Introduction

Glutamine synthetase (GS) is a key enzyme of ammonia (NH3) assimilation and catalyzes the following reaction (Lea et al. 1990):

$${\text{Glutamate }}+{\text{ N}}{{\text{H}}_{\text{3}}}\,+\,{\text{ATP}}\xrightarrow{{{\text{M}}{{\text{g}}^{2+}}}}{\text{ Glutamine}}\,+\,{\text{ADP}}\,+\,{\text{Pi.}}$$

There are two major isoforms of GS: cytosolic (GS1) and chloroplastic (GS2) forms. The molecular mass of GS1 ranges from 39 to 40 kDa, whereas that of GS2 can be as large as 42 kDa in French bean (Phaseolus vulgaris L.) and hybrid poplar (Populus tremula x Populus alba) (Castro-Rodríguez et al. 2011; Lightfoot et al. 1988). The GS2 gene of bean has a sequence for long N-terminal extension that acts as a chloroplast targeting sequence (Lea 1997; Lightfoot et al. 1988). In general, GS1 is involved in nitrogen translocation from leaves (Bauer et al. 1997; Kamachi et al. 1992) and lignin synthesis (Razal et al. 1996; Sakurai et al. 1996), etc. whereas GS2 is involved in photorespiration in angiosperms (Somerville and Ogren 1980; Wallsgrove et al. 1987).

Photorespiration starts with the oxygenation of ribulose-1,5-bisphosphate (RuBP) by Rubisco. Two molecules of the oxygenation product, 2-phosphoglycolate (2-PG), are converted to the Calvin cycle intermediate 3-phosphoglycerate (3-PGA) by metabolism that involves release and re-assimilation of NH3 by GS2. GS2-deficient barley mutants show a dramatic decline of the net CO2-fixation rate and eventually necrosis after transfer from a non-photorespiratory (1% O2) to a photorespiratory condition (ambient O2 level of approximately 20%) (Blackwell et al. 1987; Wallsgrove et al. 1987). Transgenic tobacco plants overexpressing GS2 increase a tolerance to while those reducing GS2 are severely damaged to high light intensity where active photorespiration occurs (Kozaki and Takeba 1996). These results strongly support that GS2 is an essential enzyme for angiosperm photorespiration.

In contrast to angiosperm species, a conifer, Pinus sylvestris, possesses two cytosolic GS isoforms, GS1a and GS1b, but lacks GS2 (Avila et al. 1998; Cantón et al. 1993). Immunohistochemical analyses of P. sylvestris seedling have shown that GS1a localizes in chloroplast-containing tissues such as cotyledons whereas GS1b localizes in vascular tissues, suggesting an involvement of GS1a with photorespiratory NH3 assimilation (Avila et al. 2001; Suárez et al. 2002).

A phylogenetic analysis of GS genes from prokaryotes, green algae and land plants including 10 angiosperms and P. sylvestris demonstrated that GS2 evolved from a duplicated GS1 gene rather than the gene transferred from the plastid endosymbiont genome (Avila-Sáez et al. 2000). Based on this phylogenetic analysis, Cánovas et al. (2007) speculated that acquisition of GS2 is an adaptive mechanism to high levels of photorespiratory NH3 when plants encountered the present oxygen levels in the atmosphere during land plant evolution. However, there is no physiological evidence to support this hypothesis so far.

Assimilation efficiency of NH3 during photorespiration in vivo can be evaluated on the basis of leaf NH3 gas exchange. NH3 in the mesophyll cells—its amount partly reflects the balance between NH3 assimilation activity by leaf GS and NH3 production activity by photorespiration—diffuses out from the apoplastic surface of the mesophyll cells through stomata to the ambient air when air-NH3 concentration is low (Farquhar et al. 1980a; Kumagai et al. 2011; Miyazawa et al. 2014). Leaf NH3 gas exchange has been intensively studied in crops and grasses. By contrast, NH3 gas exchange studies are rarely reported in conifers.

The absence of GS2 from some other conifers in the family Pinaceae such as Pinus pinaster, Pinus pinea, Abies pinsapo, and Larix decidua have been confirmed by immunoblotting (Cánovas et al. 1991; García-Gutiérrez et al. 1998). It remains obscure whether GS2 is present or absent in conifers from the other five family members in Pinidae (Araucariaceae, Cupressaceae, Podocarpaceae, Sciadopityaceae, and Taxaceae).

Here we examined the absence of GS2 in leaves of coniferous species from all the family members. In particular, Cryptomeria japonica D.Don, “Sugi” in Japanese—a coniferous species that belongs to the family Cupressaceae—is crucial to the forestry and wood industries of Japan. More than 50,000 expressed sequence tags (ESTs) are available in the sugi database (ForestGEN). We utilized the EST database of sugi to search for the GS orthologues. We also addressed the question of whether that assimilation efficiency of photorespiratory NH3 was lower in conifers that may potentially lack GS2 than in angiosperms through leaf NH3 gas exchange.

Materials and methods

Plant materials for NH3 gas exchange, GS activity and NH3 leakiness measurements

GC-grown plants

We grew C. japonica (sugi), Phaseolus vulgaris L. cv. “Kentucky” (French bean), and Populus nigra L. (black poplar) plants in an environmentally controlled growth chamber (GC, Koito Electric Industries Ltd.; Yokohama, Japan). Bean plants were grown from seeds, and black poplar and sugi plants were propagated from cuttings. Bean and poplar plants were grown in Wagner pots (113 mm in internal diameter, 1.4-l in volume) filled with vermiculite. Sugi plants were cultured in the pots filled with a mixture (1:1 by volume) of Kanuma pumice and red granular soils.

Bean and black poplar plants were cultured in the GC under the photosynthetically active photon flux density (PPFD) of 400 µmol m− 2 s− 1. This PPFD, however, induces a yellowing symptom in sugi leaves. Therefore, sugi plants were cultured in a separate GC under the low PPFD of 170 µmol m− 2 s− 1. Day/night air temperature and relative humidity were set at 25/20 °C and 70%, respectively. Day/night periods were set at 16 h/8 h.

Potted plants were placed in the stainless-steel containers of a custom-made automatic water-supplying system. The containers had a maximum load of 20 Wagner pots. The system for bean and black poplar plants was equipped with a 60-l tank of nutrient solution, which comprised 1.8 mM NH4NO3, 0.35 mM NaH2PO4, 0.63 mM KCl, 0.36 mM CaCl2, 0.25 mM MgSO4, 0.06 mM H3BO3, 0.06 mM Fe(III)-EDTA, 0.4 µM Cu(II)-EDTA, 0.4 µM Mn(II)-EDTA, and 0.3 µM Zn(II)-EDTA. The system for sugi was supplied with the same nutrient composition at half strength. The bottoms of bean and black poplar pots were automatically soaked in nutrient solution for 15 min twice a day and those of sugi pots were soaked in nutrient solution three times a week. The nutrient solution was renewed every 2 weeks.

As described above, the sugi plants were grown under the different growth condition (low- light and low-nutrient supply, “L-condition”) from the condition where the bean and black plants were grown (high-light and high-nutrient supply, “H-condition”). We also raised black poplar plants grown under the L-condition for measurements (namely, the same conditions including soil type, PPFD and levels of nutrient supply as for sugi).

Black poplar and sugi plants had heights of 50–70 cm and 30–50 cm, respectively. We used bean plants of which the first trifoliate leaves had been just emerged. Mature leaves from the upper parts of black poplar and sugi shoots and primary leaves from bean plants were used for all experiments. Leaves were sampled from at least three plants.

Field-grown plants

Adult trees of Pinus densiflora Siebold & Zucc. (pine), Populus alba L. (silver poplar), and sugi grown in the FFPRI tree garden (Tsukuba, Japan) under field conditions were used for experiments; the sunlit branches of two to three individuals of each species were used during the periods of June to early September in 2015 or 2016. Diameters at breast height of the pine, silver poplar, and sugi trees were 37–53 cm, 33–64 cm, and 10–12 cm, respectively. We designated those samples as field-condition (“F-condition”) samples.

Plant materials for immunoblotting analysis

We collected current-year mature leaves of adult pine, silver poplar and sugi trees for immunoblotting analysis. We also collected current-year mature leaves of other coniferous species including Cedrus deodara (Roxb. ex D.Don) G.Don, Cephalotaxus harringtonia (Knight ex J.Forbes) K.Koch, Chamaecyparis obtusa Siebold & Zucc., Juniperus rigida Wall. ex Carrière, Larix kaempferi Fortune ex Gordon, Metasequoia glyptostroboides Hu & W.C.Cheng, Podocarpus macrophyllus D.Don, Sciadopitys verticillata Siebold & Zucc., Taxus cuspidata Siebold & Zucc., and Thuja standishii Carrière. We also collected current-year mature leaves of angiosperm species including Fagus crenata Blume, Firmiana simplex W.Wight, Illicium anisatum Bartr. ex Michx., Magnolia praecocissima Koidz., and Morus australis Poir. and collected mature leaves of a gymnosperm, Ginkgo biloba L. All of these species were cultivated in the FFPRI tree garden (Tsukuba, Japan) and were sampled during early September 2015.

Leaves of Cycas revoluta Thunb., Ephedra minima K.S.Hao, Gnetum gnemon L., Welwitschia mirabilis Hook.f. were collected in June 2016 and current-year leaves of Araucaria angustifolia (Bertol.) Kuntze and Wollemia nobilis W.G.Jones, K.D.Hill & J.M.Allen were collected in November 2016 in Tsukuba Botanical Garden (Tsukuba). We also collected samples of the GC-grown plants: mature primary leaves and roots of bean, current-year shoots of sugi, mature leaves of black poplar, and rosette leaves of Arabidopsis thaliana (L.) Heynh. Those samples were immediately frozen in liquid N2 and stored in a freezer at − 80 °C until use.

Phylogenetic tree

We retrieved the deduced amino acid sequences of GS genes of French bean, Populus trichocarpa, and Pinus sylvestris from the database of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). The Arabidopsis GS sequence was used as a query to search for orthologues of sugi GS in the EST database (ForestGEN; http://foresgen.ffpri.affrc.go.jp/en/info_cj.html). Two GS orthologues were found and were designated as CjGS1a and CjGS1b. A phylogenetic tree based on alignments of the deduced full-length amino acid sequences of GS genes (Fig. S1) was constructed with the neighbor-joining method using software (MEGA 6.0; Tamura et al. 2013). The number of bootstrap replicates was set at 1000. Molecular masses of sugi GS were predicted using ExPASy (http://web.expasy.org/compute_pi/). The amino acid positions of the cleavage sites for the GS2 transit peptides were predicted using ChloroP (http://www.cbs.dtu.dk/services/ChloroP/).

Immunoblotting

Soluble protein was extracted from leaf samples (root samples as well for French bean) in accordance with the method described by Tsuchida et al. (2001) using a slightly modified formulation of their reported extraction buffer. Specifically, our extraction buffer contained 50 mM HEPES-KOH (pH 7.8), 1 mM EDTA, 10% (v/v) glycerol, 10 mM MgSO4, 5 mM DTT, 0.01 mM leupeptin, and 1% (v/v) nonidet P-40.

Immunoblotting analysis was performed using a semi-dry system in accordance with the manufacturer’s protocol (Bio-Rad, Hercules, CA, USA). Four micrograms of the soluble proteins were subjected to SDS–PAGE per lane. Proteins separated through SDS–PAGE were transferred to a nitrocellulose membrane. GS polypeptides were reacted with anti-GS1 GS2 global antibody (Agrisera AB, Vannas, Sweden) then with a goat-rabbit antibody for alkaline phosphatase detection. Soluble protein content was determined by Bradford method (Bradford ULTRA; Expedeon, San Diego, CA).

Photosynthesis

Photosynthetic parameters were measured using a portable CO2/H2O gas exchange analyzer (LI-6400; LI-COR, Lincoln, NE, USA) equipped with a cylindrical transparent chamber (Model 6400-05). The parameters include net CO2 fixation (A) and transpiration (E) rates, stomatal conductance to water vapor (gs), the total conductance to water vapor (gtot, the conductance to water vapor through stomata and the boundary layer surrounding the leaf), and CO2 concentration of intercellular air apace (Ci). Light was provided by a tungsten–halogen light source (LS2; Hansatech, King’s Lynn, UK). Incident PPFD on the leaves was 800 µmol m− 2 s− 1 (an approx. light-saturated level of A for studied plants). Flow rate entering the chamber (ue) and chamber air temperature were set at 700–750 µmol s− 1 and 25 °C, respectively. Leaf-to-air vapor pressure deficit in the chamber ranged from 0.5 to 2.2 kPa. Leaf boundary layer conductance to water vapor for two-sided surfaces (gbw) was estimated through the filter paper method in reference to the LI-6400 manual and ranged from 1.5 to 2.0 mol m− 2 s− 1 for bean and black and silver poplars. The gbw of sugi and pine were set at 8 mol m− 2 s− 1, a default value of LI-6400. Leaf temperature calculated through the energy budget method of the LI-6400 program was 25–27 °C.

NH3 gas exchange and NH3 compensation point (γNH3)

Leaf NH3 absorption rate (FNH3) linearly increases with increasing atmospheric NH3 concentration surrounding the leaf (na). FNH3 is written as follows (Farquhar et al. 1980a):

$${F_{{\text{NH3}}}}{\text{~=~}}{g_{{\text{NH3}}}}\left( {{n_a}~ - {\gamma _{{\text{NH3}}}}} \right),$$
(1)

where gNH3 and γNH3 are the total conductance to NH3 diffusion and the NH3 compensation point, respectively. Leaves emit NH3 to the atmosphere when na is lower than γNH3 (Farquhar et al. 1980a).

We simultaneously measured FNH3 and CO2/H2O gas exchange rates. The air-inlet port of LI-6400 was connected to an NH3-absorbing column, which removed NH3 from the air supplied by a gas cylinder (20% O2 balanced in N2). The column contained 500 g of 5 mm diameter glass beads, which had been previously smeared with 5% (v/v) phosphoric acid and 2% (v/v) glycerol and dried overnight at 60 °C. A dew-point generator (LI-610; LI-COR) was connected between the column and cylinders to humidify the cylinder air. The outlet port, which vented air from the LI-6400 chamber, was linked to a chemiluminescence NH3 analyzer (ML9841A; Teledyne Monitor Labs, Englewood, CO, USA). All devices were connected using polytetrafluoroethylene tubing. A vent tube located between the devices was used to prevent air pressure from increasing in the analyzers. FNH3 was determined using a following equation (Miyazawa et al. 2014).

$${F_{{\text{NH3}}}}~=~{{\left( {{n_e} - {n_o}} \right){u_e}} \mathord{\left/ {\vphantom {{\left( {{n_e} - {n_o}} \right){u_e}} {{\text{LA}} - ~{n_o}E}}} \right. \kern-0pt} {{\text{LA}} - ~{n_o}E}},$$
(2)

where no is the mole fraction of NH3 in the air leaving the chamber when the chamber contains leaves. Changes in air-NH3 concentration with time were logged. ne, the NH3 mole fraction of air without leaves, was estimated from baseline values. After measurements, we took digital photographs of the leaves in the portable gas exchange chamber to estimate projected leaf area (LA) from the photographs using Image J (1.48v; NIH, Bethesda, MD, USA).

The γNH3 was determined with Eq. 1. Theoretically, gNH3 equals 0.92 times the gtot (Farquhar et al. 1980a). na was assumed equal to no. Leaf temperatures for NH3 gas exchange measurements were similar to those described above. The CO2 concentration in the portable gas exchange chamber was set at ambient level (400 ppm) throughout the measurements.

NH3 leakiness

For NH3 leakiness experiments, GC-grown plants were transferred to the laboratory before GC-light bulbs were turned on. Branches of field-grown plants were transferred to the laboratory on the evening 1 day before measurement dates. When sampling the branches, stem ends were cut in tap water to prevent air embolism. Illuminated leaves inside the LI-6400 chamber were subjected to different CO2 levels (100–800 ppm) for an hour. The steady-state values of A and Ci were obtained. For dark treatment, the chamber was covered with black clothes to make leaves inside the chamber in dark under ambient CO2 level (400 ppm) for an hour.

Leaves subjected to CO2 or dark treatments were cut while still in the chamber, immediately frozen in liquid N2, weighed and stored at − 80 °C. Leaf ammonium (NH4+) was extracted using 20 mM formic acid (Husted et al. 2000). NH4+ content was determined fluorometrically using HPLC after reaction with o-phthalaldehyde (474 Scanning Fluorescence Detector; Nihon Waters, Tokyo). We defined NH3 leakiness as:

$${\text{N}}{{\text{H}}_3}{\text{~leakiness~=~}}\frac{{{{{\text{[NH}}_{4}^{+}]}^{{\text{100}}}}~ - ~{{{\text{[NH}}_{4}^{+}]}^{{\text{800}}}}}}{{v_{o}^{{{\text{100}}}}~ - ~v_{o}^{{{\text{800}}}}}} \times \frac{1}{t},$$
(3)

where [NH4+], vo, and t were leaf NH4+ content, Rubisco oxygenation rate, and CO2 treatment period, respectively. t was 3600s. The superscript number (100 or 800) indicates CO2 concentration (ppm) for CO2 treatment. For each tested plant, we used the mean value of NH4+ content and that of vo at respective CO2 treatments for the calculations.

We did not measure LA of the leaf samples using a digital camera for NH3 leakiness measurements because taking clear photographs needs turning off the light projected on the leaves thereby possibly interrupts correct determination of the leaf NH4+. Therefore, the gas exchange parameters including vo were expressed per unit chlorophyll content. Leaf chlorophyll content per fresh weight was determined spectrophotometrically after pigment extraction with 80% (v/v) acetone (Porra et al. 1989). Chlorophyll contents per unit projected leaf area were determined using different leaf samples.

Rubisco oxygenation rate (v o)

Rubisco oxygenation rate (vo) was determined from leaf photosynthesis parameters with the FvCB model (Farquhar et al. 1980b):

$${v_o}{\text{~=~2}}{\Gamma ^*}{{\left( {A{\text{~+~}}{R_{{\text{day}}}}} \right)} \mathord{\left/ {\vphantom {{\left( {A{\text{~+~}}{R_{{\text{day}}}}} \right)} {\left( {{C_c} - ~{\Gamma ^*}} \right)}}} \right. \kern-0pt} {\left( {{C_c} - ~{\Gamma ^*}} \right)}},$$
(4)

and

$${{{\varvec{\Gamma}}}^*}~=~{\text{0.5}}{{{O_c}{\text{~}}} \mathord{\left/ {\vphantom {{{O_c}{\text{~}}} {{S_{{\text{c/o}}}}}}} \right. \kern-0pt} {{S_{{\text{c/o}}}}}},$$
(5)

where Rday is day respiration rate, Cc and Oc are CO2 and O2 concentrations in chloroplasts, respectively, and Sc/o is the relative specificity factor of Rubisco. Rday was assumed to equal the steady-state rate of respiration in the dark (Rdark) (Epron et al. 1995). The Sc/o of bean plants was obtained from the literature (Hermida-Carrera et al. 2016). Given that the Sc/o values of poplar, sugi, and pine trees are unknown, tobacco Sc/o values at a specific leaf temperature were used for these plants (Bernacchi et al. 2002). Under ambient O2 level (20% O2), calculated Γ* values (µmol mol− 1) at a measured leaf temperature for Bean_H, BPop_L, BPop_H, and Sugi_L were 40 at 26 °C, 39 at 26 °C, 37 at 25 °C, and 40 at 27 °C, respectively; those of SPop_F, Sugi_F and Pine_F were all 40 at 27 °C (see Table 1 for these abbreviations). Cc was calculated as:

Table 1 Abbreviations for the plant materials
Full size table
$${C_c}~=~{C_i} - {A \mathord{\left/ {\vphantom {A {{g_m}}}} \right. \kern-0pt} {{g_m}}},$$
(6)

where gm is mesophyll conductance to CO2 gas diffusion.

Mesophyll conductance (g m)

To determine gm by the curve-fitting method (Ethier and Livingston 2004), A–Ci curves were separately obtained from CO2 and dark treatment experiments (Fig. S2). For low Ci portions (< 220 ppm), the relationship of A against Ci is given as

$$\begin{gathered} {A~=~}\frac{{ - b{\text{~+~}}\sqrt {{b^2} - ~4{\text{ac}}} }}{{2a}} \hfill \\ a~=~{\raise0.7ex\hbox{${ - 1}$} \!\mathord{\left/ {\vphantom {{ - 1} {{g_m}}}}\right.\kern-0pt}\!\lower0.7ex\hbox{${{g_m}}$}} \hfill \\ b~=~{\raise0.7ex\hbox{${({V_{{\text{cmax}}}}~ - {R_{{\text{day}}}})}$} \!\mathord{\left/ {\vphantom {{({V_{{\text{cmax}}}}~ - {R_{{\text{day}}}})} {{g_m}}}}\right.\kern-0pt}\!\lower0.7ex\hbox{${{g_m}}$}}~+~{C_i}~+~K_{c}^{{{\text{air}}}} \hfill \\ c~=~{R_{{\text{day}}}}({C_{\text{i}}}~+~K_{{\text{c}}}^{{{\text{air}}}}) - {V_{{\text{cmax}}}}\left( {{C_{\text{i}}} - {\Gamma ^*}} \right), \hfill \\ \end{gathered}$$
(7)

where Vcmax and Kcair are the maximum Rubisco carboxylation rate and the Michaelis–Menten constants of Rubisco for CO2 under ambient O2, respectively. The curve-fitting procedure was performed with nonlinear least-squares fits (Levenberg–Marquardt algorithm) using software (Origin 9.1J; Origin Lab., Northampton, MA, USA). Bean Kcair was taken from the literature (Hermida-Carrera et al. 2016). Tobacco Kcair values at a respective leaf temperature were used as the Kcair values of sugi, pine, and the two poplar trees (Bernacchi et al. 2002). The Kcair (µmol mol− 1) values of Bean_H, BPop_L, BPop_H, and Sugi_L were 455 at 26 °C, 659 at 26 °C, 601 at 25 °C and 722 at 27 °C, respectively; those of SPop_F, Sugi_F and Pine_F were all 722 at 27 °C (see Table 1 for these abbreviations).

Leaf total GS activity

Total activity of leaf GS enzymes was measured according to the methods described by Vézina et al. (1988) and Lea et al. (1990). Leaf soluble protein extracts were incubated in a reaction buffer containing 100 mM HEPES-KOH (pH 7.8), 90 mM glutamate, 20 mM MgSO4, 9 mM ATP, 6 mM hydroxylamine, and 1 mM EDTA. After 20 min of incubation at 30 °C, a stop solution containing 20% (v/v) trichloroacetic acid, 0.5 M FeCl3, and 0.5 M HCl was added to the reaction mixture. The mixture was then centrifuged at 15,000 ×g for 10 min to separate the supernatant. The absorbance of the supernatant was measured at 540 nm using a spectrophotometer (UV-1650PC; Shimadzu, Kyoto, Japan).

Statistical analyses

All statistical analyses were performed using SAS Add-In 6.1 for Microsoft office (SAS Institute, Cary, NC, USA).

Results

The absence of GS2 in Pinidae

Orthologues of GS in Cryptomeria japonica (sugi), a conifer in Cupressaceae, were searched in the EST database (ForestGEN) based on the amino acid sequence of the Arabidopsis GS. We identified two types of GS1 genes in sugi: CjGS1a and CjGS1b (Fig. S1). As was the case for a conifer in Pinaceae, Pinus sylvestris (Cantón et al. 1993), we did not obtain a GS gene containing an extended sequence, GS2.

We constructed a phylogenetic tree using the deduced amino acid sequences of GS genes from two angiosperm species (French bean and Populus trichocarpa) together with those from both conifers (Fig. 1a). The amino acid sequences of CjGS1a and CjGS1b from sugi were similar to those of PsGS1a and PsGS1b from P. sylvestris, respectively. The sequences of conifer GS1b (CjGS1b and PsGS1b) were structurally similar to those of angiosperm GS1 rather than those of conifer GS1a (CjGS1a and PsGS1a). This phylogenetic relationship was unchanged even when truncating the sequences of the GS2 transit peptides (Fig. S1) and analyzing the alignments (data not shown).

Fig. 1
figure 1

a Phylogenetic tree constructed from alignments of the deduced amino acid sequences of GS genes from Cryptomeria japonica (sugi, Cj), Populus trichocarpa (poplar, Pt), Phaseolus vulgaris (French bean, Pv), and Pinus sylvestris (pine, Ps). Phylogenetic analysis of the full-length sequences was performed with the neighbor joining method. Bootstrap values are indicated at each branch point (1000 replicates). Bars correspond to two amino acid substitutions per 100 amino acid sites. Chloroplastic GS (GS2) are circled in green to be distinguished from the cytosolic form (GS1); conifer GS genes are in red. GenBank accession numbers are as follows: CjGS1a, LC331260; CjGS1b, LC331261; PtGS1.1-63, XP 002305885; PtGS1.1-78, XP 006372556; PtGS1.2-12, XP 002310666; PtGS1.2-66, XP 002306385; PtGS1.3-81, ABK94916; PtGS1.3-85, XP 002318305; PtGS2-14, XP 006380049; PtGS2-63, XP 002314425; PvGlnα, CAA27632; PvGlnβ, CAA27631; PvGlnγ, CAA32759; PvGlnδ, CAA31234; PsGS1a, CAA52448; PsGS1b, CAA06383. The names of the GS proteins for P. trichocarpa and those for P. vulgaris were assigned according to Castro-Rodríguez et al. (2011) and Lea (1997), respectively. b Immunoblotting analysis of leaf GS polypeptides from sugi, Populus alba (silver poplar) and P. nigra (black poplar). The Arabidopsis GS global antibody that recognizes both GS1 and GS2 was used. Molecular mass standards are shown on the left. 1, Field-grown sugi; 2, Growth chamber (GC)-grown sugi; 3, Field-grown silver poplar; 4, GC-grown black poplar

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To confirm the results of phylogenetic analysis, we subjected leaf soluble proteins of Populus alba (silver poplar), Populus nigra (black poplar) and sugi to immunoblotting analysis using an anti-GS1 GS2 global antibody that cross-reacts with GS1 and GS2 derived from wide range of plant species (http://www.agrisera.com). Poplar (P. trichocarpa) possesses six GS1 (39.0–39.5 kDa) and two GS2 isoforms (42.2 kDa and 42.3 kDa) (Castro-Rodríguez et al. 2011; Fig. 1a). Two polypeptide bands that correspond to the GS1 and GS2 isoforms were detected in the leaves of black and silver poplars (Fig. 1b). By contrast, sugi leaves contained only a single band (Fig. 1b). The predicted molecular masses of CjGS1a and CjGS1b from the amino acid sequence were 39.6 and 39.2 kDa, respectively. Our immunoblotting analysis is difficult to detect this 0.4 kDa difference between both isoforms. The single polypeptide band detected in sugi was therefore considered CjGS1a and/or CjGS1b.

We subjected seven angiosperm herbs and trees from seven different families and 13 coniferous species from six different families to immunoblotting analysis (Fig. 2). The leaves of all angiosperm species exhibited two clear GS1 and GS2 polypeptide bands except for French bean. Only GS2 band was clearly visible in the leaves of French bean whereas only GS1 band was obvious in the roots (Fig. 2). In French bean, identification of GS proteins through both ion exchange HPLC and immunoblotting analysis clarified that major abundance of GS2 in the bean leaves was responsible for the minor immunoblot GS1 band in their leaves (Cock et al. 1991). The opposite trend, namely, major abundance of GS1, was observed in the roots through ion exchange analysis (Lara et al. 1984). Our immunoblotting results were considered to support these previous findings. In all tested conifer leaves, a single polypeptide corresponding to GS1 was detected.

Fig. 2
figure 2

Immunoblotting analysis of GS polypeptides from angiosperm and conifer leaves. Root samples were also analyzed for Phaseolus vulgaris (French bean). Soluble proteins (4 µg per lane) extracted from the samples were subjected to SDS–PAGE (right lane). Leaf soluble proteins from silver poplar were electrophoresed together as a positive control (left lane). The upper and lower bands from silver poplar correspond to the chloroplastic (GS2) and cytosolic (GS1) forms, respectively. Immunoblotting was performed with the Arabidopsis GS global antibody that recognizes both GS1and GS2

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High γNH3 of conifer leaves

We measured the time courses of NH3 emission from leaves under low ambient NH3 concentration and used Eq. 1 to calculate NH3 compensation point (γNH3) from the measured NH3 gas exchange rates and the total conductance (Table 2). The results of French bean are shown in Fig. 3; NH3 concentration in the air exhausted from the portable gas exchange chamber increased with the increases in stomatal conductance and net CO2 assimilation rates. Similar results were found for all tested plants (data not shown). Farquhar et al. (1980a) already reported the γNH3 of bean plants; the calculated γNH3 of bean in our study was close to the value reported by Farquhar et al.

Table 2 Leaf CO2/H2O gas exchange and NH3 gas exchange properties
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Fig. 3
figure 3

Simultaneous measurements of NH3 gas exchange and photosynthesis in a primary leaf of Phaseolus vulgaris (French bean). Time course of NH3 mole fraction in air vented from the leaf chamber (solid circles), stomatal conductance to water vapor per unit leaf area (gsarea, open circles), and net CO2 fixation rate per unit leaf area (Aarea, open squares). When gsarea and Aarea reached steady state, NH3 mole fractions in the air (no and ne) were obtained for the calculation of leaf NH3 absorption rates using Eq. 2. For obtaining ne values, the straight baselines were fitted by eye. Arrows on the x-axis indicate when the leaf was inside or outside the chamber

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For the GC-grown plants, we found that the calculated γNH3 of sugi grown under L-condition (Sugi_L) was significantly higher than that of black poplar and bean plants grown under H-condition (BPop_H and Bean_H, respectively) (Fig. 4a). We did not raise sugi plants under H-condition for measurements because a yellowing symptom appears in sugi leaves under this condition (see Materials and methods). Therefore, the different γNH3 exhibited by these three species might result from differences in growth conditions. To check this possibility, we cultivated black poplar plants under L-condition (BPop_L) and compared their γNH3 values. We found that the γNH3 of BPop_L was similar to that of BPop_H (Fig. 4a). The γNH3 of Sugi_L was about twice higher than that of BPop_L, as was the case for the relationship between Sugi_L and BPop_H. Thus, growth conditions had negligible contributions to the high γNH3 of sugi.

Fig. 4
figure 4

Comparisons of leaf NH3 compensation point (γNH3) between angiosperms (open bars) and conifers (solid bars). We used plants grown in an environmentally controlled growth chamber (a GC-grown) and those grown under field conditions (b Field-grown). Abbreviations for the plant materials are shown in Table 1. Data represent mean ± SD of 3–8 leaves. Different letters indicate significant differences of means between GC- or field-grown by Tukey–Kramer’s post-hoc test at P < 0.05

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We also measured γNH3 of leaves taken from the adult trees cultivated under a field (F) condition: silver poplar (SPop_F), sugi (Sugi_F) and pine (Pine_F) trees (Fig. 4b). The γNH3 of Sugi_F was almost equal to that of the GC-grown sugi or Sugi_L (t-tests; P = 0.44). We found that the γNH3 values of Sugi_F and Pine_F were significantly higher than that of SPop_F (Fig. 4b).

Relationships of NH3 leakiness and leaf total GS activity to γNH3

To examine whether the higher γNH3 of conifers (Fig. 4) is due to higher levels of unassimilated NH3 from the photorespiration pathway, we measured changes in leaf NH4+ content in response to Rubisco oxygenation rate (vo). We calculated vo on the basis of the measured leaf CO2 gas exchange rates under different CO2 concentrations in a portable gas exchange chamber (Table 3).

Table 3 Leaf photosynthesis parameters for calculating Rubisco oxygenation rate (vo) and leaf chlorophyll (Chl) content
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The vo of all studied plants increased when CO2 concentration decreased from 800 to 100 ppm. Except for BPop_L and SPop_F, the leaf NH4+ content of all species significantly increased with the vo increases (Fig. 5). We also measured the leaf NH4+ content under dark condition at 400 ppm CO2 (Fig. 5a). The leaf NH4+ content of Sugi_L was larger than that of other three plants (BPop_H, BPop_L and and Bean_H plants) under dark treatment.

Fig. 5
figure 5

Relationship between leaf NH4+ content and Rubisco oxygenation rate (vo) in angiosperms (open symbols) and conifers (solid symbols). Leaf NH4+ content and vo are both expressed per unit chlorophyll content. We used plants grown in an environmentally controlled growth chamber (a GC-grown) and those grown under field conditions (b Field-grown). Abbreviations for the plant materials are shown in Table 1. Illuminated leaves were subjected to different CO2 concentrations ranging from 100 to 800 ppm (CO2 treatment) for one hour. For the GC-grown plants, we also plotted the data for leaves subjected to dark treatment. vo was estimated from the gas exchange data (Table 3) while it assumed to be zero for dark treatment. There were significant linear relationships using the data excluded for dark treatment (solid lines; P < 0.05). t-tests were conducted on the NH4+ content between the 100 and 800 ppm CO2 treatments (*P < 0.05, **P < 0.01, ns, not significant). Data represent mean ± SE. The number of sample leaves (n) are shown in Table 3 for CO2 treatment. n was 3 or 5 for dark treatment

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The increase in leaf NH4+ content per unit vo increment, i.e. the slope of the linear relationship between the NH4+ content and vo (Fig. 5), can be used as an index of the amount of unassimilated NH3 directly produced from the photorespiration pathway. We defined the amount of unassimilated NH3 divided by a CO2 treatment period as “NH3 leakiness” (see Eq. 3). We calculated the NH3 leakiness of each species and found that NH3 leakiness had a significant positive correlation with the γNH3 (Fig. 6a). By contrast, the differences in γNH3 were not related with the leaf total GS activities between species (Fig. 6b).

Fig. 6
figure 6

Relationships of NH3 leakiness (a) and leaf total GS activity (b) to leaf NH3 compensation point (γNH3). NH3 leakiness was calculated from both slope of the linear function of Rubisco oxygenation rate (vo) to leaf NH4+ content in Fig. 5 and a CO2 treatment period. The symbols circled in solid and dashed lines represent the conifer and angiosperm species used in this study, respectively. The total GS activity is expressed per unit soluble protein content. The scale above the left panel corresponds to a percentage of unassimilated (i.e. leaked) NH3 produced from the photorespiration pathway according to the FvCB model (Farquhar et al. 1980b). Regression coefficients (r) are shown (*P < 0.05; ns, not significant). γNH3 and GS activity data represent mean ± SD of 3–8 leaves

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Based on the FvCB model, 0.5 molecule of NH3 is produced per mol vo (Farquhar et al. 1980b). Using this model, we estimated that nearly 3% of NH3 produced per vo was unassimilated (i.e. leaked) in conifers such as sugi and pine (Figs. 6a, 7). On the other hand, in angiosperms such as bean and poplar, only less than 1% of NH3 was leaked.

Fig. 7
figure 7

Comparison of photorespiration pathways between angiosperms and conifers. The conifer photorespiration pathway is based on the pathway provided by Suárez et al. (2002). Photorespiration starts with the oxygenation of ribulose-1,5-bisphosphate (RuBP) by Rubisco. Hydroxy-Pyr, hydroxypyruvate; Gln, glutamine; Glu, glutamate; Gly, glycine; 2-OG, 2-oxoglutarate; 2-PG, 2-phosphoglycolate; 3-PGA, 3-phosphoglycerate; Ser, serine. Less than 1% and nearly 3% of the photorespiratory-produced NH3 from the mitochondria is estimated to be leaked in angiosperms and conifers, respectively (Fig. 6). GS located in chloroplasts (GS2) and ferredoxin-glutamate synthase (Fd-GOGAT) are involved in assimilation of photorespiratory NH3 in angiosperms. The cytosolic GS1a and the chloroplastic Fd-GOGAT are the candidate enzymes involved in assimilation of photorespiratory NH3 in conifers (Suárez et al. 2002)

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Discussion

Low assimilation efficiency of photorespiratory NH3 in conifers that lack GS2

Genetic information and immunoblotting analyses revealed that GS1, but not GS2, is present in sugi (Cryptomeria japonica), a member of the family Cupressaceae (Fig. 1). This is the first report showing that a non-Pinacease conifer also lacks GS2. Although the absence of GS2 needs further checking by immunolocalization analysis at the electron microscopy level as was the case for the study in Pinus pinaster (García-Gutiérrez et al. 1998), our immunoblotting analyses clearly indicated that leaves from coniferous species from all family members in Pinidae exhibited only GS1 band (Fig. 2). Thus, our results suggest that the absence of GS2 is common in conifers.

NH3 produced from the photorespiration pathway is assimilated by GS2 in angiosperms. In conifers, one of the GS1 isoforms, GS1a, is a potential isoform that might fulfill the GS2 function (Avila et al. 2001; Suárez et al. 2002) (Fig. 7). The cysteine residues of the GS2 sequences are involved in the redox regulation of this enzyme and these residues are not observed in angiosperm GS1 (Choi et al. 1999). It is noteworthy that the conifer GS1a sequences, but not the GS1b, possess one of the cysteine residues (Fig. S1). This could be an evidence that conifer GS1a has an orthologous function to GS2.

The FvCB model estimated that nearly 3% of NH3 produced per vo was unassimilated (i.e. leaked) in conifers whereas only less than 1% of the NH3 was leaked in angiosperms (Figs. 6a, 7). Higher NH3 leakiness, thereby lower assimilation efficiency of photorespiratory NH3, could be disadvantage for conifers due to high photorespiration activity under the present levels of atmospheric CO2 and O2.

Sensitivity of NH3 leakiness to g m and Γ*

As inferred from Eqs. 3–6, NH3 leakiness depends on the estimated gm and Γ* values. Several methods, such as chlorophyll fluorescence method, stable isotope method, and curve-fitting method, have been developed for gm estimation (Epron et al. 1995; Ethier and Livingston 2004; von Caemmerer and Evans 1991). The gm values estimated through the curve-fitting method are almost in good agreement with those estimated with the chlorophyll fluorescence method in tobacco (Miyazawa et al. 2008) and in gymnosperms including conifers (Veromann-Jürgenson et al. 2017). There was a significant positive relationship between the gm and the light-saturate rates of net CO2 fixation at ambient CO2 in our study (P < 0.05; Fig. S3). As compared with those relationships reported in literature, our estimated gm values were considered to be relevant. The simultaneous application of more than two methods is, however, recommended for precise gm estimation (Pons et al. 2009). We then performed a sensitivity analysis to identify to what extent changes in gm affect NH3 leakiness, and found that NH3 leakiness was almost unaffected by changes in gm (Fig. S4).

As in Eq. 5, the Γ* is determined by Sc/o. The Sc/o of bean plants is available from the literature (Hermida-Carrera et al. 2016), whereas those of poplar, sugi, and pine are unknown. We performed a sensitivity analysis to determine to what extent the uncertainty of Γ* affects NH3 leakiness (Fig. S4). We calculated Γ* range on the basis of the reported Sc/o values of purified Rubisco from 28 species of C3 crops and trees (Galmés et al. 2005; Hermida-Carrera et al. 2016). We found that the NH3 leakiness values of sugi and pine trees were always higher than those of bean and poplar within this Γ* range (Fig. S4). Taken together, NH3 leakiness was robust to changes in estimated gm and Γ* values.

Comparison with γNH3 values reported in literature

The values of γNH3 have been intensively studied in crops and grasses such as barley, bromegrass, French bean, oilseed rape, perennial ryegrass, and rice; γNH3 ranges from 0.1 to 10 nmol mol− 1 under ambient temperatures (Farquhar et al. 1980a; Hayashi et al. 2008; Miyazawa et al. 2014; Wang et al. 2013). The γNH3 is affected by the availability of soil nitrogen because NH4+ and/or NO3− are transported to leaves via the transpiration stream from soils (Hayashi et al. 2008; Husted et al. 2000). For example, bromegrass cultivated on medium with extremely high nitrogen content has a γNH3 value of approximately 10 nmol mol− 1 (6 mM NH4HCO3 in medium) (Mattsson and Schjoerring 2002). In our study, GC-sugi saplings were raised on a medium with moderate nitrogen content (0.9 mM NH4NO3), suggesting that the high γNH3 of sugi (18.4 nmol mol− 1 on average at 27 °C) cannot be attributed to high soil nitrogen.

There are only a few reports on the γNH3 of trees, particularly those of conifers. The γNH3 of mature green leaves from field-grown Fagus sylvatica, a deciduous broad-leaved tree, was 3 nmol mol− 1 at 25 °C; this γNH3 value is similar to those reported for crops and grasses (Wang et al. 2011). Geßler et al. (2002) studied changes in the FNH3 of twigs from adult conifers (spruce) fumigated with various concentrations of NH3 (2.4 to 135 nmol mol− 1) under field conditions of fluctuating light and air temperature. They estimated the γNH3 of spruce by conducting linear regression between FNH3 and air-NH3 concentration. They found that the γNH3 of spruce was approximately 2.5 nmol mol− 1, which was lower than those of sugi and pine trees in our study and close to that of crops and grasses. Geßler et al. (2002) measured the FNH3 of spruce twigs exposed to low light intensities under which photorespiration rates are reduced (< 200 µmol m− 2 s− 1 for most measurement data in Geßler et al. vs. 800 µmol m− 2 s− 1 in our study). Low photorespiration activity decreases the γNH3 in barley (Wang et al. 2013). The different light conditions in the study of Geßler et al. from that in our study might explain this inconsistency.

Is acquisition of GS2 as an adaptive mechanism to low CO2 environments on earth?

Cánovas et al. (2007) speculated that the acquisition of GS2 is an adaptive mechanism to high levels of photorespiratory NH3 when plants encountered the present oxygen levels in the atmosphere during land plant evolution. Our present study is the first report demonstrating that assimilation efficiency of photorespiratory NH3 differs between angiosperms and conifers that lack GS2 (Fig. 6).

Gymnosperm consists of four groups: Cycadidae, Ginkgoidae, Gnetidae and Pinidae (Christenhusz et al. 2011). García-Gutiérrez et al. (1998) found that leaves of a gymnosperm, Ginkgo biloba (Ginkgoidae), exhibited GS1 and GS2 polypeptide bands by immunoblotting analysis. This result was confirmed by our analysis in Ginkgo (Fig. S5). We subjected leaves of some species from the other two gymnosperm groups such as Cycas revoluta (Cycadidae), Ephedra minima (Gnetidae), Gnetum gnemon (Gnetidae) and Welwitschia mirabilis (Gnetidae) to immunoblotting, and found that all these gymnosperm species exhibited only GS1 band (Fig. S5). Therefore, except for Ginkgo, the absence of GS2 appears to be common in gymnosperms.

Angiosperms are presently the most dominant plant group on earth while gymnosperms were the dominant group during the Triassic and Jurassic periods (250 − 145 Mys ago) (Haworth et al. 2011). Atmospheric CO2 level was higher than the present average level of 400 ppm and likely fluctuated between 1200 and 1800 ppm during these periods (Haworth et al. 2011; Sage 2013). On the other hand, the estimated atmospheric O2 levels ranged from 15 to 20%, which was similar to the present level (Berner 1999; Haworth et al. 2011). Such high CO2 environment would have suppressed the NH3 production because of low photorespiration activity. In this context, the disadvantage of absence of GS2 might not have been crucial for gymnosperms under such high CO2 environments on earth. To get a clearer picture of the evolutionary significance of GS2, further studies need to confirm whether GS2 is absent from the early divergent lineage including ferns and mosses.

How does GS2 contribute to efficient assimilation of photorespiratory NH3?

Analyses of purified poplar GS2 and recombinant Pinus GS1a (PsGS1a) enzymes from previous studies indicated that Km or S0.5 values for the NH4+, glutamate and ATP were lower or similar in PsGS1a than those in poplar GS2 (de la Torre et al. 2002; Fu et al. 2003). This means that the differences in the substrate affinities between both GS enzymes fail to explain the lower assimilation efficiency of photorespiratory NH3 in conifers. Glutamate is known to be enriched in the chloroplasts (Mills and Joy 1980). It is tempting to speculate that subcellular differences in the substrate concentrations for GS such as the glutamate concentration can explain the difference in the assimilation efficiency between conifers and angiosperms.