Introduction

The effects of cold acclimation on plant morphology, physiology and biochemistry have been widely studied in cold-tolerant wheat, rye and barley as well as spinach, Arabidopsis thaliana and Brassica napus (Krause 1988; Hüner et al. 1993, 1998; Adams et al. 2002; Stitt and Hurry 2002; Öquist and Hüner 2003; Savitch et al. 2005; Ensminger et al. 2006). Cold acclimation of winter rye, winter wheat (Hüner et al. 1981, 1985; Dahal et al. 2012a), spinach (Boese and Hüner 1990) as well as Arabidopsis thaliana (Strand et al. 1999; Gorsuch et al. 2010a, b) and B. napus (Savitch et al. 2005; Dahal et al. 2012a) exhibit altered plant phenotype and growth habit. Growth and development of cold-tolerant plants at low temperature generally results in a compact, dwarf growth habit with leaves that exhibit increased thickness relative to NA controls (Hüner et al. 1981, 1985; Dahal et al. 2012a). The increased leaf thickness associated with the cold-acclimated state can be accounted for by either increases in leaf mesophyll cell size (Hüner et al. 1981; Gorsuch et al. 2010a) and/or increases in the number of palisade layers (Boese and Hüner 1990; Dahal et al. 2012a).

Cold acclimation of cold-tolerant species generally results in enhanced light-saturated rates of photosynthesis, A sat, at measuring temperatures of either 5 or 20 °C (Dahal et al. 2012a). It has been suggested that the cold acclimation-induced increase in A sat of cold-tolerant species such as wheat, rye, A. thaliana and B. napus, is correlated with a stimulation of carbon metabolism as a result of the enhanced activities of key regulatory photosynthetic enzymes such as Rubisco, cFBPase and SPS in response to low growth temperatures (Hurry et al. 2000; Stitt and Hurry 2002; Savitch et al. 2005). Our recent study (Dahal et al. 2012a) has revealed that the increased A sat of CA winter wheat and winter rye especially when measured on a leaf area basis can, by and large, be accounted for by the increased specific leaf weight in response to growth at low temperature. There is a strong, positive correlation between the cold acclimation-induced increase in A sat and the development of freezing tolerance as well as an increased resistance to low temperature-induced photoinhibition in winter rye and winter wheat (Hüner et al. 1993; Öquist et al. 1993; Sarhan et al. 1997; Pocock et al. 2001), spinach (Krause 1988; Somersalo and Krause 1989; Guy 1990; Boese and Hüner 1992; Gray et al. 1997), A. thaliana (Savitch et al. 2001) and B. napus (Savitch et al. 2005). The increased resistance to photoinhibition and decreased temperature sensitivity of photosynthetic performance associated with cold acclimation are governed by changes at the cellular and biochemical levels rather than at the level of leaf anatomy and morphology and are characterized by an increased quantum requirement to close PSII reaction centers (Dahal et al. 2012a).

What governs this complex, integrated phenomenon in cold-tolerant plant species? It has been suggested that CBFs/DREBs (cold-binding transcription factors/dehydration responsive element binding factors) appear to control the phenotypic plasticity, biochemical changes and photosynthetic performance in cold-tolerant species (Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000, 2004; Savitch et al. 2005). For instance, even at a growth temperature of 20 °C, the over-expression of BnCBF17 (Savitch et al. 2005) and AtCBF3 (Gilmour et al. 2000, 2004) mimic multiple phenotypic, physiological and biochemical changes as observed upon cold acclimation of their wild type (WT) counterparts. Similar results have been reported for over-expression of AtCBF1 in potato (Pino et al. 2008), tobacco (Yang et al. 2010), tomato CBF1 in transgenic Arabidopsis (Zhang et al. 2004) and EguCBF1 in Eucalyptus (Navarro et al. 2011). Our recent study has revealed that over-expression of BnCBF17 in B. napus grown at 20 °C mimics the effects of cold acclimation of winter rye and winter wheat with respect to increased SLW, increased leaf thickness, improved WUE, enhanced light-saturated rates of CO2 assimilation and photosynthetic electron transport coupled with a decrease in the low temperature sensitivity of these processes (Dahal et al. 2012a). Thus, over-expression of B. napus with a single transcription factor, BnCBF17, appears to cause the conversion of non-acclimated WT to a cold acclimated state without exposure to low growth temperature. CBFs/DREBs are a family of transcriptional activators required to induce the expression of cold-regulated genes (COR) that enhance plant freezing tolerance (Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000; van Buskirk and Thomashow 2006; Chinnusamy et al. 2007; Badawi et al. 2008). In addition to enhanced freezing tolerance (Savitch et al. 2005), over-expression of BnCBF17 has much broader effects on plant phenotype, leaf anatomy, photosynthetic performance, water use efficiency and dry matter accumulation typically associated with cold acclimation of winter cereals and B. napus (Dahal et al. 2012a). Thus, CBFs/DREBs appear to be critical factors that govern plant phenotypic plasticity during cold acclimation.

An immediate increase in the rates of net CO2 assimilation has been observed following short-term shift of C3 plants from ambient to elevated CO2 (Cheng et al. 1998; Long et al. 2004; Ainsworth and Rogers 2007; Dahal et al. 2012b). This CO2 stimulation of photosynthesis in C3 plants is attributed to two factors. First, at current ambient CO2 concentrations, Rubisco is CO2-limited as the K m (CO2) for Rubisco is close to the current atmospheric CO2 concentration (Long et al. 2004; Tcherkez et al. 2006). Thus, an increased CO2 substrate availability for Rubisco immediately enhances carboxylation velocity. Second, elevated CO2 competitively suppresses photorespiration because CO2 is a competitive inhibitor of the oxygenation of RuBP by Rubisco (Long et al. 2004).

However, long-term growth and development of C3 plants at high CO2 concentration may lead to an end product inhibition of photosynthetic capacity due to accumulation of non-structural carbohydrates in the cytosol (Stitt and Quick 1989; Foyer 1990). This feedback inhibition of photosynthesis in response to growth at elevated CO2 may result from the chloroplastic Pi-limitation in the short-term and downregulation of the expression and activities of key regulatory photosynthetic enzymes in the long-term (Harley and Sharkey 1991; Drake et al. 1997; Moore et al. 1999).

Previous studies have shown that over-expression of BnCBF17 in B. napus mimics the effects of cold acclimation with respect to enhanced photosynthetic performance at ambient CO2 (Savitch et al. 2005; Dahal et al. 2012a). The objective of this study is to assess whether the enhanced photosynthetic performance observed for BnCBF17-OE as well as cold acclimated WT relative to non-acclimated WT B. napus at ambient CO2 (380 μmol C mol−1) is maintained upon long-term growth and development at elevated CO2 (700 μmol C mol−1). Since cold acclimated WT B. napus exhibits enhanced sink capacity and increased activities of major photosynthetic enzymes (Savitch et al. 2005; Dahal et al. 2012a), we hypothesize first that, cold acclimation-induced increase in photosynthetic performance of B. napus at ambient CO2 is maintained under long-term growth and development at elevated CO2. Second, we hypothesized that the BnCBF17-OE grown at 20 °C should respond similarly to growth and development under elevated CO2 as does cold acclimated WT B. napus.

Materials and methods

Plant growth

Brassica napus cv. Westar (wild type) and the B. napus BnCBF17-over-expressing transgenic line (BnCBF17-OE) were used in all experiments. Seeds of WT and BnCBF17-OE were obtained from Agriculture and Agri-Food Canada, Ottawa, Canada. The BnCBF17-over-expressing line was generated as described in detail by Savitch et al. (2005). Seeds of WT and BnCBF17-OE were grown in controlled environmental growth chambers (Model: GCW15 chamber, Environmental Growth Chambers, Chargin Falls, OH, USA) at either ambient CO2 (380 ± 10 μmol C mol−1) or elevated CO2 (700 ± 25 μmol C mol−1) with a PPFD of 250 ± 20 μmol photons m−2 s−1, 50–60 % relative humidity and a 16 h photoperiod. The WT plants were grown at day/night temperature regimes of either 20/16 °C non-acclimated (NA) or 5/5 °C cold acclimated (CA) whereas the BnCBF17-over-expressing line was grown only at 20/16 °C from the seed germination. The light in the growth chambers were supplied with metal halide lamps (Philips Lighting Company, Somerset, NJ, USA) and high pressure sodium lucalox lamp (GE Lighting Inc., Cleveland, OH, USA). Each CO2 growth chamber was equipped with a computer-controlled CO2 infra-red gas analyzer (Model: WMA-4 CO2 Analyzer, PP Systems International Inc., Amesbury, MA, USA) which monitored CO2 concentrations continuously. In addition, the temperature, relative humidity, irradiance level and photoperiod in each chamber were computer-controlled and monitored continuously. The seedlings were grown in organic soil (Promix, Premier Horticulture, Quakertown, PA, USA) in 500 mL-sized plastic pots with one plant each and watered with all purpose fertilizer (Plant Prod 20–20–20, Sure-Gro IP Inc., Brantford, ON, Canada). The non-acclimated WT plants were grown for 3 weeks, the BnCBF17-OE for 4 weeks and the cold acclimated WT for 12 weeks from the seed germination. At these ages, the third leaves were fully expanded in non-acclimated WT, BnCBF17-OE and cold acclimated WT plants. Thus, all measurements of CO2 gas exchange rates and Chl-a room temperature fluorescence and biochemical analyses were carried out on fully expanded third leaves of the 3-week-old non-acclimated WT, 4-week-old BnCBF17-OE and 12-week-old cold acclimated WT B. napus.

The shoot and root fresh biomass were weighed, and the tissues were dried at 80 °C to constant weight for the determination of dry weight. Leaf blade area was measured by using a LI-COR portable area meter (LI–3000A, LI-COR Biosciences, Lincoln, NE, USA). Specific leaf weight (SLW) was calculated as leaf dry weight in g m−2 leaf blade area. The photosynthetic pigments, Chl-a and Chl-b, were quantified according to Arnon (1949).

In order to confirm that the pot size did not limit rooting volume and sink growth upon growth of plants at elevated CO2, we carried out a pot size experiment for non-acclimated WT and BnCBF17-over-expressing B. napus. Plants were grown in various pot sizes of 0.3, 0.5, 1 and 1.5 L at elevated CO2 (700 ± 30 μmol C mol−1) and at growth conditions described above. Root and shoot biomass were harvested from 3-week-old non-acclimated WT and 4-week-old BnCBF17-OE, and the tissues were dried at 80 °C in order to get dry weight.

CO2 gas exchange

Carbon dioxide gas exchange rates were measured on attached fully expanded third leaves at either 380 or 700 μmol C mol−1. In addition, the CO2 gas exchange rates for non-acclimated WT, cold-acclimated WT and BnCBF17-OE were made at the same measuring temperature of 20 °C irrespective of acclimation state. Our recent study has revealed that both cold acclimated WT and BnCBF17-OE are minimally sensitive to measuring temperatures between 5 to 25 °C for the rates of CO2 gas exchange and electron transport (Dahal et al. 2012a). Carbon dioxide gas exchange rates were measured on attached fully expanded third leaves by using the LI-COR portable infrared CO2 gas analyzer (LI-6400 XRT portable photosynthesis system, LI-COR Biosciences, Lincoln, NE, USA). Light response curves were measured using 12 irradiance values ranging from 0 to 1,300 μmol photons m−2 s−1 PPFD from high to low light intensity with 8 min of waiting time between each measurement. The apparent maximum quantum efficiency (Q) and the maximal photosynthetic capacity (A sat) were determined as the maximum initial slope and the maximum light-saturated rates, respectively from light response curves. Carbon dioxide response curves were measured by supplying 11 different CO2 values over the range of 50 to 1,200 μmol C mol−1 at a saturating irradiance of 1,300 μmol photons m−2 s−1 PPFD at 20 °C. Rubisco-limited carboxylation efficiency (CE) was calculated as the maximum initial slope and the CO2-saturated photosynthesis as the plateau of the curves. The maximum Rubisco carboxylation capacity (V cmax) and the maximum rates of electron transport (J max) were calculated using the FCB photosynthesis model (Farquhar et al. 1980). Respiration rates (R dark) were measured in the dark at either 380 or 700 μmol C mol−1 at the same measuring temperature of 20 °C irrespective of acclimation state. Measurements were made inside the CO2 enriched chambers, to reduce diffusion errors through the leaf blade and chamber seals as suggested previously (Jahnke 2001; Jahnke and Krewitt 2002). In addition, stomatal conductance and leaf transpiration rates were measured simultaneously with CO2 gas exchange measurements at growth CO2 and 20 °C regardless of acclimation state. Instantaneous leaf level water use efficiency was calculated as μmol CO2 fixed per mol of stomatal conductance (A/g s). Stomatal density was estimated on both adaxial and abaxial leaf surface using a stage micrometer (1 mm2) on a microscope (Leica ATC™ 2000, Buffalo, NY, USA). Stomata were counted at a magnification of 100×.

Room temperature Chl-a fluorescence

In vivo Chl-a fluorescence was measured to assess the effects of elevated CO2 on maximum photochemical efficiency (F v/F m), maximum potential rates of photosynthetic electron transport (ETR), excitation pressure (EP), which estimates the relative reduction state of PSII reaction centers and non-photochemical quenching (NPQ), an estimate of the capacity to dissipate energy as heat. Chl-a fluorescence was measured simultaneously with CO2 gas exchange on fully expanded third leaves using LI-COR portable photosynthesis system (LI-6400 XRT, LI-COR Biosciences). Light response curves of electron transport rates, excitation pressure and non-photochemical quenching of excess energy were measured at irradiance values ranging from 0 to 1,500 μmol PPFD photons, at 700 μmol C mol−1 and at a measuring temperature of 20 °C irrespective of growth CO2 and irrespective of growth temperature. All measurements of Chl-a fluorescence were carried out using the standard fluorescence leaf chamber (2 cm2). The leaves were dark-adapted for 20 min prior to fluorescence measurements. Minimum fluorescence (F 0) was measured by illuminating dark adapted leaves with a low irradiance measuring beam (PPFD < 1 μmol photons m−2 s−1) from a light emitting diode. Maximal fluorescence (F m) was determined by applying a flash of saturating light (PPFD > 5,000 μmol photons m−2 s−1) for pulse duration of 0.8 s. Afterwards, an actinic light (PPFD 1,300 μmol photons m−2 s−1) was applied. Superimposed on the actinic beam was a saturating light flash (PPFD > 5,000 μmol photons m−2 s−1; 0.8 s) applied repetitively at 20 s intervals to determine maximal fluorescence in the light-adapted state (\( F_{\text{m}}^{\prime } \)). Light adapted steady state fluorescence (F s) was determined by measuring the level of fluorescence during steady-state photosynthesis. Finally, minimal fluorescence (\( F_{ 0}^{\prime } \)) in the light-adapted leaf was measured by turning off the actinic light.

Determination of total leaf protein and immunodetection

The fully expanded third leaves were harvested, immediately frozen in liquid N2 and stored at −80 °C. The leaf samples for the protein determination were prepared as described in detail by Dahal et al. (2012a). Total leaf protein content was quantified using the RC-DC protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. While quantifying the total leaf protein content, the addition of 1 μg of bovine serum albumin (Invitrogen, Carlsbad, CA, USA) in the extraction buffer was used as an internal standard.

The extracted proteins were electrophoretically separated using NuPAGE Novex 10 % (W/V) Bis–Tris precast, polyacrylamide gels (Invitrogen) with MES SDS running buffer (Invitrogen) in an XCell4 SureLock Midi Cell (Invitrogen) according to manufacturer’s instructions. Samples were loaded for SDS-PAGE on either equal Chl (0.5 μg Chl per lane) or on equal protein (5 μg protein per lane) basis. Gels were electrophoresed at 80 V for about 3 h. For immunodetection, separated polypeptides were electroblotted onto nitrocellulose membranes (0.2 μM pore size, Bio-Rad) in transfer buffer for 1 h at 100 V. The membranes were blocked with 5 % (W/V) fat free, dried milk powder overnight at 4 °C and then probed with primary antibodies raised against the target proteins; rbcL (Hüner, University of Western Ontario, Canada), cFBPase and Lhcb1 (Agrisera AB, Vännäs, Sweden) at a dilution of 1:2,000–5,000. The blots were rinsed briefly and washed in TBS wash buffer at room temperature with agitation. The blots were probed with secondary antibody (anti-rabbit IgG peroxidase antibody, Sigma-Aldrich, Steinheim, Germany) at a 1:10,000–20,000 dilutions for 1 h at room temperature with agitation. The blots were washed as described above and the target proteins were visualized with enhanced chemiluminescence immunodetection (ECL Detection Kit; GE Healthcare, Buckinghamshire, UK) on X-ray film (Fujifilm, Fuji Corporation, Tokyo, Japan). Immunoblots were quantified by using a computer software program (SCION IMAGE, Scion Corporation, Frederick, MD, USA). For quantification, exposure times were varied to ensure that signals from the immunoblots were not saturating.

Statistical analysis

The non-acclimated WT, BnCBF17-OE as well as cold acclimated WT each was grown in 30 replicate pots in a completely randomized design. Out of the 30 replicate pots, three pots for each line at each growth condition were randomly selected for all measurements. Thus, all data are the means of three replicate pots. Results were subjected to analysis of variance (ANOVA). Means were compared at the 5 % level of significance (P ≤ 0.05) by using the statistical package SPSS version 17 (IBM, Armonk, NY, USA).

Results

Growth characteristics

We carried out a pot size experiment for non-acclimated WT and BnCBF17-OE to ensure that pot size was large enough for proper root and sink growth at elevated CO2 (Fig. S1). Total dry matter accumulation was significantly lower in both WT and BnCBF17-OE when grown in 0.3 L-sized pot (Fig. S1). However, no differences were observed in total dry matter accumulation for WT as well as BnCBF17-OE when grown in various pot sizes of 0.5, 1 and 1.5 L in (Fig. S1). Thus, to prevent rooting volume constraints and sink limitations, the plants were grown in 0.5 L-sized pots irrespective of growth CO2 and growth temperatures in all experiments.

The BnCBF17-OE exhibited a compact, dwarf growth habit and altered leaf morphology as compared to the typical elongated growth habit for non-acclimated WT at ambient CO2 (Fig. 1). The growth habit and plant morphology observed for BnCBF17-OE were comparable to those observed for cold acclimated WT at ambient CO2 (Fig. 1). Although the BnCBF17-OE as well as cold acclimated WT B. napus were smaller plants, they exhibited comparable total dry matter accumulation as that of non-acclimated WT at fully expanded third leaf stage at ambient CO2 (Table 1). Growth at elevated CO2 significantly increased dry matter accumulation by 20, 40 and 35 % for non-acclimated WT, BnCBF17-OE and cold acclimated WT, respectively relative to at ambient CO2 (Table 1). Thus, BnCBF17-over-expression as well as cold acclimation appeared to substantially increase the dry matter accumulation relative to that of non-acclimated WT at elevated CO2. The specific leaf weight (SLW) increased by about 85 % in BnCBF17-OE versus non-acclimated WT and by about 130 % in cold acclimated WT versus non-acclimated WT at ambient CO2 (Table 1). Elevated CO2 significantly stimulated SLW by 33 % in non-acclimated WT but had minimal effects in the BnCBF17-OE as well as in cold acclimated WT (Table 1). Consequently, at elevated CO2, the BnCBF17-OE and cold acclimated WT exhibited about 70 and 90 % higher SLW, respectively compared to non-acclimated WT (Table 1).

Fig. 1
figure 1

Effects of cold acclimation and elevated CO2 on plant morphology and growth habit of WT B. napus and BnCBF17-OE. The plants were grown at either ambient (380 μmol C mol−1) or elevated (700 μmol C mol−1) CO2 and at either 20/16 °C (NA and BnCBF17-OE) or 5/5 °C (CA Brassica WT)

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Table 1 Effects of cold acclimation and elevated CO2 on morphological and photosynthetic characteristics of WT and BnCBF17-over-expressing B. napus grown at either ambient (380 μmol C mol−1) or elevated (700 μmol C mol−1) CO2 and at either 20/16 °C (non-acclimated) or 5/5 °C (cold acclimated)
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Over-expression of BnCBF17 substantially increased Chl per unit leaf area by about 125 % as compared to non-acclimated WT at ambient CO2 (Table 1). The increase in Chl in the BnCBF17-OE was at par with the cold acclimation-induced increase in Chl per unit leaf area (105 %) for WT at ambient CO2 (Table 1). Although, elevated CO2 significantly increased Chl per unit leaf area by 16 % for non-acclimated WT, it significantly decreased the Chl per unit leaf area for BnCBF17-OE as well as cold acclimated WT by 10–15 % (Table 1). The Chl-a/b ratios decreased by about 20 % in non-acclimated WT but changed minimally in BnCBF17-OE and cold acclimated WT in response to growth at elevated CO2 (Table 1).

The BnCBF17-OE as well as cold acclimated WT B. napus exhibited about 4.8-fold higher leaf protein content per unit leaf area relative to non-acclimated WT at ambient CO2 (Table 1). Although elevated CO2 had minimal effects on leaf protein content of the BnCBF17-OE as well as cold acclimated WT, elevated CO2 appeared to significantly decrease leaf protein content by about 25 % in non-acclimated WT relative to at ambient CO2 (Table 1). The leaf protein to Chl ratio was about twofold higher in the BnCBF17-OE and cold acclimated WT B. napus relative to non-acclimated WT at ambient CO2 (Table 1). Elevated CO2 reduced this ratio by about 35 % in non-acclimated WT but had minimal effects on this ratio in the BnCBF17-OE and cold acclimated WT B. napus relative to at ambient CO2 (Table 1).

Light response curves for CO2 assimilation

Consistent with our previous report (Dahal et al. 2012a), over-expression of BnCBF17 (Table 1; Fig. 2b) mimicked cold acclimated WT Brassica (Fig. 2c) with respect to the 25 and 40 % increases in the in the apparent maximum quantum efficiency for CO2 assimilation (Q) and the light-saturated rates of gross CO2 assimilation (gross A sat), respectively as compared to WT B. napus when grown and measured at ambient CO2 (Table 1; Fig. 2a).

Table 2 Effects of cold acclimation and elevated CO2 on stomatal characteristics of WT and BnCBF17-over-expressing B. napus grown at either ambient (380 μmol C mol−1) or elevated (700 μmol C mol−1) CO2 and at either 20/16 °C (non-acclimated) or 5/5 °C (cold acclimated)
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Fig. 2
figure 2

Light response curves of gross CO2 assimilation for WT (NA) (a, d), the BnCBF17-OE (b, e) and WT (CA) (c, f) grown at either ambient (380 μmol C mol−1, square) or elevated (700 μmol C mol−1, filled square) CO2. All measurements were carried out on fully developed third leaves at 20 °C at either 380 (a–c) or 700 (d–f) μmol C mol−1. Data represent the mean of three plants from three different pots. Bars represent SD

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The sensitivity to feedback-limited rates of photosynthesis during growth at elevated CO2 condition can be assessed by comparing CO2 assimilation rates in plants grown under elevated (700 μmol C mol−1) versus ambient CO2 conditions (380 μmol C mol−1) but measured at the same CO2 concentration of either 380 or 700 μmol C mol−1. Such a comparison revealed a significant inhibition of light- saturated rates of gross CO2 assimilation in elevated versus ambient CO2-grown non-acclimated WT (Fig. 2a, d) but not in either the BnCBF17-OE (Fig. 2b, e) or cold acclimated WT (Fig. 2c, f). Compared to growth and development at ambient CO2, the growth and development of non-acclimated WT B. napus at elevated CO2 significantly inhibited the light-saturated rates of gross CO2 assimilation by 27 % when measured at an equal CO2 concentration of 700 μmol C mol−1 (Fig. 2d). A similar inhibition of gross CO2 assimilation (30 %) was observed between elevated versus ambient CO2-grown non-acclimated WT when measured at an equal CO2 concentration of 380 μmol C mol−1 (Fig. 2a). In contrast, light saturated rates of gross CO2 assimilation for the BnCBF17-OE (Fig. 2b, e) as well as cold acclimated WT B napus (Fig. 2c, f) exhibited minimal sensitivity to the elevated CO2 concentration during long-term growth and development.

CO2-response curves for CO2 assimilation

Figure 3 illustrates the effects of cold acclimation and elevated CO2 on the light-saturated CO2 response curves of net CO2 assimilation. BnCBF17-over-expression (Table 1; Fig. 3b) mimicked cold acclimation of WT Brassica (Table 1; Fig. 3c) with respect to a 26 to 37 % increase in the carboxylation efficiency (CE) and a 35 % increase in the light and CO2-saturated photosynthetic rates as well as V cmax, respectively compared to non-acclimated WT grown at ambient CO2 (Table 1; Fig. 3a). These results are consistent with our previous reports (Dahal et al. 2012a). No significant differences in the carboxylation efficiency were observed between elevated versus ambient CO2-grown plants for all Brassica lines tested (Table 1; Fig. 3). Elevated CO2, however, significantly reduced the light and CO2-saturated rates of net CO2 assimilation and V cmax by 32 % for non-acclimated WT (Table 1; Fig. 3a) but had minimal effects on these rates for BnCBF17-OE as well as cold acclimated WT (Table 1; Fig. 3b, c). The decrease in light and CO2-saturated rates of net CO2 assimilation observed for elevated versus ambient CO2-grown non-acclimated WT was consistent with the decreased rates of light and CO2-saturated electron transport in response to growth at elevated CO2 (Table 1, J max; Fig. 4a, ETR).

Fig. 3
figure 3

CO2 response curves of light-saturated net CO2 assimilation for WT (NA) (a), the BnCBF17-OE (b) and WT (CA) (c) grown at either ambient (380 μmol C mol−1, square) or elevated (700 μmol C mol−1, filled square) CO2. All measurements were carried out on fully developed third leaves at measuring temperature of 20 °C at a saturating irradiance of 1,300 μmol m−2 s−1 PPFD. Data represent the mean of three plants from three different pots. Bars represent SD

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Fig. 4
figure 4

Light response curves of electron transport rates (ETR; a–c), excitation pressures (1-qP; d–f) and non photochemical quenching of excess energy (NPQ; g–i) for non-acclimated WT (a, d, g), BnCBF17-OE (b, e, h) and cold acclimated WT (c, f, i) B. napus grown at either ambient (380 μmol C mol−1, square) or elevated (700 μmol C mol−1, filled square) CO2 and at either 20/16 °C (NA) or 5/5 °C (CA). All measurements were carried out on fully developed third leaves at 700 μmol C mol−1 and at measuring temperature of 20 °C. Data represent the mean of three plants from three different pots. Bars represent SD

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Light response curves for ETR, EP and NPQ

Light response curves for electron transport rates (ETR), excitation pressure (EP), measured as 1-qP and non-photochemical quenching (NPQ) (Fig. 4) were generated to estimate the apparent quantum requirement for ETR, the apparent quantum requirement to close PSII reaction centers by EP and the apparent quantum requirement to induce energy dissipation by NPQ, respectively (Dahal et al. 2012a). Apparent quantum requirement is the inverse of apparent quantum efficiency and is estimated as the inverse of the initial slopes calculated from the linear portion of the light response curve for either ETR (Fig. 4a–c), 1-qP (Fig. 4d–f) or NPQ (Fig. 4g–i).

In vivo Chl-a fluorescence indicated that BnCBF17-OE (Fig. 4b, open symbols) mimicked cold acclimated Brassica (Fig. 4c, open symbols) and exhibited a small (15 %) but significant increase in the light-saturated ETR but no change in the apparent quantum requirement for ETR (3.6–3.9 photons/e) compared to non-acclimated WT at ambient CO2 (Fig. 4a). The data for light saturated rates of ETR measured by room temperature Chl-a fluorescence were consistent with the J max based on CO2 gas exchange (Table 1). However, the apparent quantum requirements for closure of PSII reaction centers (1,414 photons to close 50 % of PSII reaction centers) and the induction of NPQ (883 photons/unit NPQ) under ambient CO2 conditions were 30 to 50 % greater for the BnCBF17-OE (Fig. 4e, h, open symbols) and cold acclimated WT Brassica (Fig. 4f, i, open symbols) compared with non-acclimated WT (Fig. 4d, g, open symbols).

Elevated CO2 increased the apparent quantum requirement for ETR by 18 % and significantly inhibited the light-saturated ETR by 22 % in non-acclimated WT relative to at ambient CO2 (Fig. 4a). The data for ETR were consistent with the J max based on CO2 gas exchange (Table 1). However, the apparent quantum requirements for PSII closure decreased significantly by about 25 % in elevated (826 photons to close 50 % PSII reaction centers) versus ambient CO2-grown, non-acclimated WT (1,093 photons to close 50 % PSII reaction centers) (Fig. 4d). Concomitantly, the quantum requirement to induce one unit of NPQ decreased by 30 % from 573 photons/unit NPQ to 414 photons/unit NPQ in WT grown at elevated CO2 compared at ambient CO2 (Fig. 4g). The decreased light and CO2-saturated ETR for non-acclimated WT grown at elevated CO2 was consistent with the decreased light and CO2-saturated rates of CO2 assimilation at elevated CO2 (Fig. 3a). In contrast, the light response curves of ETR, EP and NPQ for the BnCBF17-OE as well as cold acclimated WT were insensitive to growth at elevated CO2 (Fig. 4). This was consistent with the minimal changes in the CO2 response curves for the BnCBF17-over-expressing line as well as cold acclimated WT grown under elevated versus ambient CO2 conditions (Fig. 3b, c).

CO2 response curves for ETR, EP and NPQ

Carbon dioxide response curves for ETR, EP and NPQ (Fig. 5) were carried out to estimate the efficiency for ETR as a function of C i, the efficiency with which PSII reaction centers were opened by increased C i and the efficiency with which NPQ was suppressed by increased C i, respectively. The efficiency for ETR, EP and NPQ was estimated as the initial linear slopes of the C i response curve for either ETR (Fig. 5a–c), 1-qP (Fig. 5d–f) or NPQ (Fig. 5g–i).

Fig. 5
figure 5

CO2 response curves of light-saturated electron transport rates (ETR; a–c), excitation pressures (1-qP; d–f) and non photochemical quenching of excess energy (NPQ; g–i) for non-acclimated WT (a, d, g), BnCBF17-OE (b, e, h) and cold acclimated WT (c, f, i) B. napus grown at either ambient (380 μmol C mol−1, square) or elevated (700 μmol C mol−1, filled sqare) CO2 and at either 20/16 °C (NA) or 5/5 °C (CA). All measurements were carried out on fully developed third leaves at 20 °C and at a saturating irradiance of 1,300 μmol photons m−2 s−1. Data represent the mean of three plants from three different pots. Bars represent SD

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The data for the effects of C i on ETR for plants grown under ambient CO2 (Fig. 5a–c, open symbols) indicate that, under light-saturated conditions, the CO2 saturated rates of ETR were 1.2-fold higher for BnCBF17-OE as well as cold acclimated WT than for NA WT Brassica. Furthermore, the efficiency of ETR in either BnCBF17-OE or cold acclimated WT (0.81 μmol e− m−2 s−1/μmol CO2 mol−1) was 1.4-fold higher than that of NA Brassica (0.57 μmol e− m−2 s−1/μmol CO2). Growth at elevated CO2 decreased the CO2 saturated rates of ETR by about 33 % and the efficiency of ETR (μmol e− m−2 s−1/μmol CO2 mol−1) by 54 % in NA Brassica (Fig. 5a). In contrast, the CO2 saturated rates of ETR as well as the efficiency of ETR were insensitive to growth of either BnCBF17-OE (Fig. 5b) or CA Brassica (Fig. 5c) at elevated CO2. These trends were consistent with the data for the differential effects of elevated CO2 on J max in BnCBF17-OE and cold acclimated WT relative to non-acclimated WT Brassica (Table 1).

Consistent with the fact that CO2 dominates as an ultimate electron acceptor for photosynthetic carbon assimilation, increasing C i decreased excitation pressure in all plants tested (Fig. 5d–f) whether grown under ambient (open symbols) or elevated CO2 (closed symbols). However, under light saturated conditions, excitation pressure at CO2 saturation was 1.4- to 1.6-fold lower in BnCBF17-OE (Fig. 5e) and cold acclimated WT (Fig. 5f) than NA controls (Fig. 5d) grown at ambient CO2 (open symbols). In addition, the efficiency with which PSII reaction centers were opened by increased CO2 was 1.7- to 1.9-fold greater in BnCBF17-OE and cold acclimated WT than in non-acclimated WT (Fig. 5d–f).

Growth at elevated CO2 not only increased the CO2-saturated excitation pressure by about 1.4-fold in non-acclimated WT from a 1-qP of 0.48 upon growth at ambient CO2 (open symbols) to 0.68 upon growth at elevated CO2 (Fig. 5d, closed symbols) but also inhibited the efficiency with which PSII reaction centers were opened as a function of increased C i by about 1.6-fold relative to non-acclimated WT Brassica grown at ambient CO2 (Fig. 5d). In contrast, neither the CO2 saturated excitation pressure nor the efficiency with PSII reaction centers were opened by increased C i were significantly affected by growth at elevated CO2 in BnCBF17-OE (Fig. 5e) as well as cold acclimated WT Brassica (Fig. 5f).

To complement the CO2 response curves for excitation pressure, we also assessed the effects of changes in C i on NPQ (Fig. 5g–i). Consistent with the fact that CO2 is a substrate for the Rubisco-catalyzed carboxylation reaction of the Calvin cycle, increasing C i resulted in the suppression of NPQ in non-acclimated WT (Fig. 5g), BnCBF17-OE (Fig. 5h) and cold-acclimated WT Brassica (Fig. 5i) irrespective of whether plants were grown at ambient (open symbols) or at elevated CO2 (closed symbols). However, even at low C i, NPQ was about 50 % lower in BnCBF17-OE and cold acclimated WT Brassica compared to non-acclimated WT. Although the response of NPQ to increasing C i was similar whether BnCBF17-OE and cold acclimated WT were grown at ambient or elevated CO2 (Fig. 5h, i, respectively), increasing C i induced 26 % higher levels of NPQ in non-acclimated WT grown at elevated CO2 relative to non-acclimated WT grown at ambient CO2 (Fig. 5g).

Photosynthetic polypeptide abundance

The data in Table 1 indicate that either over-expression of BnCBF17 or cold acclimation of WT B. napus resulted in a doubling of the protein/Chl ratio. When SDS-PAGE gels were loaded for immunoblot analyses on equal Chl basis (Fig. 6a), BnCBF17 as well as cold acclimated WT B. napus exhibited about twofold increase in the relative amount of major stromal photosynthetic enzyme (Rubisco, rbcL) and cytosolic FBPase (cFBPase) compared to non-acclimated WT Brassica grown at ambient CO2 (Fig. 6a). However, the relative amount of Lhcb1, the major protein of PSII light harvesting complex, changed minimally in BnCBF17-OE as well as cold acclimated WT compared to non-acclimated WT at ambient CO2 (Fig. 6a). However, when gels were loaded on equal protein basis, the relative levels of rbcL, cFBPase and Lhcb1 changed minimally in BnCBF17 as well as cold acclimated WT relative to non-acclimated WT at ambient CO2 (Fig. 6b). Thus, the data in Fig. 6a and b are consistent with the increased protein/Chl ratio presented in Table 1.

Fig. 6
figure 6

Immunoblot analysis of SDS-PAGE probed with antibodies raised against: rbcL, cFBPase and Lhcb1, isolated from cold acclimated (CA) and non-acclimated (NA) wild type (WT) and BnCBF17-OE grown at either ambient (380 μmol C mol−1) or elevated (700 μmol C mol−1) CO2 and at either 20/16 °C (NA) or 5/5 °C (CA). Lanes of SDS-PAGE were loaded on either equal Chl (a, 0.5 μg/lane) or on equal protein basis (5 μg protein/lane). The bovine serum albumin (1 μg/lane) was used as an internal control. Numbers on the right indicate molecular weight (MW, kDa) of markers

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Regardless of the basis upon which the SDS-PAGE gels were loaded, growth under elevated CO2 decreased the relative levels of rbcL by about 35 % and cFBPase by 40–70 % yet increased the relative levels of Lhcb1 by about 20 % in non-acclimated WT relative to at ambient CO2 (Fig. 6). In contrast, growth at elevated CO2 appeared to have minimal effects on the abundance of rbcL, cFBPase and Lhcb1 in the BnCBF17-OE as well as cold acclimated WT relative to at ambient CO2 irrespective of loading basis (Fig. 6).

Stomatal characteristics

The results summarized in Table 2 are consistent with previous reports which indicate that overexpression of BnCBF17 and cold acclimation of B. napus resulted in a 30 and 80 % increase in instantaneous leaf level water use efficiency (WUE) compared to non-acclimated WT grown at ambient CO2 (Dahal et al. 2012a). This differential increase in instantaneous leaf level WUE was accounted for, in part, by differential changes in stomatal frequency (Table 2).

Elevated CO2 suppressed leaf stomatal conductance by 20–35 % in non-acclimated, cold acclimated and the BnCBF17-over-expressing line of B. napus and, as a consequence, leaf transpiration rates decreased by 15–25 % and instantaneous leaf level WUE increased by 80–115 % (Table 2). It appears that the increased WUE induced by either cold acclimation or elevated CO2 is primarily associated with an increase in light-saturated rates of CO2 assimilation and a decrease in stomatal conductance in response to growth at either low temperature or under elevated CO2.

The data in Table 2 indicate that the cold acclimation or elevated CO2-induced suppression of stomatal conductance can be accounted for, in part, by a decrease in stomatal density on both the abaxial and adaxial leaf surfaces in response to growth at either low temperature or under elevated CO2. For instance, cold acclimated WT exhibited about a 20 % decrease in adaxial as well as abaxial stomatal densities as compared to non-acclimated WT. Similarly, elevated CO2 decreased adaxial as well as abaxial stomatal densities by 30–75 % relative to those of at ambient CO2 for all Brassica plants tested (Table 2).

Dark respiratory rates

The BnCBF17-over-expressing line of B. napus exhibited a 35 % decrease in the dark respiratory rates (R dark) as compared to non-acclimated WT at ambient CO2 when expressed on per unit dry weight basis (Table 1). Dark respiratory rate observed for the BnCBF17-OE was comparable to that of cold acclimated WT at ambient CO2 (Table 1). Although elevated CO2 increased R dark by about 20 % in cold acclimated WT, elevated CO2 had minimal effects on R dark of non-acclimated WT as well as BnCBF17-OE relative to at ambient CO2 (Table 1).

Discussion

Compared to non-acclimated WT, BnCBF17-OE grown at 20 °C mimicked cold acclimated WT B. napus with respect to compact dwarf phenotype (Fig. 1), increased SLW (Table 1), increased photosynthetic capacity (Fig. 2), increased ETR as a function of irradiance (Fig. 4) and C i (Fig. 5), improved instantaneous leaf level WUE (Table 1) and enhanced levels of key photosynthetic enzymes and components of photosynthetic electron transport (Fig. 6). These results are consistent with an increased quantum requirement to close PSII reaction centers and to induce energy dissipation by NPQ (Fig. 4) coupled with a lower C i requirement to open PSII reaction centers and a lower propensity to dissipate absorbed energy through NPQ under CO2 saturated conditions (Fig. 5). Consequently, BnCBF17-OE and cold acclimated WT B. napus exhibited a lower excitation pressure for a given irradiance and a given CO2 concentration, and thus, a greater capacity to keep Q A oxidized compared with non-acclimated WT B. napus. This indicates that compared to non-acclimated WT, BnCBF17-OE as well as cold acclimated WT B. napus exhibit an enhanced capacity to utilize absorbed light energy and convert it to biomass as reflected in an increase in SLW with a concomitant decreased reliance on NPQ to dissipate absorbed energy for photoprotection. Thus, we show that over-expression of BnCBF17 as well as cold acclimation enhances photosynthetic energy conversion efficiency into biomass in B. napus coupled with enhanced instantaneous leaf level WUE during long-term growth at either ambient or elevated CO2 conditions.We suggest that the enhanced photosynthetic capacity and energy conversion efficiency of BnCBF17-OE as well as cold acclimated WT relative to non-acclimated WT B. napus can be explained, in part, by the enhanced abundance, on a Chl basis, of major regulatory photosynthetic enzymes such as stromal-localized Rubisco (rbcL) and cytosolic FBPase (cFBPase) important in regulating sucrose biosynthesis as well as components of the thylakoid membrane represented by Lhcb1 (Fig. 6a). We suggest that the increased protein/Chl ratio (Table 1) reflects an increase in components of the thylakoid photosynthetic electron transport chain, Calvin cycle enzymes involved in the assimilation of CO2 as well as cytosolic sucrose biosynthetic enzymes on a leaf area basis. These results for the BnCBF17-OE and cold acclimated B. napus are comparable to the enhanced photosynthetic performance reported recently for cold acclimated winter wheat and winter rye (Dahal et al. 2012a). In the cold acclimated winter cereals, the enhanced photosynthetic performance was shown to be due to increased levels of photosynthetic proteins (e.g., rbcL, psbA, psaA, cFBPase, Lhcb1) per leaf area combined with a decrease in the low temperature sensitivity of ETR and CO2 assimilation (Dahal et al. 2012a). BnCBF17-OE also exhibited a similar low temperature insensitivity for ETR and CO2 assimilation as did the cold acclimated winter cereals (Dahal et al. 2012a). This is consistent with the report of Savitch et al. (2005) who reported that the BnCBF17-OE exhibited significant enhancement for the gene expression as well as enzyme activities of Rubisco, SPS and cFBPase. Therefore, we suggest that the enhanced photosynthetic performance and energy conversion efficiency of BnCBF17-OE and cold acclimated WT B. napus is due to increased amount of photosynthetic apparatus per unit leaf area combined with a decrease in the low temperature sensitivity of ETR and CO2 assimilation. Despite the 40–50 % lower leaf area for light interception for both BnCBF17-OE and the cold acclimated WT relative to non-acclimated WT, the former exhibited a 14–18 % increase in total dry weight (Table 1). Thus, the enhanced SLW observed for BnCBF17-OE and the cold acclimated WT compensates for the decrease in total leaf area for light absorption relative to WT non-acclimated controls. Recently, Murchie et al. (2009) discussed the challenges of increasing energy conversion efficiency in order to enhance crop biomass production and harvestable yield per hectare. We suggest that CBFs/DREBs may be critical factors that govern plant phenotypic plasticity associated not only with cold acclimation from the level of gene expression and freezing tolerance but also whole plant architecture, instantaneous leaf level WUE and photosynthetic energy conversion efficiency into biomass.

The photosynthetic response of C3 plants to CO2 has been theoretically modeled by Farquhar’s group (Farquhar et al. 1980). According to their model, at higher C i, CO2 assimilation is usually limited either by the capacity of photosynthetic electron transport to supply ATP and NADPH to regenerate RuBP, or by the capacity of starch and sucrose synthesis to utilize triose phosphates and consequently regenerate Pi. The Pi regeneration-limited photosynthesis is governed by the balance between the source leaves to assimilate carbon and the sink strength to utilize photoassimilates (Arp 1991; Drake et al. 1997). It has been suggested that the increased carbon uptake resulting from initial stimulation of photosynthesis alters the balance between supply and demand due to limited sink capacity to utilize carbohydrates and concomitant retardation of carbon export to the sinks (Kramer 1981; Arp 1991; Drake et al. 1997). This results in the accumulation of sucrose in the source leaves followed by inhibition of sucrose synthesis and a short-term decrease in utilization of phosphorylated intermediates and depletion in stromal Pi. Low availability of stromal Pi triggers inhibition of ATP synthesis and thereby a decrease in the rate at which PGA is converted to triose phosphate which results in feedback inhibition of CO2 assimilation (Stitt and Quick 1989; Sharkey and Vanderveer 1989).

The light response curves (Fig. 2) as well as CO2 response curves (Fig. 3) clearly indicated that the light and CO2-saturated rates of photosynthesis were significantly lower in elevated versus ambient-CO2 grown non-acclimated WT B. napus. This was consistent with the decreased CO2-saturated rates of electron transport (Fig. 4a) combined with decreased levels of major photosynthetic enzymes such as rbcL, cFBPase (Fig. 6) as well as the maximum carboxylation velocity of Rubisco in elevated versus ambient CO2-grown non-acclimated WT (Table 1). Consequently, the EP and NPQ at any given irradiance and CO2 concentration increased significantly for non-acclimated WT in response to growth at elevated CO2 (Figs. 4, 5). In contrast, BnCBF17-OE as well as cold acclimated WT did not exhibit inhibition of light and CO2-saturated rates of photosynthesis in response to growth at elevated CO2 (Figs. 2, 3). This was consistent with no changes in the light and CO2-saturated rates of electron transport, excitation pressure and NPQ (Figs. 4, 5) and levels of rbcL and cFBPase (Fig. 6) as well as the maximum carboxylation velocity of Rubisco (Table 1) in elevated versus ambient CO2-grown BnCBF17-OE and cold acclimated WT B. napus. Thus, we report that the cold acclimated B. napus and the BnCBF17-OE are able to maintain enhanced photosynthetic performance and energy conversion into biomass even under long-term growth and development at elevated CO2. This appears to be due to a decreased sensitivity to feedback limited photosynthesis in BnCBF17-OE as well as cold acclimated B. napus relative to non-acclimated WT plants.

How does BnCBF17 over-expression and cold acclimation of WT B. napus differentially affect sensitivity to feedback inhibition of photosynthesis at elevated CO2? Our results indicate that the BnCBF17-OE as well as cold acclimated WT exhibit a 1.7–1.9-fold higher specific leaf weight relative to non-acclimated WT at elevated CO2 (Table 1). We have further shown that BnCBF17-over-expression as well as cold acclimation results in the enhanced amounts of rbcL and cFBPase (Fig. 6) when corrected on a leaf area basis not only at ambient CO2 but also at elevated CO2. This is consistent with our previous reports that cold acclimation of winter cereals induces an increase in the amount of Rubisco and cFBPase with a concomitant stimulation of respiration and plant biomass production (Dahal et al. 2012a). Furthermore, Savitch et al. (2005) reported that over-expression of BnCBF17 in B. napus not only enhanced gene expression of the triose-P translocator and cFBPase but also increased the activity of Rubisco, cFBPase and SPS. Consequently, BnCBF17-over-expression as well as cold acclimation of B. napus result in enhanced Pi cycling and increased capacity for RuBP regeneration through increased utilization of phosphorylated intermediates. Thus, we suggest that the decreased sensitivity to feedback limited photosynthesis in cold acclimated B. napus as well as the BnCBF17-OE under conditions of elevated CO2 reflects an improved capacity to maintain a high flux of carbon between source and sink even under elevated CO2 conditions. This is further supported by the fact that in cereals, carbon export rates from source leaves are also enhanced upon cold acclimation (Leonardos et al. 2003).

The improved instantaneous leaf level WUE induced by either BnCBF17-over-expression or cold acclimation is primarily associated with an increase in A sat and decrease in stomatal conductance. It appears that the suppressed stomatal conductance observed upon cold acclimation or BnCBF17-over-expression can be accounted for, in part, by a decrease in stomatal density on both the abaxial and adaxial leaf surfaces.

We conclude that the over-expression of BnCBF17 and cold acclimation of B. napus enhances photosynthetic performance, the efficiency of energy conversion and instantaneous leaf level WUE which is maintained even after long-term growth and development under elevated CO2 conditions. Thus, we suggest that the transcription factor, BnCBF17, may be a central component which governs the regulation of plant architecture, photosynthetic capacity and energy conversion efficiency of crops. This may provide important new insights into potential molecular and genetic approaches focussed on the maintenance or even the enhancement of plant productivity under suboptimal growth conditions associated with climate change.