Tree age-dependent changes in photosynthetic and respiratory CO2 exchange in leaves of micropropagated diploid, triploid and hybrid aspen

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Tree Physiology 34, 585–594doi:10.1093/treephys/tpu043

Tree age-dependent changes in photosynthetic and respiratory CO2 exchange in leaves of micropropagated diploid, triploid and hybrid aspen

Tiit Pärnik1, Hiie Ivanova, Olav Keerberg, Rael Vardja and Ülo Niinemets

Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, 51014 Tartu, Estonia; 1Corresponding author ([email protected])

Received October 12, 2013; accepted April 27, 2014; published online June 3, 2014; handling Editor Maurizio Mencuccini

The growth rate of triploid European aspen (Populus tremula L.) and hybrid aspen (P. tremula × Populus tremuloides Michx.) significantly exceeds that of diploid aspen, but the underlying physiological controls of the superior growth rates of these genotypes are not known. We tested the hypothesis that the superior growth rate of triploid and hybrid aspen reflects their greater net photosynthesis rate. Micropropagated clonal plants varying in age from 2.5 to 19 months were used to investi-gate the ploidy and plant age interaction. The quantum yield of net CO2 fixation (Φ) in leaves of young 2.5-month-old hybrid aspen was lower than that of diploid and triploid trees. However, Φ in 19-month-old hybrid aspen was equal to that in trip-loid aspen and higher than that in diploid aspen. Φ and the rate of light-saturated net photosynthesis (ANS) increased with plant age, largely due to higher leaf dry mass per unit area in older plants. ANS in leaves of 19-month-old trees was highest in hybrid, medium in triploid and lowest in diploid aspen. Light-saturated photosynthesis had a broad temperature optimum between 20 and 35 °C. Rate of respiration in the dark (RDS) did not vary among the genotypes in 2.5-month-old plants, and the shape of the temperature response was also similar. RDS increased with plant age, but RDS was still not significantly dif-ferent among the leaves of 19-month-old diploid and triploid aspen, but it was significantly lower in leaves of 19-month-old hybrid plants. The initial differences in the growth of plants with different ploidy were minor up to the age of 19 months, but during the next 2 years, the growth rate of hybrid aspen exceeded that of triploid plants by 2.7 times and of diploid plants by five times, in line with differences in ANS of 19-month-old plants of these species. It is suggested that differences in pho-tosynthesis and growth became more pronounced with tree aging, indicating that ontogeny plays a key role in the expression of superior traits determining the productivity of given genotypes.

Keywords: dry mass per leaf area, irradiance, leaf structure, net CO2 assimilation, ploidy level, respiration in the dark, temperature dependencies.

Introduction

Aspen is one of the most widely distributed trees in the tem-perate northern hemisphere forests. Thus, understanding the responses of different aspen varieties to changes in the envi-ronment provides highly valuable information for selecting and engineering aspen plants with the desired growth and economic properties. With this aim, the growth parameters of diploid, triploid (Populus tremula L.) and hybrid (P. tremula × Populus

tremuloides Michx.) aspen have been investigated, leading to demonstration of superior growth potentials of triploid and hybrid aspen over diploid aspen (Benson and Einspahr 1967, Yu et al. 2001, Tullus et al. 2012). The growth and development of plants are controlled by many factors. Among these, pho-tosynthesis is the main process supplying growth processes with energy and metabolic substrates, but photosynthetic dif-ferences among aspen genotypes with different ploidy level have not been investigated. From studies in other species, there

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is evidence that photosynthetic rate per unit leaf area does vary with plant ploidy level (Li and Zhang 2000). The differences among genotypes with different ploidy level have been shown to depend on anatomical properties of leaves such as number of cells per unit leaf area (Warner and Edwards 1993), stomatal density (Austin et al. 1982) and mesophyll surface area exposed to intercellular air spaces (Terashima et al. 2011). In addition, increases in the investment of nitrogen in rate-limiting proteins such as Rubisco (Pyke and Leech 1987, Vyas et al. 2007) and components of photosynthetic electron transport rate (Li et al. 2011, Hussain et al. 2012) have been observed in genotypes with higher ploidy level. In addition, hybrid genotypes have also been observed to possess greater photosynthetic rates, inter-preted as being indicative of a heterosis effect (Bhatt and Rao 1981, Matyssek and Schulze 1987a, 1987b, Song et al. 2010), resulting from an enhanced expression of genes coding pro-teins involved in CO2 fixation (Song et al. 2010).

Most photosynthetic investigations of trees have been performed with young plants, often with first-year seedlings (see Walters and Reich 1999 for a review). However, there are important age-dependent changes in biomass allocation (Poorter et al. 2011), leaf structure and photosynthetic capac-ity (Yoder et al. 1994, Niinemets 2002, Woodruff et al. 2007, 2009, Mullin et al. 2009, Steppe et al. 2011). To our knowl-edge, the role of age-dependent changes in photosynthetic potential among genotypes of different ploidy has not been analyzed.

The existing data on the causal relationships between growth and photosynthesis are contradictory, with growth being linked to photosynthesis in some cases, but not always (Austin et al. 1982, Sicher et al. 1984, Wachira and Ng’etich 1999, Yu 2001). The growth rates of 4-year-old and older genetically different aspen clones were not correlated with their physiological traits but with morphological and pheno-logical properties such as number of leaves on a tree, leaf area per tree, length of growth period, etc. (Yu 2001, Müller et al. 2013). Lack of a general correlation between the rates of growth and photosynthesis might imply that photosynthe-sis can limit growth only under certain environmental condi-tions or phases of development (Ceulemans et al. 1987). Differences in photosynthetic capacity may be the initial cause for the unequal size of plants. Therefore, investigating the envi-ronmental responses of different aspen genotypes with inher-ently varying growth potential can provide valuable insight into the role of photosynthetic variation as a possible determinant of genotypic differences in growth rate. Such understanding would be highly useful for engineering plants with the desired growth and economic properties. In this work, we investigate the light and temperature responses of gas-exchange charac-teristics, photosynthesis and respiration, in leaves of diploid and triploid aspen (P. tremula) and of diploid hybrid aspen (P. tremula × P. tremuloides) at different phases of development.

The results obtained were compared with the growth proper-ties of the studied genotypes to test the hypothesis that the superior growth rate of triploid and hybrid aspen reflects their greater net photosynthesis rate.

Materials and methods

Plant material

Carbon dioxide exchange was measured in leaves of micro-propagated diploid and triploid aspen (P. tremula) clones of local origin (Järvselja) and of the diploid hybrid aspen (P. tremula × P. tremuloides) clone from Lithuanian Institute of Forest Research (hybrid tree No. 39-045). Plants of different ploidy were prepared according to the standardized proto-col for micropropagation of triploid aspen (Vardja and Vardja 2001, Vardja et al. 2004). The process of micropropagation comprises five stages: (i) forcing of shoots, (ii) start, (iii) mul-tiplication, (iv) rooting and (v) acclimation (Vardja et al. 2004). As the micropropagated plants were initially very sensitive to low humidity and high light, the acclimation period is needed to induce stomatal control of transpiration and increase plant resistance to high light.

Before the end of rooting and beginning of acclimation, the transplants were illuminated for 2 weeks with fluorescent tubes BS-30 (Saransk Factory, Saransk, Russia) at a photosynthetic quantum flux density of 100 µmol m−2 s−1. After this illumina-tion period, the acclimation stage was completed by illuminat-ing plants for 1.5 months at 150 µmol m−2 s−1. These conditions were maintained until the plants were 2.5 months old, at which time the first measurements were carried out. During the accli-mation period, plants were grown in pots (diameter 15 cm) in perlite and fertilized with 1/10 diluted and acidified (pH 4.0) Murashige–Skoog basal medium (Murashige and Skoog 1962).

The plants were further grown for 2 months under fluores-cent tubes at 300 µmol m−2 s−1 light for a 16-h photoperiod and at 25 °C during day and 20 °C during night. Thereafter, the potted plants were transferred outside where they were grown for 2 months in pots under partial shade avoiding mid-day full sunlight. The trees were further transferred to full sun-light for 2 weeks, which was a sufficiently long time-period for full-light acclimation. Thus, 6.5-month-old plants were used for the second set of measurements. Finally, the oldest plants used for CO2 exchange measurements were grown for the last 13 months in the field in organic matter-rich garden soil under full sunlight (lat. 58°N, long. 24°E). The growth irradiances used for different-aged plants were the highest under which all plants survived without damage.

Gas-exchange measurements

Leaf CO2 exchange rates were measured using a multi-channel open gas-exchange system with a thermostatted leaf cham-ber described in detail by Pärnik et al. (1987) and Pärnik and

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Keerberg (2007). Carbon dioxide concentrations in ingoing and outgoing air were measured with a LI-6262 CO2/H2O analyzer (Li-Cor, Inc., Lincoln, NE, USA), and water vapor concentrations were measured with micropsychrometers calibrated against dry air. Different CO2 and O2 concentrations were prepared using a custom-made gas mixer based on manostats, and leaf temperature (TL) was calculated from the leaf energy budget (see Laisk and Oja 1998 for details). Light was provided by xenon lamps DKsEL 1000-5 (Riga, Latvia) and could be varied from 10 to 1300 µmol m−2 s−1 by neutral filters of anodized copper mesh. Light intensity was measured using a LI-190SA quantum sensor (Li-Cor, Inc.). From the CO2 exchange mea-surements, net photosynthesis rate, rate of respiration in the dark, transpiration rate and stomatal conductance were deter-mined according to Laisk and Oja (1998).

Prior to gas-exchange measurements, leaf reflectance (R) and transmittance (T) were recorded by means of a spec-trophotometer with an integrating sphere SF-10 (Lomo, St Petersburg, Russia) in the spectral region of 400–700 nm. Leaf absorbance (A) was calculated as

A R T= − +( )1 0. .

(1)

From these measurements, the absorbed quantum flux den-sity, Qabs (µmol m−2 s−1), was calculated as

Q Q Aabs i= × , (2)

where Qi is incident photosynthetic photon flux density.In 2.5-month-old potted plants, measurements were con-

ducted with attached leaves. Due to the special architecture of the leaf chamber optimized for use in radiogasometric mea-surements (Pärnik and Keerberg 2007, Timm et al. 2008, Keerberg et al. 2011), the leaf had to be cut into a rectangle (2.7 × 7.4 cm) before enclosure in the leaf chamber. As no basal major veins were damaged by this procedure, the over-all effect of leaf excision was small, <5% reduction in gas-exchange rates, in agreement with past studies (Sack et al. 2003). In 6.5- and 19-month-old plants, detached leaves har-vested in the field were used. The petiole of the selected leaf was excised under water and placed in a small plastic bag with water. After harvesting, the leaf was immediately trans-ferred to the laboratory and enclosed in the leaf chamber. After leaf enclosure, the leaf was stabilized under an ambient CO2 concentration of 400 µmol mol−1 and an O2 concentra-tion of 210 mmol mol−1 until the stomata opened and leaf gas-exchange rates reached a steady state, typically in 20–30 min after leaf enclosure. Light response curves were measured at temperatures of 20 and 32 °C. Before the measurement of temperature response curves, leaf temperature was stabilized at 25 °C. After stomatal opening, the light and temperature response curves of light-saturated net photosynthesis (ANS)

were measured. For the light response curves, ANS was first measured in the ascending sequence of Qabs of 0, 10, 25, 65 and 164 µmol m−2 s−1. Values of photosynthesis (see Figure 2c) were measured at saturating light intensities (Qabs of 400 and 1000 µmol m−2 s−1). At each light level, the measurements were conducted after the steady-state net assimilation rates were achieved.

Measurements of temperature response of ANS were car-ried out at a Qi of 1200 µmol m−2 s−1 (corresponding approxi-mately to a Qabs of 980 µmol m−2 s−1). At each temperature, measurements were recorded 20–30 min after changing the temperature, when the photosynthesis rate had reached the steady state.

Estimation of quantum yield of CO2 assimilation

The initial part of the light response curve of photosynthesis was used to determine the quantum yield of net photosynthesis (Φ, Figure 1). The curves exhibited a decrease of the slope, the Kok effect, with breaking point at a Qabs of ~25 µmol m−2 s−1 (Sharp et al. 1984). The steeper slope of the initial linear part of the curve has been explained by the suppression of a component of dark respiration by light (Sharp et al. 1984). Therefore, to eliminate the possible effects of respiration, Φ was calculated as the slope of the light response curve in the region of Qabs from 25 to 65 µmol m−2 s−1 according to the fol-lowing equation:

Φ = d dNS absA Q/ . (3)

Photosynthesis in aspen of different ploidy 587

Figure 1. Illustration of determination of the quantum yield of CO2 fixation (Φ) from the initial part of the net assimilation rate (ANS) vs absorbed light (Qabs) response curve in leaves of 2.5-month-old micro-propagated diploid aspen (P. tremula) at leaf temperatures of 19.5 and 33.5 °C, and at an ambient CO2 concentration of 400 µmol mol−1 and an O2 concentration of 210 mmol mol−1. Φ was calculated as the slope of ANS vs Qabs for the Qabs range of 25–65 µmol m−2 s−1. Lines demonstrate the Kok effect with breaking point at about Qabs = 25 µmol m−2 s−1.

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Foliage chemical and structural characteristics and growth measurements

Chlorophyll concentration was determined by the method of Arnon (1949). For determination of leaf fresh (MAF) and dry mass (MAD) per unit leaf area, five disks of 3.6 cm2 were cut from different leaves. Fresh mass was estimated immediately after cutting the disks and dry mass was estimated after oven drying at 70 °C to a constant mass.

The height of each plant used for measurements was deter-mined to assess the height growth rate. The height growth of the studied genotypes was continuously monitored until the plants were 43 months old.

Results

Size and growth of micropropagated plants

Micropropagated plants used for gas-exchange measure-ments had nearly the same average (±SD) height irrespec-tive of their ploidy: 25.0 ± 3.0 cm for 2.5-month-old plants, 60 ± 5 cm for 6.5-month-old plants and 120 ± 10 cm for 19-month-old plants. The height of genotypes was not sta-tistically different within a given age according to analysis of variance (ANOVA) (P = 0.92, F = 0.5 for 2.5-month-old, P = 0.86, F = 0.5 for 6.5-month-old and P = 0.87, F = 0.14 for 19-month-old plants).

However, during the next 2 years, the annual growth of hybrid aspen exceeded that of diploid aspen by five times and that of triploid aspen by 2.7 times. Forty-three-month-old trees planted outside in the soil had significant differ-ences in height, with the hybrid aspen being the tallest (355 ± 20 cm), followed by triploid (207 ± 11 cm) and dip-loid (165 ± 10 cm) aspen (all averages are statistically dif-ferent at P < 0.001, F = 220).

Foliage structure and chlorophyll content of micropropagated plants

The MAF and MAD per unit area were measured in micropropa-gated trees of different ploidy for almost 4 years. The leaf dry-to-fresh mass ratio was independent of plant age (Table 1). However, MAD increased with increasing plant age during the study period (Table 1). Leaf structural characteristics of plants of different ploidy did not differ significantly within the given age (Table 1).

Chlorophyll content was measured in leaves of 19-month-old trees. A higher chlorophyll content in leaves of 19-month-old hybrid and triploid aspen than in diploid aspen (Table 2) is indicative of greater light-harvesting capacity, which implies that the quantum yields for incident light are greater in the hybrid and triploid aspen. This can be an important trait for the growth in dense plantations such as often used in short-rotation forestry (Isebrands et al. 1977, Stettler et al. 1988, Perttu 1989).

Light responses of net photosynthesis in aspen plants of varying age

Our experiments started with measurements of light curves of net photosynthesis in leaves of young 2.5-month-old plants. To avoid the damage of leaves by light, the maxi-mum irradiance during the measurements did not exceed the growth light intensity. At low irradiances, up to a Qabs of 65 µmol m−2 s−1, net photosynthesis and Φ of diploid and triploid leaves were similar (Figure 2a, Table 2), but ANS and Φ in young hybrid aspen were lower than those in diploid and triploid aspen (Figure 2a, Table 2). Values of Φ in leaves of hybrid aspen remained at the same low level during the subsequent 4 months of growth (compare the values of Φ for 2.5- and 6.5-month-old hybrid aspen in Table 2). However,

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Table 1. Average (±SE) leaf fresh (MAF) and dry mass (MAD) per unit area and leaf dry-to-fresh mass ratio in micropropagated diploid and triploid aspen (P. tremula) and hybrid aspen (P. tremula × P. tremuloides) seedlings of different age. Different letters indicate statistically significant differ-ences among plants with different age according to ANOVA (P < 0.001, F = 10.9, n = 5).

6.5-month-old 19-month-old 30-month-old 43-month-old

DiploidMAF (g m−2) 142 ± 10 156 ± 11 66 ± 5b 163 ± 11MAD (g m−2) 53.0 ± 4.0a 66 ± 5b 69 ± 5b

MAD/MAF 0.37 ± 0.040 0.42 ± 0.06 0.42 ± 0.06TriploidMAF (g m−2) 158 ± 11 214 ± 13 70 ± 5b 172 ± 12MAD (g m−2) 49.0 ± 4.0a 83 ± 8b 67 ± 5b

MAD/MAF 0.31 ± 0.05 0.39 ± 0.06 0.39 ± 0.06HybridMAF (g m−2) 159 ± 11 164 ± 12 78 ± 6b 182 ± 13MAD (g m−2) 54.0 ± 4.0a 70 ± 5b 80 ± 6b

MAD/MAF 0.34 ± 0.05 0.43 ± 0.06 0.44 ± 0.06

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at high Qabs, the ANS of 6.5-month-old hybrid aspen was sig-nificantly higher than that in triploid aspen of the same age (Figure 2b, ANS in Table 3).

At saturating irradiance, the rate of photosynthesis per unit leaf area (ANS) increased with leaf age (Figure 2, Table 3). The age-dependent rise in ANS was associated with changes in leaf structure leading to an increase in dry mass per unit leaf area (Table 1, see Figure 5; see also Tosens et al. 2012). Irrespective of structural changes, the rates of light-saturated photosynthesis, calculated both per unit leaf area (ANS) and dry mass (ANM), were significantly higher in leaves of 6.5- and 19-month-old hybrid aspen than those in diploid and triploid aspen (Figure 2b and c, Table 3). This genotypic difference was correlated with differences in stomatal conductance, being 96 mmol m−2 s−1 in hybrid aspen, 65 mmol m−2 s−1 in triploid aspen and 60 mmol m−2 s−1 in diploid aspen. In hybrid aspen, quantum yield increased from 0.037 to 0.054 mol mol−1, and this value was close to that in triploid aspen, while the quantum yield in diploid aspen was lower and did not change during growth (Table 2).

Temperature responses of foliage gas-exchange rates in the light and dark in different-aged aspen plants

The quantum yield of net photosynthesis of all plants was significantly lower under high (33–37.5 °C) compared with moderate (18.5–20.5 °C) temperatures (Table 2). This decrease can reflect enhanced photorespiration and partial inactivation of Photosystem II at higher tempera-tures (Laisk et al. 1998). In accordance with this, Ku and Edwards (1978) found that under 210 mmol mol−1 O2 the quantum yield of Triticum aestivum L. decreased at higher temperatures from 0.062 at 15 °C to 0.046 (mol mol−1) at 35 °C, but at 15 mmol mol−1 O2 the quantum yield did not change with temperature. This is consistent with our

measurements carried out under non-photorespiratory con-ditions (15 mmol mol−1 O2) at which only a slight decrease of quantum yield in leaves of 6.5-month-old hybrid aspen at higher temperature was detected (Table 2).

The photosynthesis rate of young, 2.5-month-old diploid and triploid aspen leaves at 151 µmol m−2 s−1 was stable over the temperature range of 20–25 °C (Figure 3a). At temperatures >25 °C, ANS in diploid and triploid aspen gradually declined. At 40 °C, photosynthesis rates were 25–45% lower than at 25 °C (Figure 3a). The ANS of 6.5-month-old triploid aspen under 415 µmol m−2 s−1 at 25 °C was 1.5 times higher than in the younger triploid aspen (Figure 3a). In 6.5-month-old plants, the decrease of photosynthesis started at 30 °C and the decrease was steeper than in 2.5-month-old plants. At 48 °C, the rate of photosynthesis was almost three times lower than at 20 °C (Figure 3a). Rates of light-saturated CO2 assimilation of 19-month-old plants at 982 µmol m−2 s−1, irrespective of their ploidy, exhibited a broad temperature optimum ranging from 20 to 35 °C (Figure 3b). This confirms previous observations demonstrating a broad temperature optimum of light-saturated photosynthetic electron transport and net assimilation rates in aspen at current ambient CO2 concentrations (Hüve et al. 2006, Lin et al. 2012). At temperatures >35 °C, photosynthe-sis was severely suppressed, reflecting temperature-dependent increases in photorespiration and dark respiration. Diploid trees were characterized by stronger sensitivity to higher tempera-tures (Figure 3b).

In younger plants, the drop in photosynthesis was initiated at lower temperatures (Figure 3a), indicating greater heat sensi-tivity of their photosynthetic apparatus. Such higher sensitivity can be an important factor limiting the establishment of tissue-cultured plants in the field.

The response of dark respiration (RDS) to changes in tem-perature did not differ among 2.5-month-old diploid and

Photosynthesis in aspen of different ploidy 589

Table 2. Average (±SE) initial quantum yield of net CO2 fixation (Φ, mol mol−1) at different temperatures in leaves of different age, and leaf chlo-rophyll content at the end of the experiment in micropropagated diploid, triploid and hybrid aspen plants. The initial quantum yield, Φ, is given by Eq. (1). Measurements were conducted over an absorbed photosynthetic photon flux density (Qabs) range of 25–65 µmol m−2 s−1 at an ambient CO2 concentration of 400 µmol mol−1 and an O2 concentration of 210 or 15 mmol mol−1 (hybrid aspen denoted by an asterisk). Different lowercase letters indicate statistically significant differences among ages (ANOVA, P < 0.01, F = 16.8, n = 5), different uppercase letters among different tem-peratures (ANOVA, P < 0.001, F = 17.1, n = 5) and different numbers among different genotypes (ANOVA, P < 0.01, F = 10.1, n = 5).

Species 2.5-month-old 6.5-month-old 19-month-old

Leaf temperature (°C)

Φ Leaf temperature (°C)

Φ Leaf temperature (°C)

Φ Chlorophyll content (g m−2)

Diploid 19.5 0.0460 ± 0.0040aC1 19.0 0.047 ± 0.005aC1 0.340 ± 0.0301

33.5 0.0340 ± 0.0030aD 33.5 0.0280 ± 0.0030aD

Triploid 19.1 0.051 ± 0.005aC1 19.2 0.062 ± 0.007bC2 0.450 ± 0.0402

37.5 0.0320 ± 0.0040aD 36.7 0.0400 ± 0.0040bD

Hybrid 19.6 0.0380 ± 0.0040aC2 20.5 0.0370 ± 0.0030aC 18.5 0.054 ± 0.006bC2 0.49 ± 0.052

33.6 0.0290 ± 0.0030aD 33.3 0.0300 ± 0.0030aD 33.0 0.0330 ± 0.0040aD

Hybrid* 21.0 0.0480 ± 0.003032.0 0.0430 ± 0.0030

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triploid plants over the temperature range of 20–38 °C, and only a moderately greater RDS was observed at higher tem-peratures in diploids (Figure 4a). The temperature dependen-cies of RDS were similar in leaves of 19-month-old diploid and triploid aspen, but at a given temperature, RDS in hybrid aspen was lower than that in diploids and triploids (Figure 4b).

Photosynthetic capacity per leaf area and leaf dry mass dependent on genotype and plant age

The rates of net photosynthesis per unit leaf area (ANS) in leaves of 19-month-old triploid and hybrid aspen were significantly higher than ANS in leaves of 6.5-month-old plants (Table 3). Dry mass per unit leaf area (MAD) was also higher in leaves of 19-month than 6.5-month-old plants (Table 1), suggest-ing that age-related increases in ANS might have resulted from increases in MAD. Indeed, when the rates of net photosynthesis were calculated per unit leaf dry mass (ANM), no significant differences were found between 6.5- and 19-month-old trip-loid and hybrid aspen plants (Table 3). The values of ANS and RDS showed an increase in leaves with higher MAD, while the mass-based characteristics, ANM and RDM, were independent of MAD (Figure 5). These results suggest that differences in gas-exchange rates calculated per unit leaf area observed in plants with different ploidy and age were largely determined by varia-tions in MAD. Nevertheless, in hybrid aspen plants, the rates of net photosynthesis calculated on the dry mass basis (ANM) were significantly higher than in diploid and triploid plants of the same age (Table 3).

Discussion

Photosynthetic characteristics of polyploid vs diploid genotypes

In the search for possibilities to improve the photosynthetic and growth properties of plants, plant varieties with different ploidy and degree of hybridization have often been incorpo-rated in screening studies. In many cases, the increase in ploidy indeed results in a higher rate of leaf photosynthesis (Warner and Edwards 1993, Vyas et al. 2007). However, in some cases, no effect of ploidy dose or the opposite has been observed. For example, photosynthesis per unit leaf area in diploid wheat species was higher than that in tet-raploid or in hexaploid (T. aestivum) (Austin et al. 1982) species, and the rates of photosynthesis in leaves of diploid and autotetraploid barley (Hordeum vulgare L.) were not sig-nificantly different (Sicher et al. 1984). Growth traits among genotypes with different ploidy have also been observed to vary. Diploid genotypes of tea (Camellia sinensis L.) accu-mulated significantly more biomass than polyploids (Wachira and Ng’etich 1999). Analogously, heterosis effects following hybridization have been found to enhance photosynthesis and growth in several cases (Bhatt and Rao 1981, Matyssek and Schulze 1987a, 1987b, Song et al. 2010). Previous stud-ies have indicated that the height, diameter and growth rate of hybrid (P. tremula × P. tremuloides) aspen trees are signifi-cantly higher than those of triploid and diploid non-hybridized aspen trees (Benson and Einspahr 1967, Yu et al. 2001, Tullus et al. 2012).

590 Pärnik et al.

Figure 2. Representative light dependencies of net CO2 assimila-tion rate (ANS) in leaves of 2.5-month-old diploid, triploid and hybrid aspen (a), 6.5-month-old triploid and hybrid aspen (b) and 19-month-old diploid, triploid and hybrid aspen (c) under an ambi-ent CO2 concentration of 400 µmol mol−1, an O2 concentration of 210 mmol mol−1 and a leaf temperature of 20 ± 1 °C. Curve param-eters and statistical analysis for all studied leaves are provided in Tables 2 and 3.

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The focus of past studies on age-dependent changes in photosynthesis has been mainly on photosynthetic capac-ity and stomatal conductance (Yoder et al. 1994, Niinemets 2002, Woodruff et al. 2007, 2009, Mullin et al. 2009, Steppe et al. 2011). These studies have demonstrated increased leaf dry mass per unit area (Niinemets and Kull 1995, Niinemets et al. 1999, Niinemets 2002, Woodruff et al. 2007, 2009)

and reduced stomatal conductance (Yoder et al. 1994) with increasing tree age and size.

Interactive effects of plant age and ploidy level on photosynthetic characteristics

In fact, ANS may even increase with increasing tree age due to the dominating effect of MAD as was observed in our study (Table 3, Figure 5). Analogous positive effects of increases in MAD and leaf thickness with plant age on ANS have been observed in several other deciduous tree species (Malkina

Photosynthesis in aspen of different ploidy 591

Figure 4. Temperature dependencies of the rate of respiration in the dark (RDS) in leaves of micropropagated 2.5-month-old diploid and triploid aspen (a), and of 19-month-old diploid, triploid and hybrid aspen (b). Measurements were performed at an ambient [CO2] of 400 µmol mol−1 and an [O2] of 210 mmol mol−1. Error bars denote ±SE (n = 5).

Table 3. Average (±SE) rates of light-saturated net CO2 fixation in leaves of micropropagated diploid, triploid and hybrid aspen of different age expressed per unit leaf area (ANS, µmol m−2 s−1) and leaf dry mass (ANM, µmol g−1 s−1). Measurements were performed at saturated photon flux densities (Qi) of 400–1000 µmol m−2 s−1 and leaf temperatures between 20 and 30 °C (on average 25 °C). Different lowercase letters indicate statistically significant differences among ages (ANOVA, P < 0.002, F = 17.1, n = 5) and different uppercase letters among genotypes (ANOVA, P < 0.001, F = 78, n = 5).

Species 6.5-month–old 19-month-old

ANS ANM ANS ANM

Diploid 8.08 ± 0.14C 0.122 ± 0.005C

Triploid 6.56 ± 0.23aD 0.134 ± 0.005aD 9.44 ± 0.12bD 0.114 ± 0.005aC

Hybrid 8.7 ± 0.6aE 0.161 ± 0.010aE 10.47 ± 0.05bE 0.150 ± 0.005aE

Figure 3. Temperature dependencies of net assimilation rate (ANS) in leaves of micropropagated 2.5-month-old diploid and triploid aspen at a Qabs of 150 µmol m−2 s−1 (a, open symbols), of 6.5-month-old triploid aspen at a Qabs of 415 µmol m−2 s−1 (a, closed symbols) and of 19-month-old diploid, triploid and hybrid aspen at a Qabs of 980 µmol m−2 s−1 (b). Measurements were performed at an ambient [CO2] of 400 µmol mol−1 and an [O2] of 210 mmol mol−1. Error bars denote ±SE (n = 5).

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1983, Niinemets et al. 1999), and underscore the importance of age-dependent changes in mesophyll differentiation in foli-age photosynthetic capacity.

No differences in the growth rate of aspen genotypes were detected in young plants, up to the age of 19 months. Differences in the rate and quantum yield of net photosynthesis observed during this period (Figure 2c, Table 2) apparently did not affect the growth of plants. Thus, in early phases of plant development, the significance of variations in photosynthetic characteristics among genotypes was minor as plants also used the carbon derived from the culture medium. However in later phases of development, from 19 to 43 months when growth was fully autotrophic, the growth rate of hybrid aspen signifi-cantly exceeded that of triploid and diploid aspen. The absolute growth rates observed in our study are comparable to those of 3- and 4-year-old hybrid, triploid and diploid aspen in south Finland under comparable climatic conditions (Yu et al. 2001). Photosynthesis (ANS) under saturating irradiance and optimal temperatures of 19-month-old plants was highest in leaves of hybrid, medium in triploid and lowest in diploid aspen (Figure 2c, Table 3). In addition, the higher growth rate of hybrid aspen was also associated with lower respiratory losses in the dark at any

temperature measured (Figure 4b). No significant differences were detected in the photosynthetic rates of individual leaves of 5-year-old trees of hybrid, triploid and diploid aspen (data not shown; see also Yu 2001). At this phase of development, trees of different ploidy were of unequal size with strongly varying whole-plant leaf areas and different degrees of within-canopy shading. Increases in canopy shading can even out the inher-ent differences in leaf photosynthetic potentials observed in full sunlight (e.g., see Niinemets 2007 for a review). These results suggest that differences in the rate of photosynthetic CO2 assim-ilation in individual leaves become evident at certain phases of development when they importantly contribute to plant survival and formation of carbon reserves for subsequent growth.

Conclusions

This study demonstrates that the rates of net photosynthesis and dark respiration of aspen depended on hybridization, ploidy level and age of trees. The rate of net photosynthesis in leaves of 19-month-old trees at saturating irradiances and optimal tem-peratures was highest in hybrid, medium in triploid and lowest in diploid aspen, and this ranking was associated with the growth rate of studied genotypes. During the next 2 years, the growth rate of hybrid aspen exceeded that of triploid and diploid species by five and three times, respectively, confirming the superior pho-tosynthetic potential in this genotype. Overall, the differences in photosynthesis and growth became more pronounced with tree aging, indicating that ontogeny plays a key role in the expression of superior traits determining the productivity of given genotypes.

Conflict of interest

None declared.

Funding

This study was financially supported by the Estonian Ministry of Education and Science (institutional grant IUT-8-3), the Estonian Science Foundation (grants 5989 and 8927) and the European Commission through European Regional Fund (the Center of Excellence in Environmental Adaptation).

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