Photosynth Res (2015) 124:117–126 DOI 10.1007/s11120-015-0110-4
REGULAR PAPER
The arc mutants of Arabidopsis with fewer large chloroplasts have a lower mesophyll conductance Sean E. Weise • David J. Carr • Ashley M. Bourke David T. Hanson • Debbie Swarthout • Thomas D. Sharkey
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Received: 20 August 2014 / Accepted: 23 February 2015 / Published online: 3 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and 13CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (C*) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25–50 % reduction in the mesophyll conductance at ambient CO2.
Electronic supplementary material The online version of this article (doi:10.1007/s11120-015-0110-4) contains supplementary material, which is available to authorized users. S. E. Weise D. J. Carr A. M. Bourke D. Swarthout T. D. Sharkey (&) Department of Biochemistry & Molecular Biology, Michigan State University, 603 Wilson Road, Room 201, East Lansing, MI 48824, USA e-mail: [email protected] D. T. Hanson Department of Biology, University of New Mexico, Albuquerque, NM, USA
Keywords Mesophyll conductance Gamma* Arc mutants Farquhar model Carbon isotope Chloroplast size
Introduction Among the changes in photosynthetic eukaryotes as they colonized the land is the reduction in the size and increase in number of chloroplasts per cell. An exception to this is in the hornworts, an early diverging land plant lineage with variable chloroplast size and number (Hanson et al. 2014). Hornwort species with large chloroplasts normally also have pyrenoids, which are thought to be essential for active accumulation of inorganic carbon. However, species (and even cells within a species) with small chloroplasts do not have pyrenoids (Hanson et al. 2014), which may indicate that large chloroplasts interfere with diffusion of CO2 from the air to Rubisco. The mesophyll cells of later diverging land plants contain dozens to hundreds of chloroplasts per cell (Ahmadabadi and Bock 2012; Possingham and Saurer 1969; Pyke and Leech 1994). The question of why the photosynthetic cells of higher plants contain so many small chloroplasts has been asked many times (Jeong et al. 2002; Osteryoung and Pyke 2014; Pyke 1999). It has been hypothesized that mesophyll cells have a large population of small chloroplasts that can move around the cell to maximize light absorption in low light and reduce photodamage in high light (Morita and Nakamura 2012). An additional explanation is that cells with multiple smaller chloroplasts may have a reduced path length for CO2 diffusion from the intracellular air space to the chloroplast. This would be especially important as leaves became thicker and more complex.
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There is a finite resistance to CO2 diffusion from the intracellular airspaces of leaves to Rubisco in the stroma of chloroplasts. The inverse of this resistance is called mesophyll conductance (Sharkey 2012). Mesophyll conductance typically accounts for 40 % of the decrease in CO2 concentration from the atmosphere to Rubisco (Jones and Slatyer 1972; Loreto et al. 1992; Samsuddin and Impens 1979; Warren 2008). It has been shown that chloroplast size and position in the mesophyll cells has an effect on mesophyll conductance. Chloroplasts are concentrated along plasma membranes that are adjacent to intercellular airspaces and are generally not found along membranes that are in contact with neighboring cells (Busch et al. 2013; Evans et al. 1994; Psaras et al. 1996; Pyke 1999; Takagi 2003). The area of chloroplasts facing intercellular airspaces for a given leaf area is often called Sc. The requirement for chloroplasts to be adjacent to cell walls exposed to intercellular spaces can determine optimal leaf thickness because chloroplasts not adjacent to intercellular spaces will have a very reduced CO2 supply (Terashima et al. 2001). In transgenic tobacco lines in which the amount of Rubisco had been reduced by 65 %, Sc was reduced by 16–31 %, and mesophyll conductance was reduced by 19–27 % depending on growing conditions (Evans et al. 1994). Another study using transgenic tobacco plants with increased phytochrome resulted in plants with cup-shaped chloroplasts. These chloroplasts had less surface area in contact with the plasma membrane and a lower mesophyll conductance (Sharkey et al. 1991). It has also been shown that mesophyll conductance is reduced when chloroplasts move to positions away from the leaf air spaces in response to strong white light (Tholen et al. 2008). The accumulation and replication of chloroplasts (arc) mutants are Arabidopsis mutants in which the chloroplast division machinery has been compromised resulting in plants with one or few chloroplasts per cell compared to wild type, which typically has over 100 chloroplasts per cell (Pyke and Leech 1992). The arc mutants provide an ideal system for testing the effects of plastid size on mesophyll conductance. It has been shown that in these mutants the total chloroplast compartment size and even the number of starch granules per volume of chloroplast is constant, although the mechanisms of regulation of chloroplast number, volume, and size are still unknown (CrumptonTaylor et al. 2012; Pyke 1999; Pyke and Leech 1994). Mesophyll conductance can be estimated by fitting the photosynthetic model of Farquhar, von Caemmerer, and Berry (1980) (Farquhar model) to measurements of CO2 assimilation with varying internal leaf CO2 concentrations (Ci) (Ethier and Livingston 2004; Harley et al. 1992a; Loreto et al. 1992; Sharkey et al. 2007). Another method
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with fewer assumptions is based on 13C isotope discrimination (Evans et al. 1986; Harley et al. 1992a). The Farquhar model fitting method is the easiest, but most prone to error. One large source of error can come from using incorrect Rubisco kinetics. The ratio of carboxylation to oxygenation can be predicted using the parameter C*. This is the CO2 partial pressure at which the rate of carboxylation by Rubisco is equal to two times the rate of oxygenation. An accurate estimate of C* is critical for characterizing the specificity of Rubisco for CO2 and O2, and its value is embedded in models used to estimate mesophyll conductance. The estimate of C* made by Bernacchi et al. (2002) using tobacco is often used for other plant species, including for Arabidopsis. There are two reports of C* estimates specifically for Arabidopsis. The value of C* determined by Flexas et al. (Flexas et al. 2007b) of 4.9 Pa at 25 °C is much higher than the C* for tobacco of 3.74 Pa. The C* determined by Walker et al. (2013) of 3.64 Pa at 25 °C is lower. Mesophyll conductance can also be measured using 13C isotope discrimination although this requires the use of a mass spectrometer or, more recently, tunable diode laser (TDL) capable of differentiating 12CO2 from 13CO2 (Barbour et al. 2007; Flexas et al. 2006). This method is based on the fact that enzymatic action of Rubisco is ordered binding RuBP first, and then preferentially catalyzing the carboxylation reaction with 12CO2 as opposed to 13CO2. If CO2 can freely diffuse between Rubisco and the atmosphere, then this preferential usage of 12CO2, called discrimination (D), causes an accumulation of 13CO2 in air surrounding a photosynthesizing leaf. Limitations to the total conductance of CO2 reduce the observed D. The effects of boundary layer and stomatal conductance limitations on D are estimated using standard gas exchange techniques, and the remaining D is attributed to the effects of mesophyll conductance with a correction for decarboxylations (Evans et al. 1986). All methods for measuring mesophyll conductance have sources of error and methodological biases, however the largest uncertainty in 13 C discrimination is the fractionation by Rubisco (Gu and Sun 2014) and this should not be different between the wild-type and arc mutants. In this study, we estimated the mesophyll conductance using Farquhar model fitting and the 13CO2 discrimination methods in wild-type arc3, 5, 6, and 10 mutants of Arabidopsis with variable chloroplast numbers, sizes, and shapes. In order to properly fit the Farquhar model to the data, we determined the day respiration rate (Rd) and C* for Arabidopsis. The lower mesophyll conductances we estimated in the arc mutants by gas exchange were confirmed by measuring 13CO2 discrimination using an online TDL and measuring relative 13C content (d) in rosette tissue.
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Materials and methods Determination of C* for Arabidopsis Plant material Wild-type, accession Wassilewskija (WS), seeds were cold treated at 4 °C in distilled water for 3 days to ensure uniform germination. Seeds were then germinated and grown in 1.500 9 5.500 (3.8 9 14 cm) SC7 Cone-tainers (Stuewe and Sons, Tangent, OR) containing Sun Gro Redi-earth plug and seedling mix (Sun Gro, Bellevue WA, USA). Plants were watered with deionized water for the first 4 weeks, and then quarter-strength Hoagland’s solution thereafter. Plants were grown in a Percival AR4 growth chamber (Percival, Perry IA, USA) with an 8-h photoperiod. Day temperature was 22 °C and night temperature was 18°C. The photon flux density (PFD), measured at leaf level, was 120 lmol m-2 s-1. Humidity was maintained at a minimum of 60 % RH. Plants were 7–8 weeks old when used for experiments. Gas exchange Gas exchange measurements were conducted on plants 1 h after the start of the photoperiod and were finished at least 1 h before the end of the photoperiod to minimize complications due to possible circadian effects on photosynthesis. A single fully expanded leaf was placed in a standard 2 9 3 cm LI-COR 6400-02B red/blue LED light source head connected to a LI-COR 6400 photosynthesis system (LI-COR Biosciences, Lincoln NB, USA). The LICOR 6400 used was modified as recommended by LI-COR according to application note seven in order to be able to obtain CO2 concentrations less than 50 ppm (http:// envsupport.licor.com/docs/AppNote7_LowCO2.pdf). The photosynthetic rates at low CO2 were corrected for diffusion according to the LI-COR 6400 operator’s manual version five, Sects. 4–43. The determination of C* for Arabidopsis was made using the Laisk method (1977) and described in Brooks and Farquhar (1985) as modified by Ethier and Livingston (2004) and Flexas et al. (2007a, b). The PFDs used were 1000, 75, 50, and 30 lmol m-2 s-1, and leaf temperatures were 16, 21, 25, 28, 32, and 36 °C. High and low temperatures were achieved using the 6400-88 expanded temperature control water jacket connected to a recirculating water bath. Humidified nitrox, 79 % N2 and 21 % O2, was provided to the LI-COR 6400 using a custom built mixing apparatus with MKS mass flow controllers (MKS Instruments, Andover, MS, USA). The following precautions were taken in an attempt to minimize potential error when using the Laisk method. Four light levels were used, and gas exchange equipment was
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modified to be capable of operating at low CO2, so that points both above and below the crossover point of the A/Ci lines at low CO2 could be measured, eliminating the need for extrapolation. This eliminated a concern with nonlinearities of the relationships described by Gu and Sun (2014). To select A/Ci lines that crossed at one single point, the following criteria were used. If the average of the cross over points of the four lines varied by a standard error of more than ± 0.26 Pa or ± 0.05 lmol m-2 s-1 one of the lines (frequently the lowest light level) was thrown out. If the standard error still exceeded these parameters, the measurement of all the A/Ci lines at a particular temperature was repeated. Determination of C* requires knowing mesophyll conductance. C ¼ ðRd =gm Þ þ Ci
ðð1ÞÞ
where Rd is the nonphotorespiratory respiration in the light, gm is the mesophyll conductance, and Ci* is the CO2 partial pressure at which lines at different light levels cross over. The mesophyll conductance was determined iteratively by fitting the Farquhar model with the spreadsheet provided in Sharkey et al. (2007) to an A/Ci curve generated with the same leaf just before the determination of Ci*. The new gm was used to estimate a new C* until C* was constant. The A/Ci curve was measured using partial pressure of CO2 between 5 and 119 Pa, and a PFD of 1000 lmol m-2 s-1. C* was estimated this way over a range of temperature. Theoretically, it is possible to estimate C* and gm with one fitting but we chose the iterative method. An Arrhenius plot was used to determine the enthalpy of activation (DHa) as the slope of this regression multiplied by the ideal gas constant of 0.008314 kJ K-1 mol-1. The c term was determined using the Solver mathematical package in Microsoft Excel to minimize the sum of the squares of the differences between the measured C* at 16, 21, 25, 28, 32, and 36 °C and the mathematically computed DHa C using the equationC ¼ eðcR T Þ . *
Mesophyll conductance Plant material Wild-type (Col-0) Arabidopsis and four tDNA-insert lines: arc3 (SALK_062123C), arc5 (SAIL_71-D11), arc6 (SAIL_693-G04), and arc10 (SALK_073878) were used. All the SALK and SAIL lines were verified by PCR to be homozygous for tDNA insert. The wild-type and arc mutant chloroplast number phenotype were verified by microscopy. Arc6 is the most severe chloroplast division mutant containing only one or two large chloroplasts per cell (Pyke et al. 1994). Arc3 and arc5 are intermediate with
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15–18 chloroplasts per cell (Pyke and Leech 1992; Pyke and Leech 1994), and arc10 has one to two chloroplasts per cell (Yoder et al. 2007). Plants were grown in 1.500 9 5.500 (3.8 9 14 cm) SC7 Cone-tainers (Stuewe and Sons, Tangent, OR) using Metro-Mix 360 potting soil (Sun Gro, Bellevue, WA) and watered with tap water. Plants were grown for 7 weeks in a fluorescently lit growth chamber with an 8 h photoperiod to ensure large leaves conducive to gas exchange measurements. The day temperature was 22 °C, and the night temperature was 18 °C with a minimum of 60 % relative humidity. The PFD was 190 lmol m-2 s-1 measured at leaf level. Gas exchange Gas exchange measurements were conducted on plants 1 h after the start of the photoperiod and were finished at least 1 h before the end of the photoperiod to minimize complications due to possible circadian effects on photosynthesis. For estimates of mesophyll conductance made by fitting the Farquhar model, a single fully expanded leaf was placed in a standard 2 9 3 cm LI-COR 6400-02B red/blue LED light source head connected to a LI-COR 6400 photosynthesis system. Humidified synthetic air, 79 % N2 and 21 % O2, was provided to the LI-COR 6400 using a custom built mixing apparatus with MKS mass flow controllers (MKS Instruments, Andover, MS, USA). The leaf was allowed to acclimate in the system at an ingoing CO2 concentration of 400 ppm for half an hour prior to beginning measurements. Light was provided by a 6400-02B RGB light source with a PFD of 1200 lmol m-2 s-1 at leaf level. The reference CO2 level was then set to 1200 ppm using the LI-COR CO2 controller, and the assimilation rate was measured. A dew point of 16–18 °C was maintained in the air surrounding the leaf to minimize errors in mesophyll conductance measurements associated with a high vapor pressure deficient. Assimilation rates were measured at CO2 levels that were incrementally decreased in 50 or 100 ppm increments to 0 ppm CO2. The Farquhar model was then fit to the data with the spreadsheet provided in Sharkey et al. (2007). Rd was held constant at 0.45 lmol m-2 s-1, and a C* was fixed using a DHa of 18.707 kJ mol-1 and c of 9.044. For 13C discrimination measurements, a single fully expanded leaf was placed in a standard LI-COR 2 x 3 cm clear top chamber connected to a LI-COR 6400 photosynthesis system. Light was provided by a 6400-18 RGB light source with a PFD of 500 lmol m-2 s-1 at leaf level. Humidified synthetic air, 79 % N2 and 21 % O2 or 98 % N2 and 2 % O2, was provided to the LI-COR 6400 using a custom built mixing apparatus with MKS mass flow controllers (MKS Instruments, Andover, MS, USA). A dew
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point of 16–18 °C was maintained in the air surrounding the leaf, so the vapor pressure difference was constant. CO2 was supplied at 38 and 128 Pa with d13C of approximately -4 % through the LI-COR 6400 mixer and an external CO2 cylinder. Exhaust air from the leaf sample chamber was used for online measurements of photosynthetic 13CO2 discrimination from a combined IRGA-Tunable Diode Laser system (Barbour et al. 2007; Bickford et al. 2009; Flexas et al. 2006; Uehlein et al. 2008). Mesophyll conductance was calculated using the equations of Evans et al. (1986). The TGA-100 (Campbell Scientific, Logan, UT, USA) measures absolute concentrations of 13CO2 and 12 CO2 at a frequency of 10 Hz from dry air before and after exposure to a photosynthesizing leaf. The 10 Hz data were averaged over 10 s to calculate the isotopic composition (d13C) of sampled air with a precision of 0.05–0.09 %. d13C values were calculated using the R software package (Erhardt and Hanson 2013) in which periodic measurements of the calibration gasses (high and low) were interpolated using a cubic smoothing spline to account for slow drifts throughout the measurement period; then the sample values were calibrated using a gain and offset determined from the mean interpolated tank values. Isotopic pointbased calculations of gm (Bickford et al. 2009) at a PFD of 500 lmol m-2 s-1 were included, as gm may change with light intensity. Leaf temperature was maintained at 25 °C. The point-based calculations assumed no significant effects of fractionation by photorespiration and respiration. We also made measurements under non-photorespiratory conditions to test whether differences between plant lines were the result of differences in photorespiration. 13
C analysis of leaf tissue
Whole Arabidopsis rosettes were harvested after 5 weeks of growth and dried at 70 °C for 24 h. Plants were harvested in the morning to minimize the contribution of starch to the analysis. Dried plant material was ground in a 50 ml Falcon tube using a glass marble and vigorous shaking. A subset of material was then placed in a 2 ml tube and sent to the Stable Isotope Ratio Facility for Environmental Research (SIRFER) at the University of Utah (Salt Lake City, UT, USA) for analysis. Statistics Statistical significance of differences in values of mesophyll conductance was determined using a one-tailed Student’s t tests with a cut-off value of p = 0.05. A one-tailed test was used because we were testing the hypothesis that the mean of the arc mutant mesophyll conductance was less than the mean of the WT mesophyll conductance.
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Results C* in Arabidopsis We determined C* using the Laisk method with the same equipment used to determine mesophyll conductance of the arc mutants (Fig. 1). The C* determined in Arabidopsis at 25 °C was 4.47 Pa with a DHa of 18.685 kJ mol-1 and c of 9.036. The dependence of C* on temperature from this and three other commonly used DHa
studies was compared using the equation C ¼ ec RT (Fig. 2). This equation can be found in Harley et al.(1992b) and Sharkey et al. (2007).The value of R (ideal gas constant) used was 0.008314 kJ K-1 mol-1. Rd remained constant with temperature at 0.45 lmol m-2 s-1 with a standard error of 0.04 lmol m-2 s-1. A sensitivity analysis of the Farquhar model using an A/Ci curve generated with Arabidopsis at a leaf temperature of 25 °C was conducted. Values of C* from 3.5 Pa to 5.0 Pa were used with Rd held constant at 0.45 lmol m-2 s-1. All parameters of the model were extremely sensitive to changes in C*. Estimates of the maximum rate of carboxylation by Rubisco (Vcmax) and rate of electron transport (J) were highest, and the sum of squares error terms (measure of how well the model fit the data) was lowest at a C* between 4 and 4.5 Pa (Fig. 3). The mesophyll conductance increased significantly at values of C* greater than 4.3 Pa (Fig. 3). When Rd was allowed to change, Vcmax, J, and Rd all declined while mesophyll
Fig. 2 Comparisons of reported C* with temperature in Arabidopsis and tobacco. The green squares are the actual C* measured in this study. The green line was fit using a DHa 18.685 kJ mol-1 a c of 9.036. The Walker et al. 2013 line was calculated using their DHa of 33.7 kJ mol-1 and a C* at 25 °C of 3.64 Pa. The tobacco line from Bernacchi et al. (2002) was calculated using their DHa of 24.46 and a C* at 25 °C of 3.74 Pa
Fig. 3 Sensitivity of Farquhar model to C*. An A/Cc curve of Arabidopsis at 25 °C was fit. Rd was held constant at 0.45 lmol m-2 s-1. Vcmax = maximum velocity of carboxylations allowed by Rubisco, J = rate of photosynthetic electron transport (based on NADPH use). Arrows indicate mesophyll conductance (gm) and sum of square error terms at the following C* values: 3.64 Pa Walker et al. (2013), 4.47 Pa this study, and 4.90 Pa Flexas et al. (2007b)
Fig. 1 Measuring Ci* in Arabidopsis at 25 °C. Ci* is the internal leaf CO2 concentration at which photosynthesis is balanced by photorespiration. At Ci* an increase in light driven electron transport will increase both photosynthesis and photorespiration by the same amount. Ci* is lower than C* because the internal leaf CO2 concentration is lowered by the conductance of CO2 from the site of production into the internal leaf airspace. In order to minimize potential errors if the average of the cross over points of the four lines varied by a standard error of more than ±0.26 Pa or ±0.05 lmol m-2 s-1 one of the lines (in this case the line at a PFD of 30 lmol m-2 s-1) was not used in the determination of Ci*
conductance rose with increasing values of C* (Supplemental Fig. 1). Photosynthesis and mesophyll conductance of the arc mutants Both instantaneous photosynthesis and photosynthetic capacity of the arc mutants were greater than or similar to wild type (Fig. 4 and Supplemental Table 1). All three capacities for photosynthesis, Vcmax of Rubisco, J (electron
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Fig. 5 Estimates of mesophyll conductance (gm) from Farquhar model fitting of A/Ci curves. The model was fit using the C* for Arabidopsis measured in this study. Values are mean ± SE, n = 4. Asterisks indicate statistical difference p = 0.05
Fig. 4 Estimates of photosynthetic parameters from Farquhar model fitting of A/Ci curves. The parameters include: the maximum velocity of carboxylations allowed by Rubisco (Vcmax), the rate of photosynthetic electron transport based on NADPH use (J), and the minimum rate of triose phosphate usage (TPU). The model was fit using the C* for Arabidopsis measured in this study. Values are mean ± SE, n = 4
transport), and triose phosphate use capacity (TPU) indicated a balanced increase in photosynthetic capacity in this mutant, where the chloroplast number was approximately 15–18 per cell. In contrast, the photosynthetic capacity of the arc6 mutant was similar to the wild type (Fig. 4), despite the presence of only one large chloroplast in this mutant line. An independent experiment on arc10 mutants, also containing only one to two chloroplasts per cell, at a different location (Albuquerque versus East Lansing), showed no difference in the photosynthetic capacity of this mutant line and the wild-type Arabidopsis plants (displayed as a different experiment in Fig. 4). Mesophyll conductance was estimated by curve fitting using the same data used to determine photosynthetic capacity. Mesophyll conductance in arc 3, 6, and 10 mutants was lower than the wild type by 24–42 % (Fig. 5) even though photosynthetic capacities were similar or higher than the wild type.
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Fig. 6 Measurements of mesophyll conductance (gm) using 13CO2 measured with a TDL. Measurements were taken at ambient CO2 and O2 (400 ppm and 21 %) and high CO2 and low O2 (1500 ppm and 2 %). Values are mean ± SE, n = 4. Asterisks indicate statistical difference from wild type p = 0.05
Mesophyll conductance in the wild-type arc 3, 5, 6, and 10 mutants was also determined by 13CO2 discrimination using a TDL. In the TDL measurements, arc 3, 6, and 10 were on average 39 % lower than wild type while arc 5 was only 7 % lower at ambient CO2 levels (Fig. 6). At high CO2, the relationship between the wild-type and arc mutant mesophyll conductance was similar to what was observed at ambient CO2 but all mesophyll conductances were reduced (Fig. 6). The d13C value of plant material provides an integrated measure of discrimination. The d13 C content was less negative for arc3 and arc6 plants (Fig. 7). No statistically
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significant difference in stomatal conductance was observed (Supplemental Table 1). The data for elevated CO2 are especially clear that Ci was the same but Cc was reduced in the arc mutants. Because the arc 6 mutant had the lowest estimated and measured mesophyll conductance as well as the least negative d13C, we examined the location of the chloroplasts in the cells. Each cell in the leaf mesophyll of the arc6 mutant had one large chloroplast. The single chloroplast surrounded the periphery of each cell but did not appear to be thicker than the individual chloroplasts of the wild type (Fig. 8).
Discussion Mutants of Arabidopsis with fewer large chloroplasts have lower mesophyll conductances even though the photosynthetic capacities were the same or higher. The two methods for determining mesophyll conductance used here, curve fitting and stable isotope discrimination, indicated similar degrees of reduction in mesophyll conductance even though the two methods have very few assumptions in common. The short-term (TDL) and long-term (d13C) stable isotope methods also agreed. The arc 5 mutant has about the same number of chloroplasts, 13 per cell, as arc 3 yet the arc 5 mutant had a mesophyll conductance almost as large as wild type (Fig. 6). This demonstrates that the lower mesophyll conductance in the arc mutants was not solely determined by chloroplast number. One major difference between arc 3 and arc 5 is the shape of the chloroplasts. The arc 5 mutant has large dumbbell-shaped chloroplasts (Pyke and Leech 1994). This difference in morphology might increase the area available for diffusion for a portion of the chloroplast resulting in a lower mesophyll conductance. If true, this could help explain some of the vast diversity of chloroplast morphology among large chloroplasts found in many algae (Graham and Wilcox 2000). The position of chloroplasts in
Fig. 7 d13C content of Arabidopsis rosettes. Values are mean ± SE, n=4
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the cell is, in part, determined by the amount of light. For example, the optimum location of chloroplasts in high light is in a stacked configuration at the edges of the cell (Wada 2013). This minimizes the light interception and protects against photo damage but it is not necessarily the best position for CO2 diffusion and uptake. It has been found when blue light is used to induce the chloroplast light avoidance response, which is normally observed when plants are moved from low to high light, the surface area of chloroplasts adjacent to intercellular airspaces is reduced (Tholen et al. 2008). The dumbbell shape of the arc 5 chloroplasts could hamper the light avoidance response thus maintaining a higher mesophyll conductance. The Arabidopsis plants were grown in low light, 190 lmol m-2 s-1, but were then placed in high light, 500 lmol m-2 s-1, to measure mesophyll conductance, possibly causing chloroplast movements. It is not known what proportion of the chloroplasts in the arc mutants is adjacent to intercellular airspaces and whether chloroplast position can change with light intensity. In cases like arc6 with one large chloroplast surrounding the cell, there will be parts of the chloroplast not adjacent to air spaces (Fig. 8) and this could lead to reduced mesophyll conductance (Terashima et al. 2001). A number of effects of internal cell architecture on estimated mesophyll conductance have been discussed in recent papers (Griffiths and Helliker 2013; Scafaro et al. 2011; Tholen et al. 2012). Chloroplast movements in high light tend to decrease mesophyll conductance (Tholen et al. 2008) so if the arc plants cannot respond to light by chloroplast movement, the difference in mesophyll conductance between the arc plants and wild-type plants could be even greater at low light. The mesophyll conductance for both mutants and wild type was lower when measured at high CO2 and low O2, but arc 3, 6, and 10 mutant lines were significantly lower than wild type (Fig. 6). Changes in the physical resistance, perhaps through changes in aquaporin function (Flexas et al. 2007a; Flexas et al. 2006; Heckwolf et al. 2011) have been proposed while Evans and von Caemmerer (2013) and Tholen et al. (2012) indicate that many apparent effects can be explained the changes in fluxes associated with photorespiration (Lanigan et al. 2008). Gu and Sun (2014) propose that the apparent CO2 sensitivity of mesophyll conductance can result from methodological errors and biases. While the absolute value of mesophyll conductance may be difficult to determine with a high degree of accuracy, the relative differences between the wild-type and arc mutants in this study are likely robust since they were done at both high CO2 (1500 ppm) and low O2 (2 %) eliminating most photorespiration and many of the biases associated with D13C analysis. Photosynthetic capacities were greater than (arc3) or similar to wild type in these experiments (Fig. 4).
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Fig. 8 Chloroplast autofluorescence in WT and arc6 mutants of Arabidopsis. Chloroplasts were observed in free hand sections using autofluorescence in a confocal microscope
Therefore, biases that can result from having very different photosynthetic capacities are unlikely to explain the differences in mesophyll conductance between the wild type and arc mutants. Austin and Webber (2005) have reported reduced photosynthesis in the arc 3, 5 and 6 mutants; it is not clear why our results are different from theirs. The relationship observed between lower mesophyll conductance and lower photosynthetic capacity (for example Li et al., (2013) and von Caemmerer and Evans (1991), was not observed in the arc mutants. The value of C* used in the Farquhar model had a large effect on the estimated mesophyll conductance, especially if the C* value was high (Fig. 2). The values of C* for Arabidopsis found in this study are higher than those of Walker et al. 2013 but lower than Flexas et al. (2007b) (Fig. 3). Ci* was determined by the Laisk (1977) method in all these studies. Because the estimate of C* from Ci* depends on mesophyll conductance, iteration of the estimates for both can be carried out or an independent method of measuring mesophyll conductance used. C* was determined using a mesophyll conductance determined by 13CO2 discrimination in the Walker et al. (2013) study while it was determined according to Warren and Dreyer (2006) in the Flexas et al. (2007b) study. Here, we used an iterative approach, estimating mesophyll conductance from some of the same data used to determine Ci*. Gu and Sun (2014) pointed out some of the biases that can be introduced when using the ‘‘Laisk C*’’ which is Ci*, therefore the C* we used was as described by Ethier and Livingston (2004) which accounts for mesophyll conductance. Despite the precautions outlined in the materials and methods section in our use of the Laisk method, the reasons for the differences in values found for C* in Arabidopsis are unclear. The value of C* as a measure of the Rubisco compensation point depends on several assumptions including a constant Rd and a release of one CO2 for every two oxygenations. We suspect that
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these assumptions may not always hold at the lowest light intensity. The estimate of mesophyll conductance is very sensitive to Rd. Allowing Rd to vary resulted in an estimate of mesophyll conductance that was higher, and Vcmax and J that were lower at a C* of 4.5 Pa (Fig. 2 and Supplemental Fig. 1). The outputs of the model were more consistent when Rd was held constant. Day respiration is not trivial to measure and thus few measurements have been made. Previous studies have found no consistent relationship between Rd and temperature. Rd has been reported to increase exponentially with temperature in tobacco (Bernacchi et al. 2001). It has been found to increase linearly with temperature in Heteromeles and Lepechinia (Villar et al. 1995). Rd has also been shown to be relatively constant between 15 and 25 °C even decreasing at temperatures above 25 °C in Eucalyptus pauciflora (Atkin et al. 2000). In this study with Arabidopsis, Rd was fairly constant with temperatures between 15 and 36 °C. In summary, chloroplast number and shape have a significant impact on mesophyll conductance with more but smaller chloroplasts conferring a higher mesophyll conductance than fewer but larger chloroplasts. The reduced number of chloroplasts is not the sole determinant of lowered mesophyll conductance; shape plays a large role as well. Fitting of the Farquhar model of leaf photosynthesis to A/Ci data can provide an effective estimate for mesophyll conductance that is in close agreement with 13CO2 discrimination based methods. Use of model fitting requires that the model is parameterized with the correct C* and Rd. Acknowledgments We thank Dr. Katherine Osteryoung for providing the arc mutant seeds and for thoughtful discussions throughout the project. This work was supported by DOE grant DE-SC0008509 to TDS and NSF grant IOS-0719118 and NIH grant NIH-NCRR P20RR18754 to DTH. Partial salary support for TDS comes from Michigan State University AgBioResearch.
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126 and wild Oryza species with contrasting mesophyll cell wall thickness. Plant Cell Environ 34:1999–2008 Sharkey TD (2012) Mesophyll conductance: constraint on carbon acquisition by C3 plants. Plant Cell Environ 35:1881–1883 Sharkey TD, Vassey TL, Vanderveer PJ, Vierstra RD (1991) Carbon metabolism enzymes and photosynthesis in transgenic tobacco (Nicotiana tabacum L.) having excess phytochrome. Planta 185:287–296 Sharkey TD, Bernacchi CJ, Farquhar GD, Singsaas EL (2007) Fitting photosynthetic carbon dioxide response curves for C3 leaves. Plant Cell Environ 30:1035–1040 Takagi S (2003) Actin-based photo-orientation movement of chloroplasts in plant cells. J Exp Biol 206:1963–1969 Terashima I, Miyazawa S-I, Hanba YT (2001) Why are sun leaves thicker than shade leaves? —Consideration based on analyses of CO2 diffusion in the leaf. J Plant Res 114:93–105 Tholen D, Boom C, Noguchi KO, Ueda S, Katase T, Terashima I (2008) The chloroplast avoidance response decreases internal conductance to CO2 diffusion in Arabidopsis thaliana leaves. Plant Cell Environ 31:1688–1700 Tholen D, Boom C, Zhu X-G (2012) Opinion: prospects for improving photosynthesis by altering leaf anatomy. Plant Sci 197:92–101 Uehlein N, Otto B, Hanson DT, Fischer M, McDowell N, Kaldenhoff R (2008) Function of Nicotiana tabacum aquaporins as
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REGULAR PAPER
The arc mutants of Arabidopsis with fewer large chloroplasts have a lower mesophyll conductance Sean E. Weise • David J. Carr • Ashley M. Bourke David T. Hanson • Debbie Swarthout • Thomas D. Sharkey
•
Received: 20 August 2014 / Accepted: 23 February 2015 / Published online: 3 March 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and 13CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (C*) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25–50 % reduction in the mesophyll conductance at ambient CO2.
Electronic supplementary material The online version of this article (doi:10.1007/s11120-015-0110-4) contains supplementary material, which is available to authorized users. S. E. Weise D. J. Carr A. M. Bourke D. Swarthout T. D. Sharkey (&) Department of Biochemistry & Molecular Biology, Michigan State University, 603 Wilson Road, Room 201, East Lansing, MI 48824, USA e-mail: [email protected] D. T. Hanson Department of Biology, University of New Mexico, Albuquerque, NM, USA
Keywords Mesophyll conductance Gamma* Arc mutants Farquhar model Carbon isotope Chloroplast size
Introduction Among the changes in photosynthetic eukaryotes as they colonized the land is the reduction in the size and increase in number of chloroplasts per cell. An exception to this is in the hornworts, an early diverging land plant lineage with variable chloroplast size and number (Hanson et al. 2014). Hornwort species with large chloroplasts normally also have pyrenoids, which are thought to be essential for active accumulation of inorganic carbon. However, species (and even cells within a species) with small chloroplasts do not have pyrenoids (Hanson et al. 2014), which may indicate that large chloroplasts interfere with diffusion of CO2 from the air to Rubisco. The mesophyll cells of later diverging land plants contain dozens to hundreds of chloroplasts per cell (Ahmadabadi and Bock 2012; Possingham and Saurer 1969; Pyke and Leech 1994). The question of why the photosynthetic cells of higher plants contain so many small chloroplasts has been asked many times (Jeong et al. 2002; Osteryoung and Pyke 2014; Pyke 1999). It has been hypothesized that mesophyll cells have a large population of small chloroplasts that can move around the cell to maximize light absorption in low light and reduce photodamage in high light (Morita and Nakamura 2012). An additional explanation is that cells with multiple smaller chloroplasts may have a reduced path length for CO2 diffusion from the intracellular air space to the chloroplast. This would be especially important as leaves became thicker and more complex.
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There is a finite resistance to CO2 diffusion from the intracellular airspaces of leaves to Rubisco in the stroma of chloroplasts. The inverse of this resistance is called mesophyll conductance (Sharkey 2012). Mesophyll conductance typically accounts for 40 % of the decrease in CO2 concentration from the atmosphere to Rubisco (Jones and Slatyer 1972; Loreto et al. 1992; Samsuddin and Impens 1979; Warren 2008). It has been shown that chloroplast size and position in the mesophyll cells has an effect on mesophyll conductance. Chloroplasts are concentrated along plasma membranes that are adjacent to intercellular airspaces and are generally not found along membranes that are in contact with neighboring cells (Busch et al. 2013; Evans et al. 1994; Psaras et al. 1996; Pyke 1999; Takagi 2003). The area of chloroplasts facing intercellular airspaces for a given leaf area is often called Sc. The requirement for chloroplasts to be adjacent to cell walls exposed to intercellular spaces can determine optimal leaf thickness because chloroplasts not adjacent to intercellular spaces will have a very reduced CO2 supply (Terashima et al. 2001). In transgenic tobacco lines in which the amount of Rubisco had been reduced by 65 %, Sc was reduced by 16–31 %, and mesophyll conductance was reduced by 19–27 % depending on growing conditions (Evans et al. 1994). Another study using transgenic tobacco plants with increased phytochrome resulted in plants with cup-shaped chloroplasts. These chloroplasts had less surface area in contact with the plasma membrane and a lower mesophyll conductance (Sharkey et al. 1991). It has also been shown that mesophyll conductance is reduced when chloroplasts move to positions away from the leaf air spaces in response to strong white light (Tholen et al. 2008). The accumulation and replication of chloroplasts (arc) mutants are Arabidopsis mutants in which the chloroplast division machinery has been compromised resulting in plants with one or few chloroplasts per cell compared to wild type, which typically has over 100 chloroplasts per cell (Pyke and Leech 1992). The arc mutants provide an ideal system for testing the effects of plastid size on mesophyll conductance. It has been shown that in these mutants the total chloroplast compartment size and even the number of starch granules per volume of chloroplast is constant, although the mechanisms of regulation of chloroplast number, volume, and size are still unknown (CrumptonTaylor et al. 2012; Pyke 1999; Pyke and Leech 1994). Mesophyll conductance can be estimated by fitting the photosynthetic model of Farquhar, von Caemmerer, and Berry (1980) (Farquhar model) to measurements of CO2 assimilation with varying internal leaf CO2 concentrations (Ci) (Ethier and Livingston 2004; Harley et al. 1992a; Loreto et al. 1992; Sharkey et al. 2007). Another method
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with fewer assumptions is based on 13C isotope discrimination (Evans et al. 1986; Harley et al. 1992a). The Farquhar model fitting method is the easiest, but most prone to error. One large source of error can come from using incorrect Rubisco kinetics. The ratio of carboxylation to oxygenation can be predicted using the parameter C*. This is the CO2 partial pressure at which the rate of carboxylation by Rubisco is equal to two times the rate of oxygenation. An accurate estimate of C* is critical for characterizing the specificity of Rubisco for CO2 and O2, and its value is embedded in models used to estimate mesophyll conductance. The estimate of C* made by Bernacchi et al. (2002) using tobacco is often used for other plant species, including for Arabidopsis. There are two reports of C* estimates specifically for Arabidopsis. The value of C* determined by Flexas et al. (Flexas et al. 2007b) of 4.9 Pa at 25 °C is much higher than the C* for tobacco of 3.74 Pa. The C* determined by Walker et al. (2013) of 3.64 Pa at 25 °C is lower. Mesophyll conductance can also be measured using 13C isotope discrimination although this requires the use of a mass spectrometer or, more recently, tunable diode laser (TDL) capable of differentiating 12CO2 from 13CO2 (Barbour et al. 2007; Flexas et al. 2006). This method is based on the fact that enzymatic action of Rubisco is ordered binding RuBP first, and then preferentially catalyzing the carboxylation reaction with 12CO2 as opposed to 13CO2. If CO2 can freely diffuse between Rubisco and the atmosphere, then this preferential usage of 12CO2, called discrimination (D), causes an accumulation of 13CO2 in air surrounding a photosynthesizing leaf. Limitations to the total conductance of CO2 reduce the observed D. The effects of boundary layer and stomatal conductance limitations on D are estimated using standard gas exchange techniques, and the remaining D is attributed to the effects of mesophyll conductance with a correction for decarboxylations (Evans et al. 1986). All methods for measuring mesophyll conductance have sources of error and methodological biases, however the largest uncertainty in 13 C discrimination is the fractionation by Rubisco (Gu and Sun 2014) and this should not be different between the wild-type and arc mutants. In this study, we estimated the mesophyll conductance using Farquhar model fitting and the 13CO2 discrimination methods in wild-type arc3, 5, 6, and 10 mutants of Arabidopsis with variable chloroplast numbers, sizes, and shapes. In order to properly fit the Farquhar model to the data, we determined the day respiration rate (Rd) and C* for Arabidopsis. The lower mesophyll conductances we estimated in the arc mutants by gas exchange were confirmed by measuring 13CO2 discrimination using an online TDL and measuring relative 13C content (d) in rosette tissue.
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Materials and methods Determination of C* for Arabidopsis Plant material Wild-type, accession Wassilewskija (WS), seeds were cold treated at 4 °C in distilled water for 3 days to ensure uniform germination. Seeds were then germinated and grown in 1.500 9 5.500 (3.8 9 14 cm) SC7 Cone-tainers (Stuewe and Sons, Tangent, OR) containing Sun Gro Redi-earth plug and seedling mix (Sun Gro, Bellevue WA, USA). Plants were watered with deionized water for the first 4 weeks, and then quarter-strength Hoagland’s solution thereafter. Plants were grown in a Percival AR4 growth chamber (Percival, Perry IA, USA) with an 8-h photoperiod. Day temperature was 22 °C and night temperature was 18°C. The photon flux density (PFD), measured at leaf level, was 120 lmol m-2 s-1. Humidity was maintained at a minimum of 60 % RH. Plants were 7–8 weeks old when used for experiments. Gas exchange Gas exchange measurements were conducted on plants 1 h after the start of the photoperiod and were finished at least 1 h before the end of the photoperiod to minimize complications due to possible circadian effects on photosynthesis. A single fully expanded leaf was placed in a standard 2 9 3 cm LI-COR 6400-02B red/blue LED light source head connected to a LI-COR 6400 photosynthesis system (LI-COR Biosciences, Lincoln NB, USA). The LICOR 6400 used was modified as recommended by LI-COR according to application note seven in order to be able to obtain CO2 concentrations less than 50 ppm (http:// envsupport.licor.com/docs/AppNote7_LowCO2.pdf). The photosynthetic rates at low CO2 were corrected for diffusion according to the LI-COR 6400 operator’s manual version five, Sects. 4–43. The determination of C* for Arabidopsis was made using the Laisk method (1977) and described in Brooks and Farquhar (1985) as modified by Ethier and Livingston (2004) and Flexas et al. (2007a, b). The PFDs used were 1000, 75, 50, and 30 lmol m-2 s-1, and leaf temperatures were 16, 21, 25, 28, 32, and 36 °C. High and low temperatures were achieved using the 6400-88 expanded temperature control water jacket connected to a recirculating water bath. Humidified nitrox, 79 % N2 and 21 % O2, was provided to the LI-COR 6400 using a custom built mixing apparatus with MKS mass flow controllers (MKS Instruments, Andover, MS, USA). The following precautions were taken in an attempt to minimize potential error when using the Laisk method. Four light levels were used, and gas exchange equipment was
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modified to be capable of operating at low CO2, so that points both above and below the crossover point of the A/Ci lines at low CO2 could be measured, eliminating the need for extrapolation. This eliminated a concern with nonlinearities of the relationships described by Gu and Sun (2014). To select A/Ci lines that crossed at one single point, the following criteria were used. If the average of the cross over points of the four lines varied by a standard error of more than ± 0.26 Pa or ± 0.05 lmol m-2 s-1 one of the lines (frequently the lowest light level) was thrown out. If the standard error still exceeded these parameters, the measurement of all the A/Ci lines at a particular temperature was repeated. Determination of C* requires knowing mesophyll conductance. C ¼ ðRd =gm Þ þ Ci
ðð1ÞÞ
where Rd is the nonphotorespiratory respiration in the light, gm is the mesophyll conductance, and Ci* is the CO2 partial pressure at which lines at different light levels cross over. The mesophyll conductance was determined iteratively by fitting the Farquhar model with the spreadsheet provided in Sharkey et al. (2007) to an A/Ci curve generated with the same leaf just before the determination of Ci*. The new gm was used to estimate a new C* until C* was constant. The A/Ci curve was measured using partial pressure of CO2 between 5 and 119 Pa, and a PFD of 1000 lmol m-2 s-1. C* was estimated this way over a range of temperature. Theoretically, it is possible to estimate C* and gm with one fitting but we chose the iterative method. An Arrhenius plot was used to determine the enthalpy of activation (DHa) as the slope of this regression multiplied by the ideal gas constant of 0.008314 kJ K-1 mol-1. The c term was determined using the Solver mathematical package in Microsoft Excel to minimize the sum of the squares of the differences between the measured C* at 16, 21, 25, 28, 32, and 36 °C and the mathematically computed DHa C using the equationC ¼ eðcR T Þ . *
Mesophyll conductance Plant material Wild-type (Col-0) Arabidopsis and four tDNA-insert lines: arc3 (SALK_062123C), arc5 (SAIL_71-D11), arc6 (SAIL_693-G04), and arc10 (SALK_073878) were used. All the SALK and SAIL lines were verified by PCR to be homozygous for tDNA insert. The wild-type and arc mutant chloroplast number phenotype were verified by microscopy. Arc6 is the most severe chloroplast division mutant containing only one or two large chloroplasts per cell (Pyke et al. 1994). Arc3 and arc5 are intermediate with
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15–18 chloroplasts per cell (Pyke and Leech 1992; Pyke and Leech 1994), and arc10 has one to two chloroplasts per cell (Yoder et al. 2007). Plants were grown in 1.500 9 5.500 (3.8 9 14 cm) SC7 Cone-tainers (Stuewe and Sons, Tangent, OR) using Metro-Mix 360 potting soil (Sun Gro, Bellevue, WA) and watered with tap water. Plants were grown for 7 weeks in a fluorescently lit growth chamber with an 8 h photoperiod to ensure large leaves conducive to gas exchange measurements. The day temperature was 22 °C, and the night temperature was 18 °C with a minimum of 60 % relative humidity. The PFD was 190 lmol m-2 s-1 measured at leaf level. Gas exchange Gas exchange measurements were conducted on plants 1 h after the start of the photoperiod and were finished at least 1 h before the end of the photoperiod to minimize complications due to possible circadian effects on photosynthesis. For estimates of mesophyll conductance made by fitting the Farquhar model, a single fully expanded leaf was placed in a standard 2 9 3 cm LI-COR 6400-02B red/blue LED light source head connected to a LI-COR 6400 photosynthesis system. Humidified synthetic air, 79 % N2 and 21 % O2, was provided to the LI-COR 6400 using a custom built mixing apparatus with MKS mass flow controllers (MKS Instruments, Andover, MS, USA). The leaf was allowed to acclimate in the system at an ingoing CO2 concentration of 400 ppm for half an hour prior to beginning measurements. Light was provided by a 6400-02B RGB light source with a PFD of 1200 lmol m-2 s-1 at leaf level. The reference CO2 level was then set to 1200 ppm using the LI-COR CO2 controller, and the assimilation rate was measured. A dew point of 16–18 °C was maintained in the air surrounding the leaf to minimize errors in mesophyll conductance measurements associated with a high vapor pressure deficient. Assimilation rates were measured at CO2 levels that were incrementally decreased in 50 or 100 ppm increments to 0 ppm CO2. The Farquhar model was then fit to the data with the spreadsheet provided in Sharkey et al. (2007). Rd was held constant at 0.45 lmol m-2 s-1, and a C* was fixed using a DHa of 18.707 kJ mol-1 and c of 9.044. For 13C discrimination measurements, a single fully expanded leaf was placed in a standard LI-COR 2 x 3 cm clear top chamber connected to a LI-COR 6400 photosynthesis system. Light was provided by a 6400-18 RGB light source with a PFD of 500 lmol m-2 s-1 at leaf level. Humidified synthetic air, 79 % N2 and 21 % O2 or 98 % N2 and 2 % O2, was provided to the LI-COR 6400 using a custom built mixing apparatus with MKS mass flow controllers (MKS Instruments, Andover, MS, USA). A dew
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point of 16–18 °C was maintained in the air surrounding the leaf, so the vapor pressure difference was constant. CO2 was supplied at 38 and 128 Pa with d13C of approximately -4 % through the LI-COR 6400 mixer and an external CO2 cylinder. Exhaust air from the leaf sample chamber was used for online measurements of photosynthetic 13CO2 discrimination from a combined IRGA-Tunable Diode Laser system (Barbour et al. 2007; Bickford et al. 2009; Flexas et al. 2006; Uehlein et al. 2008). Mesophyll conductance was calculated using the equations of Evans et al. (1986). The TGA-100 (Campbell Scientific, Logan, UT, USA) measures absolute concentrations of 13CO2 and 12 CO2 at a frequency of 10 Hz from dry air before and after exposure to a photosynthesizing leaf. The 10 Hz data were averaged over 10 s to calculate the isotopic composition (d13C) of sampled air with a precision of 0.05–0.09 %. d13C values were calculated using the R software package (Erhardt and Hanson 2013) in which periodic measurements of the calibration gasses (high and low) were interpolated using a cubic smoothing spline to account for slow drifts throughout the measurement period; then the sample values were calibrated using a gain and offset determined from the mean interpolated tank values. Isotopic pointbased calculations of gm (Bickford et al. 2009) at a PFD of 500 lmol m-2 s-1 were included, as gm may change with light intensity. Leaf temperature was maintained at 25 °C. The point-based calculations assumed no significant effects of fractionation by photorespiration and respiration. We also made measurements under non-photorespiratory conditions to test whether differences between plant lines were the result of differences in photorespiration. 13
C analysis of leaf tissue
Whole Arabidopsis rosettes were harvested after 5 weeks of growth and dried at 70 °C for 24 h. Plants were harvested in the morning to minimize the contribution of starch to the analysis. Dried plant material was ground in a 50 ml Falcon tube using a glass marble and vigorous shaking. A subset of material was then placed in a 2 ml tube and sent to the Stable Isotope Ratio Facility for Environmental Research (SIRFER) at the University of Utah (Salt Lake City, UT, USA) for analysis. Statistics Statistical significance of differences in values of mesophyll conductance was determined using a one-tailed Student’s t tests with a cut-off value of p = 0.05. A one-tailed test was used because we were testing the hypothesis that the mean of the arc mutant mesophyll conductance was less than the mean of the WT mesophyll conductance.
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Results C* in Arabidopsis We determined C* using the Laisk method with the same equipment used to determine mesophyll conductance of the arc mutants (Fig. 1). The C* determined in Arabidopsis at 25 °C was 4.47 Pa with a DHa of 18.685 kJ mol-1 and c of 9.036. The dependence of C* on temperature from this and three other commonly used DHa
studies was compared using the equation C ¼ ec RT (Fig. 2). This equation can be found in Harley et al.(1992b) and Sharkey et al. (2007).The value of R (ideal gas constant) used was 0.008314 kJ K-1 mol-1. Rd remained constant with temperature at 0.45 lmol m-2 s-1 with a standard error of 0.04 lmol m-2 s-1. A sensitivity analysis of the Farquhar model using an A/Ci curve generated with Arabidopsis at a leaf temperature of 25 °C was conducted. Values of C* from 3.5 Pa to 5.0 Pa were used with Rd held constant at 0.45 lmol m-2 s-1. All parameters of the model were extremely sensitive to changes in C*. Estimates of the maximum rate of carboxylation by Rubisco (Vcmax) and rate of electron transport (J) were highest, and the sum of squares error terms (measure of how well the model fit the data) was lowest at a C* between 4 and 4.5 Pa (Fig. 3). The mesophyll conductance increased significantly at values of C* greater than 4.3 Pa (Fig. 3). When Rd was allowed to change, Vcmax, J, and Rd all declined while mesophyll
Fig. 2 Comparisons of reported C* with temperature in Arabidopsis and tobacco. The green squares are the actual C* measured in this study. The green line was fit using a DHa 18.685 kJ mol-1 a c of 9.036. The Walker et al. 2013 line was calculated using their DHa of 33.7 kJ mol-1 and a C* at 25 °C of 3.64 Pa. The tobacco line from Bernacchi et al. (2002) was calculated using their DHa of 24.46 and a C* at 25 °C of 3.74 Pa
Fig. 3 Sensitivity of Farquhar model to C*. An A/Cc curve of Arabidopsis at 25 °C was fit. Rd was held constant at 0.45 lmol m-2 s-1. Vcmax = maximum velocity of carboxylations allowed by Rubisco, J = rate of photosynthetic electron transport (based on NADPH use). Arrows indicate mesophyll conductance (gm) and sum of square error terms at the following C* values: 3.64 Pa Walker et al. (2013), 4.47 Pa this study, and 4.90 Pa Flexas et al. (2007b)
Fig. 1 Measuring Ci* in Arabidopsis at 25 °C. Ci* is the internal leaf CO2 concentration at which photosynthesis is balanced by photorespiration. At Ci* an increase in light driven electron transport will increase both photosynthesis and photorespiration by the same amount. Ci* is lower than C* because the internal leaf CO2 concentration is lowered by the conductance of CO2 from the site of production into the internal leaf airspace. In order to minimize potential errors if the average of the cross over points of the four lines varied by a standard error of more than ±0.26 Pa or ±0.05 lmol m-2 s-1 one of the lines (in this case the line at a PFD of 30 lmol m-2 s-1) was not used in the determination of Ci*
conductance rose with increasing values of C* (Supplemental Fig. 1). Photosynthesis and mesophyll conductance of the arc mutants Both instantaneous photosynthesis and photosynthetic capacity of the arc mutants were greater than or similar to wild type (Fig. 4 and Supplemental Table 1). All three capacities for photosynthesis, Vcmax of Rubisco, J (electron
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Fig. 5 Estimates of mesophyll conductance (gm) from Farquhar model fitting of A/Ci curves. The model was fit using the C* for Arabidopsis measured in this study. Values are mean ± SE, n = 4. Asterisks indicate statistical difference p = 0.05
Fig. 4 Estimates of photosynthetic parameters from Farquhar model fitting of A/Ci curves. The parameters include: the maximum velocity of carboxylations allowed by Rubisco (Vcmax), the rate of photosynthetic electron transport based on NADPH use (J), and the minimum rate of triose phosphate usage (TPU). The model was fit using the C* for Arabidopsis measured in this study. Values are mean ± SE, n = 4
transport), and triose phosphate use capacity (TPU) indicated a balanced increase in photosynthetic capacity in this mutant, where the chloroplast number was approximately 15–18 per cell. In contrast, the photosynthetic capacity of the arc6 mutant was similar to the wild type (Fig. 4), despite the presence of only one large chloroplast in this mutant line. An independent experiment on arc10 mutants, also containing only one to two chloroplasts per cell, at a different location (Albuquerque versus East Lansing), showed no difference in the photosynthetic capacity of this mutant line and the wild-type Arabidopsis plants (displayed as a different experiment in Fig. 4). Mesophyll conductance was estimated by curve fitting using the same data used to determine photosynthetic capacity. Mesophyll conductance in arc 3, 6, and 10 mutants was lower than the wild type by 24–42 % (Fig. 5) even though photosynthetic capacities were similar or higher than the wild type.
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Fig. 6 Measurements of mesophyll conductance (gm) using 13CO2 measured with a TDL. Measurements were taken at ambient CO2 and O2 (400 ppm and 21 %) and high CO2 and low O2 (1500 ppm and 2 %). Values are mean ± SE, n = 4. Asterisks indicate statistical difference from wild type p = 0.05
Mesophyll conductance in the wild-type arc 3, 5, 6, and 10 mutants was also determined by 13CO2 discrimination using a TDL. In the TDL measurements, arc 3, 6, and 10 were on average 39 % lower than wild type while arc 5 was only 7 % lower at ambient CO2 levels (Fig. 6). At high CO2, the relationship between the wild-type and arc mutant mesophyll conductance was similar to what was observed at ambient CO2 but all mesophyll conductances were reduced (Fig. 6). The d13C value of plant material provides an integrated measure of discrimination. The d13 C content was less negative for arc3 and arc6 plants (Fig. 7). No statistically
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significant difference in stomatal conductance was observed (Supplemental Table 1). The data for elevated CO2 are especially clear that Ci was the same but Cc was reduced in the arc mutants. Because the arc 6 mutant had the lowest estimated and measured mesophyll conductance as well as the least negative d13C, we examined the location of the chloroplasts in the cells. Each cell in the leaf mesophyll of the arc6 mutant had one large chloroplast. The single chloroplast surrounded the periphery of each cell but did not appear to be thicker than the individual chloroplasts of the wild type (Fig. 8).
Discussion Mutants of Arabidopsis with fewer large chloroplasts have lower mesophyll conductances even though the photosynthetic capacities were the same or higher. The two methods for determining mesophyll conductance used here, curve fitting and stable isotope discrimination, indicated similar degrees of reduction in mesophyll conductance even though the two methods have very few assumptions in common. The short-term (TDL) and long-term (d13C) stable isotope methods also agreed. The arc 5 mutant has about the same number of chloroplasts, 13 per cell, as arc 3 yet the arc 5 mutant had a mesophyll conductance almost as large as wild type (Fig. 6). This demonstrates that the lower mesophyll conductance in the arc mutants was not solely determined by chloroplast number. One major difference between arc 3 and arc 5 is the shape of the chloroplasts. The arc 5 mutant has large dumbbell-shaped chloroplasts (Pyke and Leech 1994). This difference in morphology might increase the area available for diffusion for a portion of the chloroplast resulting in a lower mesophyll conductance. If true, this could help explain some of the vast diversity of chloroplast morphology among large chloroplasts found in many algae (Graham and Wilcox 2000). The position of chloroplasts in
Fig. 7 d13C content of Arabidopsis rosettes. Values are mean ± SE, n=4
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the cell is, in part, determined by the amount of light. For example, the optimum location of chloroplasts in high light is in a stacked configuration at the edges of the cell (Wada 2013). This minimizes the light interception and protects against photo damage but it is not necessarily the best position for CO2 diffusion and uptake. It has been found when blue light is used to induce the chloroplast light avoidance response, which is normally observed when plants are moved from low to high light, the surface area of chloroplasts adjacent to intercellular airspaces is reduced (Tholen et al. 2008). The dumbbell shape of the arc 5 chloroplasts could hamper the light avoidance response thus maintaining a higher mesophyll conductance. The Arabidopsis plants were grown in low light, 190 lmol m-2 s-1, but were then placed in high light, 500 lmol m-2 s-1, to measure mesophyll conductance, possibly causing chloroplast movements. It is not known what proportion of the chloroplasts in the arc mutants is adjacent to intercellular airspaces and whether chloroplast position can change with light intensity. In cases like arc6 with one large chloroplast surrounding the cell, there will be parts of the chloroplast not adjacent to air spaces (Fig. 8) and this could lead to reduced mesophyll conductance (Terashima et al. 2001). A number of effects of internal cell architecture on estimated mesophyll conductance have been discussed in recent papers (Griffiths and Helliker 2013; Scafaro et al. 2011; Tholen et al. 2012). Chloroplast movements in high light tend to decrease mesophyll conductance (Tholen et al. 2008) so if the arc plants cannot respond to light by chloroplast movement, the difference in mesophyll conductance between the arc plants and wild-type plants could be even greater at low light. The mesophyll conductance for both mutants and wild type was lower when measured at high CO2 and low O2, but arc 3, 6, and 10 mutant lines were significantly lower than wild type (Fig. 6). Changes in the physical resistance, perhaps through changes in aquaporin function (Flexas et al. 2007a; Flexas et al. 2006; Heckwolf et al. 2011) have been proposed while Evans and von Caemmerer (2013) and Tholen et al. (2012) indicate that many apparent effects can be explained the changes in fluxes associated with photorespiration (Lanigan et al. 2008). Gu and Sun (2014) propose that the apparent CO2 sensitivity of mesophyll conductance can result from methodological errors and biases. While the absolute value of mesophyll conductance may be difficult to determine with a high degree of accuracy, the relative differences between the wild-type and arc mutants in this study are likely robust since they were done at both high CO2 (1500 ppm) and low O2 (2 %) eliminating most photorespiration and many of the biases associated with D13C analysis. Photosynthetic capacities were greater than (arc3) or similar to wild type in these experiments (Fig. 4).
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Fig. 8 Chloroplast autofluorescence in WT and arc6 mutants of Arabidopsis. Chloroplasts were observed in free hand sections using autofluorescence in a confocal microscope
Therefore, biases that can result from having very different photosynthetic capacities are unlikely to explain the differences in mesophyll conductance between the wild type and arc mutants. Austin and Webber (2005) have reported reduced photosynthesis in the arc 3, 5 and 6 mutants; it is not clear why our results are different from theirs. The relationship observed between lower mesophyll conductance and lower photosynthetic capacity (for example Li et al., (2013) and von Caemmerer and Evans (1991), was not observed in the arc mutants. The value of C* used in the Farquhar model had a large effect on the estimated mesophyll conductance, especially if the C* value was high (Fig. 2). The values of C* for Arabidopsis found in this study are higher than those of Walker et al. 2013 but lower than Flexas et al. (2007b) (Fig. 3). Ci* was determined by the Laisk (1977) method in all these studies. Because the estimate of C* from Ci* depends on mesophyll conductance, iteration of the estimates for both can be carried out or an independent method of measuring mesophyll conductance used. C* was determined using a mesophyll conductance determined by 13CO2 discrimination in the Walker et al. (2013) study while it was determined according to Warren and Dreyer (2006) in the Flexas et al. (2007b) study. Here, we used an iterative approach, estimating mesophyll conductance from some of the same data used to determine Ci*. Gu and Sun (2014) pointed out some of the biases that can be introduced when using the ‘‘Laisk C*’’ which is Ci*, therefore the C* we used was as described by Ethier and Livingston (2004) which accounts for mesophyll conductance. Despite the precautions outlined in the materials and methods section in our use of the Laisk method, the reasons for the differences in values found for C* in Arabidopsis are unclear. The value of C* as a measure of the Rubisco compensation point depends on several assumptions including a constant Rd and a release of one CO2 for every two oxygenations. We suspect that
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these assumptions may not always hold at the lowest light intensity. The estimate of mesophyll conductance is very sensitive to Rd. Allowing Rd to vary resulted in an estimate of mesophyll conductance that was higher, and Vcmax and J that were lower at a C* of 4.5 Pa (Fig. 2 and Supplemental Fig. 1). The outputs of the model were more consistent when Rd was held constant. Day respiration is not trivial to measure and thus few measurements have been made. Previous studies have found no consistent relationship between Rd and temperature. Rd has been reported to increase exponentially with temperature in tobacco (Bernacchi et al. 2001). It has been found to increase linearly with temperature in Heteromeles and Lepechinia (Villar et al. 1995). Rd has also been shown to be relatively constant between 15 and 25 °C even decreasing at temperatures above 25 °C in Eucalyptus pauciflora (Atkin et al. 2000). In this study with Arabidopsis, Rd was fairly constant with temperatures between 15 and 36 °C. In summary, chloroplast number and shape have a significant impact on mesophyll conductance with more but smaller chloroplasts conferring a higher mesophyll conductance than fewer but larger chloroplasts. The reduced number of chloroplasts is not the sole determinant of lowered mesophyll conductance; shape plays a large role as well. Fitting of the Farquhar model of leaf photosynthesis to A/Ci data can provide an effective estimate for mesophyll conductance that is in close agreement with 13CO2 discrimination based methods. Use of model fitting requires that the model is parameterized with the correct C* and Rd. Acknowledgments We thank Dr. Katherine Osteryoung for providing the arc mutant seeds and for thoughtful discussions throughout the project. This work was supported by DOE grant DE-SC0008509 to TDS and NSF grant IOS-0719118 and NIH grant NIH-NCRR P20RR18754 to DTH. Partial salary support for TDS comes from Michigan State University AgBioResearch.
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