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. 2014 Mar 27;65(13):3637–3647. doi: 10.1093/jxb/eru106

Comparative studies of C3 and C4 Atriplex hybrids in the genomics era: physiological assessments

Jason C Oakley 1, Stefanie Sultmanis 1, Corey R Stinson 1, Tammy L Sage 1, Rowan F Sage 1,*
PMCID: PMC4085961  PMID: 24675672

Summary

Leaf anatomy and physiology are characterized in newly generated hybrids between C3 and C4 species of Atriplex, thus re-establishing the classic system exploited by Björkman and colleagues 45 years ago.

Key words: C4 engineering, C4 photosynthesis, CO2 concentrating mechanism, photosynthetic hybrids, Rubisco.

Abstract

We crossed the C3 species Atriplex prostrata with the C4 species Atriplex rosea to produce F1 and F2 hybrids. All hybrids exhibited C3-like δ13C values, and had reduced rates of net CO2 assimilation compared with A. prostrata. The activities of the major C4 cycle enzymes PEP carboxylase, NAD-malic enzyme, and pyruvate-Pi dikinase in the hybrids were at most 36% of the C4 values. These results demonstrate the C4 metabolic cycle was disrupted in the hybrids. Photosynthetic CO2 compensation points (Г) of the hybrids were generally midway between the C3 and C4 values, and in most hybrids were accompanied by low, C3-like activities in one or more of the major C4 cycle enzymes. This supports the possibility that most hybrids use a photorespiratory glycine shuttle to concentrate CO2 into the bundle sheath cells. One hybrid exhibited a C4-like Г of 4 µmol mol–1, indicating engagement of a C4 metabolic cycle. Consistently, this hybrid had elevated activities of all measured C4 cycle enzymes relative to the C3 parent; however, C3-like carbon isotope ratios indicate the low Г is mainly due to a photorespiratory glycine shuttle. The anatomy of the hybrids resembled that of C3-C4 intermediate species using a glycine shuttle to concentrate CO2 in the bundle sheath, and is further evidence that this physiology is the predominant, default condition of the F2 hybrids. Progeny of these hybrids should further segregate C3 and C4 traits and in doing so assist in the discovery of C4 genes using high-throughput methods of the genomics era.

Introduction

C4 photosynthesis is a carbon-concentrating mechanism that evolved from C3 progenitors at least 65 times (Sage et al., 2012). During C4 evolution, a coordinated series of anatomical and biochemical adjustments established the compartmentation and enzyme activities required to efficiently concentrate CO2 around Rubisco (Monson and Rawsthorne, 2000). In the process, dozens to hundreds of genes have been altered (Bräutigam et al., 2011a, b; Gowik et al., 2011). A number of the modifications to key biochemical enzymes such as PEP carboxylase have been identified, although most remain unknown, particularly the genes controlling the anatomical modifications (Kajala et al., 2011; Ludwig, 2013). Identification of these elements is essential in the effort to improve C4 photosynthesis and potentially engineer the C4 pathway into C3 crops, as is now being attempted with rice (von Caemmerer et al., 2012; http://c4rice.irri.org/).

Gene discovery is most efficient when researchers can apply forward and reverse genetic approaches using genetic model organisms (Meinke et al., 1998). Unfortunately, in the case of the C4 pathway, ideal model organisms have not been developed, although Setaria viridis is a potential candidate (Li and Brutnell, 2011; Covshoff et al., 2014). The lack of tractable genetic models for C4 photosynthesis requires that alternative means of gene discovery be considered. One option is to generate hybrids between closely related C3 and C4 species, and then use a genetic mapping strategy to associate genes with segregating traits. A number of congeneric pairs of C3 and C4 species have been hybridized since the discovery of the C4 pathway. The first C3 × C4 hybrids were produced by Malcolm Nobs and Olle Björkman (Fig. 1) between Atriplex rosea (C4) and Atriplex prostrata (C3, formerly termed A. patula ssp. hastata and A. triangularis; Kadereit et al., 2010), and A. rosea and A. glabriuscula (C3) (Björkman et al., 1969; Osmond et al., 1980). Subsequent efforts created hybrids between C3 and C4-like Flaveria species (Apel et al., 1988), and C3-C4 intermediate and C4 Flaveria species (Brown et al., 1986; Brown and Bouton, 1993). Hybrids have also been generated between C3 and C3-C4 intermediate Panicum species (Bouton et al., 1986). In many of the Flaveria crosses, the F1 hybrids were sterile (Brown and Bouton, 1993). In cases where F2 hybrids were generated and segregation of traits observed, problems associated with chromosome abnormalities and pairing were evident, such that mapping populations could not be formed (Osmond et al., 1980; Covshoff et al., 2014). All hybrid studies were abandoned, and the hybrids eventually perished.

Fig. 1.

Fig. 1.

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(A) Malcolm Nobs pollinating Atriplex rosea, with the pollen donor, Atriplex prostrata, to his right. Photo supplied by Olle Björkman, with kind permission. (B) Olle Björkman standing behind a clump of Atriplex prostrata (arrow) at the collection site, December 15, 2010 (Photo by R.F. Sage). (This figure is available in colour at JXB online.)

With the advent of high-throughput sequencing and bioinformatics, the ability to evaluate genetic differences between hybrid offspring has dramatically improved, such that the requirement for a mapping population can be relaxed. Of particular promise is sequencing of transcriptomes (RNA-Seq), which can quantify gene expression over a large dynamic range and does not require prior knowledge of the genome sequence (Bräutigam and Gowik, 2010). Comparative transcriptomics has already been used to identify genes that are differentially expressed in leaves of closely related C3 and C4 plants (Bräutigam et al., 2011a, b; Gowik et al., 2011). By using a comparative transcriptomics approach with segregating F2 hybrids, the C4 genes controlling the segregating traits may be identified.

C3 × C4 hybrids can also provide novel insights for understanding C4 structure, function, and evolution. With advances in photosynthetic methodology, the development of theoretical models of C3 and C4 photosynthesis, and an improved appreciation of how structural adaptations enhance C4 function, we are now in a much better position to interpret patterns observed in C3 × C4 hybrid lines then was the case a generation ago (Dengler and Nelson, 1999; von Caemmerer, 2000; Sage et al., 2013). Predictions from theoretical models of C4 photosynthesis developed since the hybrid era can also provide valuable insights that will aid the interpretation of C3 × C4 hybrid studies (von Caemmerer, 2000; Ubierna et al., 2013). In addition, models describing the function of C3-C4 intermediate species (Rawsthorne et al., 1988; von Caemmerer, 1989 and 1992) appeared near the end of the hybrid studies (Brown and Bouton, 1993). With the modern understanding of C3-C4 intermediacy, it is now possible to address the degree to which C3 × C4 hybrids express the physiology of C3, C4, or C3-C4 intermediate species (Sage et al., 2012). In C3-C4 intermediates, the major physiological trait is a CO2-concentrating mechanism (CCM) that shuttles photorespiratory glycine from mesophyll (M) to bundle sheath (BS) tissues where the photorespiratory enzyme glycine decarboxylase is localized (Monson and Rawsthorne, 2000). This CCM is now termed C2 photosynthesis (Sage et al., 2012).

In reviewing the literature on C3 × C4 hybrids, the most attractive system seems to be the cross between A. rosea and A. prostrata (Björkman et al., 1969). An appealing aspect of this system is that the axile inflorescences of A. rosea are entirely composed of female flowers. This facilitates cross-pollination with A. rosea as the maternal parent because the bisexual inflorescences at the branch tips can be easily removed (Osmond et al., 1980). The F1 offspring of the A. rosea × A. prostrata cross are fertile, although with reduced pollen fertility and seed set. The F2 offspring exhibit a gradation in many C4 traits, with independent assortment (Boynton et al., 1970). For example, no correlation is apparent between leaf anatomy and expression of C4 enzymes (Boynton et al., 1970). These findings were the first to demonstrate that multiple genes are involved in the expression of C4 photosynthesis, and show that the loss of any one C4 trait leads to breakdown of the C4 CCM (Björkman, 1976; Osmond et al., 1980). However, chromosomal abnormalities were observed, with only four out of nine chromosomes regularly pairing at meiosis (Nobs, 1976). This precluded traditional genetic analysis, as forming a linkage map was impossible. The use of high-throughput genomics can potentially overcome this constraint (Bräutigam and Gowik, 2010).

To exploit the potential of C3 × C4 hybrids in the genomics era, it is necessary to produce new hybrid lines to replace those lost decades ago. We therefore regenerated hybrids between A. rosea and A. prostrata through to the F2 generation. Here, we describe the physiology and leaf anatomy of these hybrids using gas exchange and biochemical assays, and interpret the results in light of current theory for the function of C3-C4 intermediate and C4 systems.

Materials and methods

Generation of F1 and F2 hybrids

With the assistance of Olle Björkman (Fig. 1B), seeds of A. prostrata were collected from a salt marsh along San Francisco Bay in Baylands Park, Palo Alto, California USA (37°27’38.65”N × 122°06’19.63”W). This is the same collection site for this species in the first hybrid trials (Björkman et al., 1969). Seeds of A. rosea were collected in a corral along Ball’s Canyon road, 30 km northwest of Reno, Nevada, USA by Chris Root (39°39’20.68”N × 120°03’19.89”W). All plants used for crosses were grown from these collections in a rooftop greenhouse located at the University of Toronto. Plants were grown in a mixture of sand, Pro-Mix (Premier Tech Ltd., Rivière-du-Loup, Québec, Canada), and sterilized topsoil (2:2:1 by volume) in either 7.6 l or 3.8 l pots. Plants were watered as necessary to avoid drought and fertilized weekly with a mixture containing 1.8g l–1 of 24-8-16 Miracle-Gro All Purpose fertilizer, 1.2g l–1 30-10-10 Miracle-Gro Evergreen Tree and Shrub fertilizer (Scotts Miracle-Gro Co., Marysville, Ohio, USA), 4.0mM Ca(NO3)2, and 1.0mM MgSO4. The daytime temperature during growth was 26– 32 ºC depending on outdoor temperature and solar insolation, and night temperature was approximately 23 ºC.

In A. rosea, bisexual inflorescences are produced at the branch tips, whereas only female inflorescences are produced in the leaf axils of mature stems (Osmond et al., 1980). A. prostrata has only bisexual inflorescences. By removing the bisexual inflorescences from A. rosea, we were able to protect the axile flowers from self-pollination and ensure they would receive only pollen produced by A. prostrata. Flowers of A. rosea were pollinated using an extra-fine paintbrush from August to October, 2011. F1 hybrid seed was mature when plants senesced in mid-to-late November, 2011. F1 hybrids were grown in identical environments as the parents, using high-pressure sodium lamps to maintain photoperiod at 14h. These plants flowered beginning in mid-August and were allowed to self-pollinate, with seeds maturing by late October.

The F2 hybrids, along with F1, A. rosea and A. prostrata plants were grown in a plant growth chamber (Conviron PGC-20, Conviron Ltd., Winnipeg, Manitoba, Canada) at 27 ºC day/22 ºC night using the same soil, watering, and fertilizer regime as described above. Photoperiod was 18h with a light intensity near 700 µmol m–2 s–1 during the central 8-h portion of the photoperiod, and 200 µmol m–2 s–1 for 4h on each side of the high light period. One hour of incandescent light provided 20 µmol m–2 s–1 during the first and last hour of the photoperiod. We selected this photoperiod after preliminary trials showed plants flowered in a 14h photoperiod.

Gas exchange, leaf nitrogen, and enzyme assays

Gas exchange measurements were conducted on 6–10-week-old plants, using a recently expanded leaf for all measurements. Leaf disks for enzyme and nitrogen assays were sampled from the leaves used for gas exchange. Carbon isotope ratios of leaf disks from adjacent leaves were determined by the University of Washington Isotope Facility (http://depts.washington.edu/isolab/). Whole-leaf gas exchange parameters were measured using a LI-6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, Nebraska, USA) at a leaf temperature of 30 ºC (Vogan et al., 2007). For determination of the response of net CO2 assimilation rate (A) to intercellular CO2 content (C i), a saturating light intensity of 1500 µmol m–2 s–1 was used for A. prostrata and 1800 µmol m–2 s–1 for A. rosea. In the measurement of the A/C i response, leaves were first equilibrated to saturating light (1500 µmol m–2 s–1 for A. prostrata and 1800 µmol m–2 s–1 for A. rosea) and then measurements were recorded. Subsequently, ambient CO2 concentration was raised to almost 1000 µmol mol–1 to determine the maximum assimilation rate and then reduced in steps to 35 µmol mol–1 for A. prostrata, 10 µmol mol–1 for A. rosea and 20 µmol mol–1 for the F1 and F2 hybrids. The CO2 compensation point was calculated using the x-intercept of a linear regression through the lowest 4–6 CO2 concentrations that fell on a linear response of A versus C i. This regression was also used to calculate the initial slope of the A/C i curve, which is an estimate of carboxylation efficiency (CE). Leaf nitrogen was assayed using a Costech ESC 4010 C:N analyzer by the University of Nebraska Ecosystem Analysis lab, Lincoln, Nebraska (biosci.unl.edu/facilities).

Enzyme assays were conducted at 30 ºC for Rubisco and three C4 cycle enzymes: phosphoenolpyruvate carboxylase (PEPCase), NAD malic enzyme (NAD-ME), and pyruvate phosphate dikinase (PPDK) (Sage et al., 2011). Leaf samples were extracted into 50mM HEPES buffer (pH 7.8) containing 10mM MgCl2, 2mM MnCl2, 1mM EDTA, 2% PVPP (w/v), 1% PVP, 1% BSA, 10mM DTT, 0.5% (v/v) Triton X-100, 10mM 6-aminocaproic acid, and 2mM benzamide. Enzyme activities were assayed with a diode array spectrophotometer by measuring at 340nm the reduction of NAD+ (for NAD-ME), or the oxidation of NADH in a coupled enzyme assay (Rubisco, PEPCase, PPDK). NAD-malic enzyme and PEP carboxylase were assayed according to Sage et al., (2011). Rubisco was assayed according to Ashton et al. (1990), with the extract being incubated in the reaction mixture for 10min before the assay to ensure full activation of the enzyme. The PPDK assay was modified from Ashton et al. (1990), with 10mM KHCO3 replacing NaHCO3 and the concentration of PEPCase being increased to 3 units ml–1. All chemicals for enzymes assays with the exception of PEPCase were obtained from Sigma-Aldrich, St. Louis, USA. PEPCase was obtained from Bio-Research Products, North Liberty, Iowa, USA.

Leaf anatomy

For light and transmission microscopy, 2mm2 samples were cut from the middle region of recently expanded leaves and prepared for microscopy as described by Sage and Williams (1995). Briefly, sections were fixed in 2% glutaraldehyde and 0.5M sodium cacodylate buffer solution (pH 6.9) and post-fixed with a 2% osmium tetroxide solution. Samples were then dehydrated in ethanol increments and embedded in Spurr’s resin. The microscopy samples were obtained from leaves adjacent to those used for gas exchange analyses, and were harvested in the middle of the four-week period when gas exchange data were acquired.

Results

Generation and growth of the F1 and F2 hybrids

Approximately 80% of A. rosea flowers that were hand-pollinated with A. prostrata pollen yielded seed. By contrast, Nobs et al., (1970) reported seed set near 10%. Seedlings of F1 plants were easy to identify as they lacked the red colour present on the bottom of A. rosea leaves. The F1 hybrids produced 50–100 F2 seeds each, similar to the results of Nobs et al., (1970). The germination rate for F1 seeds was over 80%. The growth habit and leaf shape of the F1 hybrids was intermediate between that of the parents and uniform compared with each other, whereas the F2 hybrids were also intermediate in growth habit, but exhibited variable leaf shape (Supplementary Fig. S1). Notably, all F1 and F2 hybrids retained female-only inflorescences in the leaf axils, as seen in the maternal parent A. rosea.

Gas exchange results

The F1 hybrids exhibited a CO2 compensation point (Γ) near 30 µmol mol–1, in contrast to nearly 0 µmol mol–1 in A. rosea and 50 µmol mol–1 in A. prostrata, at 30 °C (Fig. 2). Representative A/C i responses for the parents and all hybrids are presented in Supplementary Fig. S2. In Fig. 2A, we show normalized A/C i responses of the C3 and C4 parents, three hybrids, and for comparison, the C3-C4 intermediate species Flaveria floridana. The normalized curves demonstrate the F1 and F2 hybrids had a similar qualitative response as A. prostrata and F. floridana, with the major exception being that the hybrids had a lower carboxylation efficiency (CE) and CO2 compensation point (Γ) than A. prostrata (Fig. 2B; Fig 3A). The Γ values of the F2 hybrids ranged from a C4-like value of 4 µmol mol–1 in F2-114 to 45 µmol mol–1 in F2-123; Γ in most F2 hybrids clustered between 25–35 µmol mol–1 (Table 1; Fig. 3). At current air levels of CO2 (about 400 µmol mol–1 in Toronto), A 400 values in the F2 hybrids ranged between 48% and 67% (average 57%) of the A. prostrata value (Table 1). At CO2 saturation, the difference between the mean A value (A max) of the F2 hybrid lines and A. prostrata was less: A max in the hybrids ranged between 68% and 89% (mean 77%) of the C3 values (Table 1). The difference in the A 400 values between the hybrids and A. prostrata was largely due to reduced carboxylation efficiency in the hybrids. The CE values ranged from 32–53% (average 44%) of the C3 value in the F1 and F2 hybrids (Table 1), and exhibited no relationship with variation in Γ (Fig 3A). The δ13C of the F1 and F2 hybrids ranged from –29.3 to –27.6‰, and were consistently more positive than the C3 mean of –32.2‰ (Fig. 3B). These values were shifted more negative by approximately 2‰ units owing to an enriched fossil fuel signature in downtown Toronto, where the growth facilities are located. No relationship was apparent between δ13C and either Γ, A 400, or A max, and the CE value (not shown).

Fig. 2.

Fig. 2.

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The response of net CO2 assimilation rate, A, to intercellular CO2 (C i) at 30 °C and 1500 µmol photons m–2 s–1 for the two Atriplex parents, an F1 hybrid and F2 hybrids. (A) Normalized net CO2 assimilation rate for the Atriplex parents, the C3-C4 intermediate Flaveria floridana, an F1 hybrid, and the F2 hybrids 114 and 123. (B) The low CO2 portion of the A versus C i response illustrating CO2 compensation points and initial slopes for all hybrids in the study. Results shown are representative responses of 3–6 A versus C i measurements for the hybrids and A. prostrata, and two measurements of A. rosea. See Supplementary Fig. S2 for the non-normalized A/C i responses of each hybrid.

Fig. 3.

Fig. 3.

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The relationship between the CO2 compensation point of the net CO2 assimilation rate and (A) the carboxylation efficiency of photosynthesis and (B) the carbon isotope ratio of leaves in Atriplex prostrata (C3, ■), Atriplex rosea (C4, ▲), an F1 hybrid (♦), and F2 hybrids (●). “114” indicates the datapoint for the F2-114 hybrid. Some error bars are obscured by the symbols.

Table 1.

Summary of leaf gas exchange, nitrogen and nitrogen-use efficiency parameters for C3 × C4 hybrids and their parents grown in plant growth chambers

Means ± SE. n=3–6 for gas exchange except for A. rosea (n=2). Abbreviations: ATPR, A. prostrata; ATRO, A. rosea; A 400, net CO2 assimilation rate at an ambient CO2 of 400 µmol mol–1; A max, net CO2 assimilation rate at 800 µmol mol–1 CO2; Ci/Ca, ratio of intercellular to ambient CO2 concentration; N, nitrogen. Carboxylation efficiency is equal to the initial slope of the A versus C i response. Superscripted x, y, or z indicate differences at P<0.05 between the ATPR, ATRO, F1 hybrid, and the pooled mean of all F2 hybrids. The a, b, c, or d letters after each value indicate statistical groups at P< 0.05 when all genotypes were compared. Statistical differences were tested using a one-way ANOVA followed by a Student-Newman-Keuls post-hoc test.

A 400 A max Ci/Ca @ 400 CO2 compensation point (Γ) Carboxylation efficiency Leaf N content Leaf nitrogen-use efficiency (NUE) (=A 400/leaf N)
 Genotype µmol m–2 s–1 µmol m–2 s–1 mol mol–1 µmol mol–1 mol m–2 s–1 mmol m–2 mmol mol–1 s–1
ATPR-C3 31.6±1.2ax 37.7±0.7ax 0.80±0.02ax 50.5±0.3az 0.191±0.007by 175±12ax 182±7abx
ATRO–C4 31.2±0.0ax 32.8±0.6abcxy 0.57±0.09by –2.2±0.2dx 0.783±0.085ax 143±14ax 221±22ax
F1 16.0±1.0bcy 25.6±2.0cy 0.81±0.01ax 32.1±4.9cy 0.065±0.008cz 156 (n=1) 115 (N=1)
F2-107 20.2±1.1bc 31.2±1.1bc 0.80±0.02a 25.1±1.5c 0.100±0.007c 130±7a 131±5b
F2-108 17.9±1.0bc 30.7±1.0bc 0.76±0.02a 27.8±1.2c 0.088±0.004c 164±14a 110±19b
F2-109 20.5±0.8bc 32.1±0.9abc 0.81±0.01a 31.1±2.3c 0.094±0.004c 130±18a 164±26ab
F2-112 15.3±2.1c 27.7±2.8bc 0.79±0.07a 31.1±1.5c 0.069±0.007c 147±25a 104±47b
F2-114 16.0±0.4bc 26.6±0.3c 0.80±0.01a 3.8±2.4d 0.061±0.004c 125±4a 125±3.6b
F2-118 16.2±1.0bc 25.4±1.3c 0.76±0.06a 31.2±1.1c 0.084±0.007c 133±6a 124±15b
F2-119 15.9±3.1bc 28.6±4.2bc 0.71±0.06a 38.3±0.8bc 0.077±0.010c 141±15a 110±16b
F2-120 17.5±1.1bc 26.6±1.2c 0.82±0.02a 26.5±3.8c 0.083±0.004c 154±6a 115±8b
F2-123 21.3±0.6b 33.5±0.7ab 0.81±0.01a 44.6±1.0ab 0.102±0.004c 149±7a 147±8b
All F2 18.0±0.5y 29.2±0.6y 0.79±0.01x 29.5±2.0y 0.08±0.003z 145±4x 126±5y
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Leaf nitrogen content and nitrogen-use efficiency

Although the C4 parent and all hybrids lines exhibited lower leaf nitrogen content than the C3 parent, none of their means were significantly different (Table 1). Differences in leaf nitrogen-use efficiency (NUE) between the C3 and C4 species could not be statistically resolved, whereas each hybrid line except F2-109 has a significantly lower NUE than the C4 parent (Table 1). On average, the mean NUE of all the F2-hybrids was 31% less than the C3 mean and 43% less than the C4 value.

Enzyme activity

The Rubisco activity of the F1 and F2 hybrids was 30–50% of the C3 value (Table 2). When the CE of each hybrid was plotted against its corresponding Rubisco activity, the hybrid values cluster around the theoretical relationship between Rubisco and CE in a C3 species (Fig. 4). The activities of the three major C4 cycle enzymes — PEPC, NAD-ME, and PPDK — were generally low in the hybrids and in many cases approached the activity of the C3 parent (Table 2). The F1 hybrid had significantly higher NAD-ME, PEPC, and PPDK activity than the C3 parent. Five of the nine F2 hybrids had significantly higher NAD-ME activities than the C3 parent, whereas just three had significantly higher PPDK activities than A. prostrata. Differences in PEPC between the F2 hybrids and the C3 parent could not be resolved using a one-way ANOVA at P<0.05; however, low statistical power in the test weakened our ability to resolve differences in PEPC activity. Four hybrids exhibited mean PEPC activities that were over twice the C3 value, and one of these, the F2-114 with the low, C4-like Г value, also had elevated activities of NAD-ME and PPDK (Table 2).

Table 2.

The in vitro activity of NAD-malic enzyme (NAD-ME), PEP carboxylase (PEPC), pyruvate-phosphate dikinase (PPDK) and Rubisco at 30 °C

Mean ± SE, n=4. Abbreviations: ATPR, A. prostrata; ATRO, A. rosea. Statistical differences between ATPR, ATRO, F1 and the pooled F2 means at P<0.05 were tested using one-way ANOVA followed by a Student-Newman-Keuls post-hoc test and are shown as superscripts x, y and z. * beside a value indicates means are significantly different from the ATPR activity using a one-way ANOVA followed by a Holm-Sidak post-hoc test where the ATPR mean was treated as the control value.

Genotype Enzyme Activity, µmol m–2 s–1
NAD-ME PEPC PPDK Rubisco
ATPR-C3 2.0±1.1z 12.5±1.4z 2.2±1.2z 156.7±5.1x
ATRO-C4 39.9±4.3x* 223.2±19.1x* 43.0±5.1x* 42.3±3.4z*
F1 7.8±0.8y* 55.9±7.9y* 16.0±0.6y* 74.0±6.3y*
F2-107 11.2±0.2* 32.9±6.3 3.1±1.4 79.9±6.8*
F2-108 11.8±1.1* 27.4±5.9 2.8±0.7 69.5±8.9*
F2-109 8.2±1.6 15.8±3.8 3.2±0.9 48.4±7.9*
F2-112 9.7±0.8* 15.3±4.7 4.0±1.7 65.7±12.5*
F2-114 9.6±1.3* 26.8±3.0 15.3±1.8* 68.2±5.4*
F2-118 4.5±1.8 20.7±2.1 11.7±3.7* 68.4±6.0*
F2-119 9.1±1.1 28.8±6.6 2.8±1.0 64.5±4.8*
F2-120 12.0±0.8* 27.7±2.0 3.7±0.6 77.3±2.1*
F2-123 4.6±0.7 23.9±5.4 13.2±3.3* 72.7±0.3*
All F2 9.1±0.6y 24.7±1.7z 6.9±1.1z 69.5±2.9y
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Fig. 4.

Fig. 4.

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The carboxylation efficiency of photosynthesis as a function of in vitro Rubisco activity for the C3 species Atriplex prostrata, an F1 hybrid and all F2 hybrids in the study. Carboxylation efficiencies were calculated as the initial slope of the A versus C i response for each genotype. Mean ± 3–6. The line is the theoretical carboxylation efficiency predicted for C3 Rubisco activity using the model of von Caemmerer (2000) and assuming the Rubisco activation state is 80%, Γ* equals that of spinach at 30 °C, (Brooks and Farquhar, 1985) and the Rubisco kinetics and activation energies for the C3 Atriplex glabriuscula equal those of A. prostrata (von Caemmerer and Quick, 2000). “114” and “109” indicate the data points for F2-114 and F2-109.

Leaf anatomy

The leaf anatomy (Fig. 5A, B) and ultrastructure (Fig. 6A, B) of A. prostrata and A. rosea were typical for C3 and C4 members of the genus (Downton et al., 1969; Dengler et al., 1995). Atriplex rosea has well-developed BS cells that are discontinuous on the abaxial side of the vein (see also Liu and Dengler, 1994). In cross-section, BS cells are triangular in shape, which allows them to be tightly packed against the vein. Enlarged chloroplasts occupy the centripetal half of the BS cells in A. rosea, whereas no chloroplasts occur in the outer-most region of the cells (Figs. 5B, 6B). This is typical for the Atriplicoid-type of Kranz anatomy (Dengler and Nelson, 2000). In A. prostrata, BS chloroplasts are smaller than in the C4 plants and the chloroplasts are generally positioned along the outer periphery of the BS cell opposite intercellular air spaces. In cross section, chloroplasts were infrequent along the inner, centripetal wall of the BS cells of A. prostrata, and the individual BS cells were generally circular in outline. The BS cells of the F1 and F2 hybrids were variable in size and shape yet typically intermediate in structure between the C3 and C4 condition (Figs 5, 6, and Supplementary Fig. 3). Many of the BS cells of both F1 and F2 hybrids were oval in cross section, in contrast to the circular BS cells of A. prostrata and the triangular BS cells of A. rosea. This pattern resembles that observed in an immature leaf in A. rosea (see Fig. 4 in Liu and Dengler, 1994). In all hybrids, BS chloroplasts were numerous and arrayed all around the BS cell periphery (Figs 5 and 6). Chloroplast size and shape in the BS of the F2 hybrids was similar to what was observed in the BS of A. prostrata (Figs 5 and 6). In the BS cells of the hybrids, mitochondria occurred between chloroplasts, but did not form distinct ranks between elongated chloroplasts as observed in A. rosea (Fig. 6).

Fig. 5.

Fig. 5.

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Light micrographs of cross-sections through leaves of (A) Atriplex prostrata, (B) Atriplex rosea, (C) their F1 hybrid, (D) F2-108, (E) F2-114, and (F) F2-123. See Supplementary Fig. S3 for light micrographs of leaf cross sections for the other six hybrids in the study. “*” delineates bundle sheath cells; C, a crystal containing cell; m, mesophyll cells; and V, vascular bundles. Bars=50 µm.

Fig. 6.

Fig. 6.

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Transmission electron micrographs of bundle sheath cells in cross section of (A) Atriplex prostrata, (B) Atriplex rosea, (C) F2-114, and (F) F2-123. Arrows delineate mitochondria. Abbreviations: c, chloroplasts; m, mesophyll cells; n, nucleus; and v, vascular tissue. Bars=0.5 µm.

Discussion

Results from this study and previous research with C3 × C4 hybrids demonstrate that C4 photosynthesis is disrupted in the hybrids, as shown by a general increase in the Γ, a reduction in CE and NUE, and the expression of a C3-like δ13C (Björkman et al., 1971b, Björkman, 1976; Osmond et al., 1980; Brown and Bouton, 1993). In the hybrids generated here, we observed that the F1 and most F2 hybrids exhibited Γ values in the mid-range between C3 and C4 species. However, one F2 line (#114) exhibited Γ values that overlap with those of C4-like species such as Flaveria brownii that have a fully functional C4 cycle (Ku et al., 1991). A second F2 (#123) had Γ approaching the C3 range. In previous studies, F1 hybrids exhibit intermediate Γ values; these were interpreted to reflect a mix of C3 and C4 biochemistry in the F1 leaf (Pearcy and Björkman, 1971). The F1 hybrids are diploid with one set of chromosomes from each parent, and therefore have one C3 copy and one C4 copy of each gene, resulting in the mixed physiology (Osmond et al., 1980). In F2 lines, trait segregation is apparent, and hybrids probably lose one or more of the genes essential for C4 function (Osmond et al., 1980; Brown and Bouton, 1993). In F3 lines, further segregation leads to most hybrids exhibiting C3-like photosynthetic characteristics (Björkman et al., 1971a, b). Occasionally, however, F3 hybrids exhibit Γ values close to the C4 value (Björkman, 1976), which is consistent with results from F2-114. Previous hybrid studies indicate that all parts of the C4 biochemical cycle and Kranz anatomy must be present for efficient C4 function (Björkman, 1976; Brown and Bouton, 1993). As these traits independently segregate (Brown and Bouton, 1993), the probability of an F2 hybrid acquiring all of the C4 traits is low, and hence, it is unlikely that full C4 photosynthesis can occur. However, Γ values in the mid-range between C3 and C4 plants demonstrate the existence of a CCM in the F2 lines. This could result from either a modest C4 metabolic pump or a C2-type CCM where photorespiratory glycine is shuttled into the BS cells (Brown and Bouton, 1993). With new hybrids, we are now in a position to evaluate these possibilities and develop working hypotheses to guide future hybrid studies. In our discussion of the F2 hybrids, we mainly focus on F2-114, whose C4-like Γ value indicates greater CCM activity.

In F2-114, the low, C4-like Γ is indicative of significant C4 cycle activity and/or a highly effective C2 CCM. F2-114 had activities of PEPC, PPDK and NAD-ME that were 12–30% of the C4 values, indicating the potential for a modest C4 cycle that could contribute to a reduction in Γ by supplying some CO2 to the BS. All other F2 hybrids in this study had C3-like activities in at least one of these enzymes, indicating low potential for more than minor C4 cycle activity. As shown by Type II C3-C4 intermediates (those with significant C2 photosynthesis and C4 metabolism; Edwards and Ku, 1987), modest C4 cycle activity combined with a C2-type of glycine shuttle is sufficient to reduce Γ below 10 µmol mol–1. In the Type II C3-C4 intermediate F. ramosissima, for example, a Γ of 7 µmol mol–1 was associated with C4 enzyme activities between 12% and 18% of C4 values (Ku et al., 1983). A 3.5‰ increase in δ13C in F2-114 relative to A. prostrata is also evidence for modest C4 cycle activity, and is consistent with observed δ13C values of Type II intermediates such as F. ramossissima, and with modelled increases in δ13C assuming a 20–30% contribution by PEPC to the BS CO2 pool and moderate CO2 leakage (Monson et al., 1988; von Caemmerer, 1992; Sudderth et al., 2007). However, C4 metabolism could not contribute a large amount of carbon to the final pool of photosynthate in F2-114, because the δ13C values would shift more towards the C4 values than observed (von Caemmerer, 1992). We therefore hypothesize that the low Γ in F2-114 reflects a large contribution of a glycine shuttle to the CO2 pool of its BS cells.

Because C2 species with no C4-cycle activity (the type I C3-C4 intermediates; Edwards and Ku, 1987) exhibit Г values above 15 µmol mol–1 (Edwards and Ku, 1987; Ku et al., 1991; Vogan et al., 2007) it seems unlikely that a C2-type of glycine shuttle could reduce Γ to 4 µmol mol–1 by itself. However, according to von Caemmerer’s model of C3-C4 intermediate photosynthesis (von Caemmerer, 1989), a C4-like Γ could occur in a pure C2 species if there is an elevated (20%) fraction of leaf Rubisco in the BS cells, the conductance to CO2 leakage in the BS is low, and nearly all of the photorespired CO2 is released into the BS cells. Given the segregation of traits in the F2 lines (Osmond et al., 1980), it is probable that these criteria could be met in a few hybrids. All of the F2 hybrids here exhibited Rubisco activities that are a third to a half that of the A. prostrata parent, indicating some C4-type control over Rubisco expression is present in the hybrid lines. C4 species produce 25–35% as much Rubisco as C3 species (Sage et al., 1987), as is demonstrated by lower Rubisco activity in A. rosea relative to A. prostrata. Although we do not know where the Rubisco is distributed in our hybrids, previous work demonstrates F1 and F3 hybrids of A. rosea × A. prostrata express Rubisco in all chlorenchymatous cells (Hattersley et al., 1977). The high number of plastids in the BS of the F2 hybrids also indicates significant amounts of Rubisco are present in their BS chloroplasts. With respect to BS conductance, we hypothesize that some hybrids, perhaps including F2-114, have inherited traits contributing to low, C4-like conductance in the BS, such as thick BS cell walls (von Caemmerer and Furbank, 2003). It is also likely that there is a high fraction of photorespiratory CO2 released in the BS of most hybrids. In C4 plants, photorespiratory glycine decarboxylase (GDC) is localized to BS cells, whereas in C3 plants, GDC and the photorespiratory cycle is expressed in both BS and M tissues (Muhaidat et al. 2011; Sage et al. 2011; Schulze et al. 2013). In an F2 hybrid, there is a good chance that one or more of the GDC subunits exhibit a C4 pattern and are not expressed in the M cells, whereas their expression in the BS cells would occur if either the C4 or C3 pattern were inherited. Hence, it is probable that GDC activity is low in the M tissues of the F2 hybrids and high in the BS, so that much of the photorespiratory glycine would have to migrate into the BS for decarboxylation. This would explain why most of the F2 lines have C2-like Γ values. Certain lines, such as F2-123 with a more C3-like Γ may have a leakier BS or relatively less Rubisco in the BS, whereas other lines with low Γ such as F2-114 may have proportionally more BS Rubisco or less BS leakiness, plus some contribution from a C4 cycle. These possibilities point to a need for enzyme localization and leakage assessments in future hybrid studies.

In most hybrids, it is apparent that the BS Rubisco is adequately supplied with CO2. When carboxylation efficiency is plotted as a function of Rubisco activity, the hybrid values clustered around the theoretical relationship between Rubisco activity and carboxylation efficiency of a C3 Atriplex-like plant (Fig. 4). This demonstrates that in most hybrids, Rubisco is on average operating with the same efficiency as in a C3 leaf. The CE of F2-109 sits well above the CE versus Rubisco activity plot, which would occur if much of its Rubisco is in a CO2-enriched environment. The low PEPC and PPDK activity in F2-109 indicates the reduction of Γ below C3 values is predominately due to CO2 influx into the BS via C2 photosynthesis. Hybrid F2-114 exhibits the lowest CE relative to the theoretical CE versus Rubisco plot, demonstrating that at least some of its Rubisco is operating with reduced efficiency. Low CO2 levels in the BS would reduce CE, but this would not result in the low Γ value of F2-114 because Rubisco oxygenase activity would increase at low CO2 and raise Γ. Alternatively, Rubisco may be limited by low RuBP regeneration capacity, or a low activation state owing to a lack of Rubisco activase. Low RuBP regeneration might result if a C4 pattern of thylakoid protein expression corresponded to a C3 pattern of Calvin cycle expression, in which case one of the C3 compartments could be energy limited. The potential lack of activase expression is an intriguing possibility that could not be considered in the first era of Atriplex hybrid studies, as activase was unknown at the time. In C4 plants, activase expression is four times higher in the BS than M tissue (Majeran et al., 2005). In the hybrids, a C4-like pattern of activase expression could leave Rubisco in the M cells in a partially deactivated state. This would explain the low CE in F2-114, as the M Rubisco could be deactivated and unable to contribute to the CE values.

Anatomical patterns

Anatomically, all of the hybrid lines failed to express the well-developed Atriplicoid-type of Kranz anatomy, as has been noted before (Boynton et al., 1970). Atriplicoid Kranz anatomy consists of enlarged BS cells with a surrounding layer of M cells (Liu and Dengler, 1994; Dengler and Nelson, 1999). Chloroplasts in the BS cells of A. rosea are elongated and fill the inner two-thirds of the BS, and have many mitochondria distributed along the sides of the chloroplasts (Fig. 6). No chloroplasts or mitochondria occur along the outer BS wall of C4 Atriplex species. This arrangement allows for rapid re-assimilation of CO2 released by NAD-ME in the mitochondria, with the vacuole of the outer BS providing significant resistance to CO2 efflux (von Caemmerer and Furbank, 2003). By contrast, the C3 A. prostrata produces small BS chloroplasts that are similar to M cell chloroplasts; these occur along the outer wall of the BS cell against the intercellular air spaces (Boynton et al., 1970). In all the hybrids, the BS chloroplasts are similar in size and shape to those of the C3 parent, yet their positioning resembles a pattern that is often observed in C2-type species, where chloroplasts can occur in both a centripetal and centrifugal position (Muhaidat et al., 2011; Sage et al., 2013). Mitochondria still occur between chloroplasts, but to less of a degree than seen in A. rosea. Many of the mitochondria in the hybrids also appear between the inner BS wall and the chloroplasts, resembling a pattern apparent in C3-C4 intermediate plants using the C2-type of CCM (Monson and Rawsthorne, 2000; Sage et al., 2011; 2013). These observations further indicate that the BS cells of the F2 hybrids use the C2 mode of photosynthesis, although this will depend upon whether enough GDC is present in the BS mitochondria to create a strong sink for glycine produced in the M tissue.

Stomatal control

Previous work with C3 × C4 hybrids did not emphasize stomatal control, due in part to incomplete understanding at the time of stomatal regulation in C3 and C4 species. It is now known that non-stressed C3 species regulate C i/C a to generally be between 0.7–0.8 under humid conditions, whereas in C4 plants, C i/C a is maintained between 0.4–0.6 (Wong et al., 1979; Taylor et al. 2011; Vogan and Sage, 2011). The lower C i/C a in C4 species reflects tighter stomatal control and increased carboxylation efficiency of the C4 pathway relative to C3 photosynthesis; this explains the greater water-use efficiency of C4 plants (Huxman and Monson, 2003; Vogan and Sage, 2011). Under the relatively low vapour pressure difference between leaf and air in this study, we observed C i/C a to be 0.57 in A. rosea and 0.80 in A. prostrata. In the hybrids, the C i/C a values have largely reverted to the C3 value (C i/C a of 0.71–0.81), indicating that a full complement of C4 machinery is required for a C4 pattern of stomatal control.

Conclusions

With the new C3 × C4 hybrids in Atriplex, we have re-established an important system for investigating the genetic control and physiological function of C4 photosynthesis. In the F2 lines, we demonstrate a loss of efficient C4 function, further supporting the hypothesis that all of the components of the C4 pathway must be in place for C4 photosynthesis to occur. Although impairment of C4 photosynthesis in the F2 hybrids is no surprise, an intriguing observation is that improper assembly of the C3 pathway is also apparent in most F2 hybrids. This may reflect incomplete expression of the photorespiratory pathway in the M cells of the hybrids, or mismatched compartmentalization of C3 photosynthetic components. Ironically, with the incomplete assembly of the C3 and C4 conditions in the F2 lines, the default state seems to be C2 photosynthesis, for what may be a rather simple reason. Because both C3 and C4 plants express GDC in the BS (Schulze et al., 2013), the probability is high that GDC of the F2 hybrids is abundant in the BS cells, whereas GDC levels in the M cells may be low owing to inheritance of C4 expression patterns for at least one of the four GDC subunits. Hence, glycine would have to flow to the BS for decarboxylation, to the benefit of Rubisco in the BS chloroplasts.

We have now successfully generated the F3 hybrids and will be producing F4 lines and beyond to further segregate traits and possibly create near isogenic lines. With the analytical capabilities provided by modern tools and theory, we are better positioned to evaluate genetic, biochemical, and structural limitations affecting photosynthesis in the hybrids and hence provide critical information that can be utilized to engineer C4 photosynthesis into C3 crops as well as understand the evolution of C4 photosynthesis. These were the initial goals of Olle Björkman, John Boynton, Malcolm Nobs, and Bob Pearcy in the late 1960s when the initial hybrids were created. In the near future, these goals may be realized.

Supplementary data

Supplementary data are available at JXB online.

Supplementary Figure S1. Photographs of the Atriplex parents, F1 hybrid and F2 hybrids from this study.

Supplementary Figure S2. The response of net CO2 assimilation rate to intercellular CO2 partial pressure for the Atriplex parents and C3 x C4 hybrids in this study.

Supplementary Figure S3. Light micrographs of cross- sections through leaves of six Atriplex prostrata x Atriplex rosea F2 hybrids from this study.

Supplementary Data
supp_65_13_3637__index.html (982B, html)

Acknowledgements

We dedicate this work to the late Malcolm Nobs (1916–1992; Fig. 1A), and to Professor Olle Björkman (Fig. 1B), who created the first Atriplex hybrids within just a few years of the C4 pathway discovery. Their hybrid research has long been an inspiration to the C4 research community and it is the feeling of many that such studies should continue. We also thank Patrick Friesen for assistance on the gas exchange measurements, and Roxana Khoshravesh and Jeff Harsant for assistance with the imaging. We are grateful to Professor Björkman and Dr Chris Root for their help in acquiring seeds of A. prostrata and A. rosea. This work was supported by Discovery grant # RGPIN 154273 from the National Science and Engineering Research Council to RFS.

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