8 h, i, k, l). cell (Fig. 4g, h). In the Kranz-like type, the preferential localization of these organelles in the centripetal region was most pronounced Immunogold localization of GDC‑P (Fig. 5h, i, S8n, o). The ratio of chloroplasts and mito- chondria in the inner half of BS cells was higher in both In the non-Kranz and proto-Kranz types, GDC-P was detected proto-Kranz and Kranz-like types than in the non-Kranz in the mitochondria of both M and BS cells (Fig. S9a–d). In type (Table 5). In the M cells of the non-Kranz type, most both types, the labeling density did not difer signifcantly mitochondria were located on the vacuolar side of chloro- between M and BS mitochondria (Fig. 6). In the Kranz-like plasts (in the inner position) (Fig. 5a; Table 5; see Fig. 5d, type, GDC-P was detected almost exclusively in BS mitochon- g for the proto-Kranz and Kranz-like types). The ratio of dria (Fig. 6, S9e, f). mitochondria in the inner position decreased from the non- Kranz to the proto-Kranz to the Kranz-like type (Table 5). There were signifcant correlations between Г and the cen- Discussion tripetal positioning of chloroplasts and mitochondria in BS cells (Fig. 4c, d) and between Г and the inner positioning of Photosynthetic types in Chenopodium M mitochondria (Fig. 4e). In M cells, the size of chloroplasts and mitochondria and Although Chenopodium is considered to be a C­ 3 genus, our the number of chloroplasts and mitochondria per cell did not study revealed that Chenopodium species show great varia- difer among the three types, and their numbers per unit area tion in leaf anatomy, and some species have biochemical and were higher in the proto-Kranz type than in the other two physiological traits characteristic of C­ 3–C4 intermediates. types (Table 6). In BS cells, the number of chloroplasts per Light microscopy showed a diference in chloroplast number cell was highest in the Kranz-like type, and the number of in the centripetal region of BS cells, an indication of the mitochondria was lowest in the non-Kranz type (Table 6). The existence of non-Kranz, proto-Kranz, and Kranz-like species numbers of these organelles per unit area tended to be lowest in this genus (Table 2). Gas exchange measurements showed in the non-Kranz type (Table 6). In BS cells, the size of chlo- that the non-Kranz type had the highest Г values, typical roplasts did not difer signifcantly among the three types, and of ­C3 plants, whereas the Kranz-like type had the lowest Г the mitochondria were smallest in the non-Kranz and largest values, typical of ­C3–C4 intermediate plants (Edwards and in the Kranz-like type (Table 6). There were signifcant nega- Ku 1987). GDC-P was found mainly in M cells in the non- tive correlations between Г and the size of BS mitochondria Kranz type and exclusively in BS cells in the Kranz-like

1 3 Journal of Plant Research (2019) 132:839–855 849 0.05;

<

* ), the ratio P: c ), number of chloroplasts per BS cell ), number of chloroplasts f ), size of BS mitochondria ( e ), the ratio of chloroplasts located in the inner half of BS cells ( ), the of chloroplasts ratio b ), M/BS cell size ratio ( ), M/BS cell size ratio a species. Filled circles, non-Kranz type; white circles, proto-Kranz type; triangles, type. Signifcant at Kranz-like proto-Kranz type; white circles, non-Kranz circles, species. Filled ) and M/BS tissue area ratio ( ratio ) and M/BS tissue area Г ), the of mitochondria located in the ratio inner position of M cells ( d Chenopodium ) in h compensation point ( compensation 2 ­ CO 0.001

<

Relationships between between Relationships

0.01; ***

4

<

), and number of mitochondria per BS cell ( g Fig. of mitochondria located in the inner half of BS cells ( ( **

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Fig. 5 Ultrastructure of mesophyll cells (a, d, g) and bundle-sheath C. album (Arizona, USA). BSC bundle-sheath cell, ICS intercellular cells at a low (b, e, f) and at a high (c, f, i) magnifcation in Che- space, MC mesophyll cell, V vascular bundle, c chloroplast, mt mito- nopodium species. (a–c) C. vulvaria; (d–f) C. album (India); (g–i) chondrion, n nucleus. Bars = 3 µm type. The latter GDC-P distribution is responsible for the immunostaining was detected in both M and BS cells, but operation of the glycine shuttle (Monson and Rawsthorne the relative intensity of staining between these cells var- 2000; Rawsthorne 1992; Rawsthorne et al. 1988). These data ied among species (Table 2). These Chenopodium species suggest that the Arizona accession of C. album and C. stric- resemble the proto-Kranz plants previously found in some tum (Kranz-like type) are ­C3–C4 intermediates. genera, such as Heliotropium (Muhaidat et al. 2011; Vogan We found numerous proto-Kranz species, intermediate et al. 2007), Flaveria (Sage et al. 2013), Salsola (Schüssler between the non-Kranz and Kranz-like types (Table 2). et al. 2017; Voznesenskaya et al. 2013), and Steinchisma The BS cells of proto-Kranz species also contained a con- (Brown et al. 1983; Khoshravesh et al. 2016). siderable number of chloroplasts in the centripetal region, In comparison with the C­ 4 Amaranthus species, Chenopo- but the number was somewhat lower than in the BS cells dium species had higher activities of the ­C3 enzyme Rubisco of the Kranz-like type. In the proto-Kranz type, GDC-P and lower activities of the ­C4 enzymes PEPC, NADP-ME

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Table 5 Intracellular position of M cells BS cells mitochondria and chloroplasts in mesophyll and bundle sheath Species (locality) Mitochondria Chloroplasts Mitochondria cells of Chenopodium species Inner position (%) Inner half of cell (%) Inner half of cell (%)

C. atrovirens 81.6 ± 2.5bc 22.2 ± 7.9a 56.3 ± 5.7b C. pallidicaule 87.2 ± 6.3c 19.7 ± 4.0a 49.1 ± 8.3ab C. vulvaria 83.5 ± 2.5bc 24.7 ± 10.4a 34.4 ± 11.6a Non-Kranz type average 84.1 ± 4.4z 22.2 ± 2.5x 46.6 ± 11.1x C. album (India) 66.6 ± 7.9ab 49.3 ± 2.8b 82.2 ± 2.6c C. album (Poland) 75.7 ± 9.8abc 54.7 ± 0.5b 83.4 ± 4.3c C. nevadense 75.6 ± 2.6abc 48.6 ± 1.4b 82.3 ± 3.2c Proto-Kranz type average 72.6 ± 7.9y 50.9 ± 3.3y 82.6 ± 3.0y C. album (Arizona) 64.2 ± 8.6a 63.1 ± 11.0b 91.5 ± 4.2c C. strictum 59.1 ± 3.0a 52.3 ± 6.9b 91.5 ± 4.3c Kranz-like type average 61.6 ± 6.4x 57.9 ± 7.3y 91.5 ± 3.8y

The values for each species/accession are given as the mean ± SD of three plants. Diferent letters indicate signifcant diference at P < 0.05 M mesophyll, BS bundle sheath

Table 6 Quantitative traits of chloroplasts and mitochondria in mesophyll and bundle sheath cells of Chenopodium species Organelles and species (locality) M cells BS cells Size (μm2) No. per cell No. per cell area Size (μm2) No. per cell No. per cell area (μm−2×10−3) (μm−2×10−3)

Chloroplasts C. atrovirens 9.4 ± 1.3abc 19.9 ± 3.6b 11.5 ± 2.1a 7.8 ± 2.1a 12.7 ± 1.1ab 9.8 ± 0.9a C. pallidicaule 6.5 ± 0.9a 16.8 ± 0.6ab 23.6 ± 0.8c 6.7 ± 0.3a 9.9 ± 1.3a 17.0 ± 2.3abc C. vulvaria 7.7 ± 0.8ab 16.8 ± 1.2ab 22.9 ± 1.6bc 7.5 ± 1.5a 9.7± 1.2a 15.7 ± 2.0abc Non-Kranz type average 7.9 ± 1.5x 17.8 ± 1.8x 19.4 ± 6.8x 7.3 ± 0.6x 10.7 ±1.7x 14.2 ± 3.8x C. album (India) 7.9 ± 1.0ab 15.9 ± 2.7ab 54.3 ± 9.3e 6.6 ± 0.7a 11.9 ± 1.9ab 23.3 ± 3.7bc C. album (Poland) 9.8 ± 1.3bc 16.7 ± 1.1ab 24.5 ± 1.6c 9.0 ± 1.9a 18.3 ± 2.4bc 15.0 ± 2.0abc C. nevadense 9.0 ± 1.3abc 14.7 ± 0.5a 38.1 ± 1.2d 9.8 ± 2.0a 14.6 ± 2.6ab 23.7 ± 4.1c Proto-Kranz type average 8.9 ± 1.0x 15.8 ± 1.7x 39.0 ± 14.9y 8.5 ± 2.0x 14.9 ± 3.2x 20.6 ± 5.2xy C. album (Arizona) 12.0 ± 0.8c 17.5 ± 0.8ab 27.4 ± 1.3c 7.3 ± 0.7a 28.1 ± 5.5d 35.1 ± 0.9d C. strictum 8.6 ± 0.9ab 19.5 ± 0.5ab 13.4 ± 0.3ab 9.2 ± 0.9a 26.0 ± 3.7cd 13.7 ± 2.0ab Kranz-like type average 10.3 ± 2.5x 18.5 ± 1.2x 20.4 ± 9.9x 8.2 ± 1.3x 27.1 ± 1.5y 24.4 ± 12.6y Mitochondria C. atrovirens 1.0 ± 0.1a 13.9 ± 8.8a 8.1 ± 5.1ab 0.7 ± 0.2a 11.6 ± 2.2a 9.0 ± 1.7a C. pallidicaule 0.6 ± 0.1a 12.8 ± 2.5a 18.0 ± 3.5ab 0.7 ± 0.0a 9.7 ± 1.4a 16.8 ± 2.4ab C. vulvaria 0.7 ± 0.2a 7.8 ± 1.6a 10.6 ± 2.2ab 0.6 ± 0.1a 9.3 ± 3.0a 15.0 ± 4.9a Non-Kranz type average 0.8 ± 0.2x 11.5 ± 3.3x 12.2 ± 5.2x 0.7 ± 0.1x 10.2 ± 2.3x 13.6 ± 4.1x C. album (India) 0.7 ± 0.1a 7.5 ± 2.9a 25.4 ± 10.0b 1.6 ± 0.3bc 16.0 ± 3.5ab 31.4 ± 6.8bc C. album (Poland) 0.8 ± 0.3a 17.1 ± 7.8a 25.0 ± 11.4b 1.3± 0.4abc 28.9 ± 9.2bc 23.6 ± 7.5ab C. nevadense 0.7 ± 0.3a 10.7 ± 1.9a 27.6 ± 5.0c 0.9 ± 0.4ab 18.9 ± 3.2abc 30.7 ± 5.1bc Proto-Kranz type average 0.7 ± 0.1x 11.7 ± 4.9x 26.0 ± 1.4y 1.3 ± 0.5y 21.3 ± 6.7y 28.5 ± 4.3y C. album (Arizona) 1.0 ± 0.2a 7.5 ± 1.8a 11.8 ± 2.8ab 1.9 ± 0.2c 32.2 ± 6.4c 40.2 ± 8.0c C. strictum 0.8 ±0.2a 8.3 ± 3.4a 5.7 ± 2.3a 1.8 ± 0.1c 21.1 ± 5.6abc 11.2 ± 3.0a Kranz-like type average 0.9 ± 0.2x 7.9 ± 0.6x 8.8 ± 4.3x 1.8 ± 0.2z 26.7 ± 8.1y 25.7 ± 16.8xy

The values for each species/accession are given as the mean ± SD of three plants. Diferent letters indicate signifcant diference at P < 0.05 M mesophyll, BS bundle sheath

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2014), but as far as we know no ­C4 species has been identi- fed in Chenopodium. A few genera include ­C3–C4 interme- diate species with C­ 3 species (e.g., Moricandia, Diplotaxis, and Brassica in Brassicaceae, Schlüter et al. 2017; Ueno 2011; Parthenium in Compositae, Moore et al. 1987).

Transition of leaf structural and photosynthetic traits in Chenopodium

Our study revealed a gradation of structural and photosyn- thetic traits from ­C3 to proto-Kranz to ­C3–C4 intermediate type in Chenopodium. Muhaidat et al. (2007) found no sig- nifcant diference in vein density between closely related ­C3 and ­C4 species of eudicots. In Chenopodium, we also found no signifcant diferences in vein density among the three types, but the great variation in size of M and BS cells among Chenopodium species might afect the tendency of changes in vein density (Table 4). The M/BS tissue area ratio is higher in ­C species than in ­C species (Hattersley Fig. 6 3 4 Quantifcation of immunogold labeling of GDC-P in the mito- 1984; Muhaidat et al. 2011). In Chenopodium, the M/BS chondria of mesophyll cells (MC) and bundle-sheath cells (BSC) of Chenopodium species. Numbers of gold particles per unit area of tissue area ratio decreased from C­ 3 (non-Kranz) to proto- mitochondria are given as mean ± SD of three plants. * signifcant Kranz to C­ 3–C4 intermediate (Kranz-like) type (Table 4; diferences between MC and BSC at P < 0.05. NS not signifcant. Fig. 4a). The M/BS cell size ratio showed a similar trend Labeling densities in the cell area excluding mitochondria were (Table 4; Fig. 4b). Therefore, volume changes at the tissue between 0.05 and 0.15 µm−2 and cell levels appear to occur during the transition from ­C3 to proto-Kranz to ­C3–C4 intermediate species in Chenopo- and NAD-ME (Table 3). Western blot analysis showed dium, although the diferences between the proto-Kranz and that Chenopodium species accumulated smaller amounts ­C3–C4 intermediates were somewhat indistinct. of PEPC and PPDK but greater amounts of Rubisco and In the BS cells of C­ 3–C4 intermediate plants, the amount GDC than did Amaranthus species. These data suggest that and positioning of chloroplasts and mitochondria are criti- Chenopodium species perform photosynthesis without con- cal structural traits involved in photosynthesis (Brown and tribution of the ­C4 cycle. Rubisco accumulated in all chlo- Hattersley 1989; Edwards and Ku 1987; Muhaidat et al. roplasts of M and BS cells. We concluded that the Arizona 2011; Rawsthorne 1992; Sage et al. 2013; Ueno et al. 2003; accession of C. album and C. strictum (Kranz-like anatomy) Ueno 2011; Voznesenskaya et al. 2013). In the BS cells are type I ­C3–C4 intermediates, Chenopodium species with of Chenopodium, the size of mitochondria increased from non-Kranz anatomy are ­C3, and other species of Chenopo- ­C3 to proto-Kranz to ­C3–C4 intermediate species (Fig. 4f), dium and the remaining accessions of C. album are of the but there was no signifcant diference in chloroplast size proto-Kranz type. This conclusion is also supported by δ13C (Table 6). The numbers of chloroplasts and mitochondria values of Chenopodium species (Table 2). Previous studies per cell and per unit cell area tended to be lowest in ­C3 spe- reported that type I C­ 3–C4 intermediates and proto-Kranz cies (Table 6; Fig. 4g, h). The distribution of chloroplasts 13 type have ­C3-like δ C values (Edwards and Ku 1987; Vogan and mitochondria to the inner half of BS cells (centripetal et al. 2007), as in Chenopodium species. This is due to these positioning) was also lowest in C­ 3 species (Table 5; Fig. 4c, plants originally fxing ­CO2 via Rubisco, not via the ­C4 cycle d), as reported for other genera (Brown et al. 1983; Kho- (von Caemmerer and Hubick 1989). shravesh et al. 2016; Muhaidat et al. 2011; Rawsthorne 1992; The genus Chenopodium is polyphyletic and combines Sage et al. 2013; Ueno 2011; Ueno et al. 2003; Voznesens- species from three clades of Chenopodioideae (Kadereit kaya et al. 2013). The mitochondria in BS cells were located et al. 2003). Our study investigated 15 species, which is between the centripetally located chloroplasts and the inner only about 10% of the known Chenopodium species. From tangential walls. These structural features would help to cap- these limited data, it would be difcult to reliably deduce on ture photorespiratory ­CO2 released from mitochondria and the phylogenetic relationships among photosynthetic types to suppress the escape of ­CO2 from BS cells (Rawsthorne in Chenopodium, and a more extensive survey would be 1992; Sage et al. 2014). required. In general, most ­C3–C4 intermediate species have In M cells, the size of chloroplasts and mitochondria been found in genera that include C­ 4 species (Sage et al. and number of chloroplasts and mitochondria per cell did

1 3 Journal of Plant Research (2019) 132:839–855 853 not difer among the three types, but the degree of inner suggest that C. album may include diferent photosynthetic positioning of mitochondria gradually decreased from ­C3 types within a species. However, it is well known that C. to proto-Kranz to ­C3–C4 intermediate species (Table 6; album is a heterogenous assemblage of many taxonomic Fig. 4e). In contrast, the immunogold labeling density of entities with cosmopolitan distribution, probably because GDC did not difer signifcantly between the M and BS mito- many weedy and semi-domesticated forms have arisen by chondria of ­C3 and proto-Kranz species, but in ­C3–C4 inter- hybridization and polyploidization (Bhargava et al. 2006; mediates, GDC accumulated exclusively in BS mitochondria Ohri 2015). It has been recently suggested that hybridization (Fig. 6). Most mitochondria in M cells are located on the may also be involved in the occurrence of C­ 3–C4 intermedi- vacuolar side of chloroplasts (inner position) in ­C3 grasses ates (Kadereit et al. 2017; Ueno et al. 2006). Strict genetic (Busch et al. 2013; Hatakeyama and Ueno 2016; Sage and and taxonomic studies will be required to ascertain whether Sage 2009) but are adjacent to the cell wall (outer position) diferent photosynthetic types occur within C. album. On the in ­C4 grasses (Hatakeyama and Ueno 2017). This diference other hand, it cannot be ruled out that environmental factors is associated with the diference in localization of GDC and may infuence the expression level of ­C3–C4 intermediate Rubisco (these enzymes are present in ­C3 M cells but absent traits (Teese 1995). Oono et al. (2017) have recently reported in C­ 4 M cells) and thereby the diference in the requirement that high growth temperature and low nitrogen level in soil for scavenging of photorespiratory CO­ 2 released from mito- induce a decrease in Г and stronger expression of GDC-P in chondria (Hatakeyama and Ueno 2017). This relationship BS cells relative to M cells in the Tsukuba accession of C. between mitochondria positioning and photosynthetic types album. Further research on C. album would provide better in grasses appears to be also applicable to Chenopodium understanding of the ecological and adaptive aspects and the species. expression of ­C3–C4 intermediate traits. Our data suggest that the main structural and biochemical Acknowledgements We thank the Plant Introduction Station, ARS, events during the transition from ­C3 to proto-Kranz to ­C3–C4 Chenopodium USDA and the NARO Genebank, Tsukuba for their gifts of seeds and intermediate type in are (1) the increase in Prof. N. Furuya, Faculty of Agriculture, Kyushu University, for the use BS relative to M tissue area (volume); (2) the increase in of an electron microscope. This study was supported by Japan Society the number of chloroplasts and mitochondria in BS cells; for the Promotion of Science KAKENHI (Grant No. JP15K14638) to (3) the increase in the distribution of these organelles to O.U. the centripetal region of BS cells; (4) the enlargement of Open Access This article is licensed under a Creative Commons Attri- BS mitochondria; and (5) the increase in the level of GDC bution 4.0 International License, which permits use, sharing, adapta- in BS relative to M tissue. These changes allow limited tion, distribution and reproduction in any medium or format, as long operation of the glycine shuttle in the proto-Kranz type and as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes full operation of the shuttle in the C­ 3–C4 intermediate type, Г were made. The images or other third party material in this article are resulting in decreasing values (Fig. 4). Our data on the included in the article’s Creative Commons licence, unless indicated proto-Kranz type also clearly indicate that a complete sup- otherwise in a credit line to the material. If material is not included in pression of GDC-P expression in M cells is not required the article’s Creative Commons licence and your intended use is not for the reduction in Г, consistent with our previous stud- permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a ies on artifcial hybrids with diferent genome constitution copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. between ­C3–C4 intermediate and C­ 3 species of Brassicaceae (Ueno et al. 2003). The average Г value in the proto-Kranz type of Chenopodium was reduced by 23% relative to that of References the ­C3 species. In proto-Kranz species from other genera, a 5–15% reduction in Г has been reported (Sage et al. 2012). Because Chenopodium includes numerous proto-Kranz spe- Bauwe H (2011) Photorespiration: the bridge to ­C4 photosynthesis. 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