1.                  Introduction

Environmental changes caused by increased emissions of greenhouse gasses have influenced the stability of ecosystems worldwide[1]. The increase of atmospheric carbon dioxide (CO₂) is one of the most important environmental changes in the world in the past and the future. Especially, human activities also increased the concentration of CO₂ in the atmosphere, which is expected to reach 700 μL/L by the middle of the next century [2-5]. Current evidence showed the increase of CO₂ concentration will influence on plant growth, development biological yield. In general, high CO₂ concentrations increase photosynthetic rates, which promoted the growth

of plants. 

However, there are most reports on the response of crop plant species to elevatedatmospheric CO₂, but few of them have given account of the response of woody species to high CO₂ levels. In this study, we compared the effect of elevated CO₂ on the growth and photosynthetic characteristics of four Populus (PopulusL.) species.

Populus spp., as poplar hybrids, belongs to a fastest-growing tree. For their rapid growth, their wood can be used for construction, pulp, paper, and as a renewable, cost-effective alternative to fossil fuels. Previous reports showed that poplars are sensitive to environmental stress compared with other tree specise[6,7]. To date, the effects of high CO₂ levels on Populusphysiology and growth have been studied in numerous researches. For example, in birch, elevated CO₂ treatments had no major impacts on wood anatomy or wood density [8]. Lee et al., (2014) found that the cuttings of Populus alba × glandulosaunder the elevated CO₂treatment showed reduced tree height and photosynthetic pigment contents such as chlorophyll and carotenoid. In particular, the elevated treatment resulted in a marked reduction in the chlorophyll a. However, some works also showed that increased CO₂ delayed leaf fall, but this effect is species-specific. However, there are few tests of whether differences in photosynthetic abilityexist between different Populusspecies in response to elevated CO₂. Generally, the photosynthetic ability was reported to a reliable indirect indicator of tree performances under different environmental stress [9,10].

As a rapid and non-destructive technique, this technology is very useful to investigate the photosynthetic function, and has been used to study the effects of drought, high temperature, salt stress on plant species. Additionally, this technology has also provided valuable information on the functional and structural attributes of components involved in photosynthetic electron transport and especially to study photosystem II (PSII) behavior[11- 21]. In general, a sequence of phases (labelled as O, K, J, I, P) can be obtained in the fluorescence rise during the first second of illumination from the initial (F_o) to the maximal (F_m) fluorescence value. This mathematical model was named as JIP-test, which can provide quantum yields, biophysical parameters and probabilities characterizing structure and function of PSII [22,23]. PSⅡ is very sensitive to environmental stresses [9,10]. However, a complex study comparing influences of high CO₂ levels on PSII behaviors of woody species is still lack. Therefore, a detailed comparison of high CO₂-induced changes in PSII photochemistry in different Populus spp. species was carried out by using the fast Chl fluorescence records.

In our study, we have examined photosynthetic responses of fourPopulus spp. species to high CO₂ levels using fast Chlorophyll (Chl) a fluorescence transient (O-J-I-P) technology. In this study, we analyzed the transient fluorescence using JIP-test in four Populusspecies (PopulusX, Populusdeltoides ×cathayana, Poplus alba ′Berdinensis ′ L,Populuseuramericana′N3016′ ×Populusussuriensis) under atmospheric CO₂ (1500 ± 50 μmol/mol). Our objectives were to determine that whether differentPopulusspeciesvary in their PSⅡ traits under elevated atmospheric CO₂.

2.                  Materials and Methods

2.1.              Plant Material and Stress Treatment

Experiments were performed at College of Life Science, Northeast Forestry University, which is located in Harbin, Heilongjiang Province. Four Populus species including PopulusX,Populusdeltoides × cathayana,Populus alba ′Berdinensis′ Land Populuseuramericana′N3016′ × Populusussuriensiswere used in the experiments.

The cuttings were planted in plastic pots (60 cm in length, 25 cm in breadth and 15 cm in depth) filled with 1.5 kg of soil and sand (2: 1). The experiments were carried out in the close gas-exchange system consisting of chambers of 300 L volume (71cm in length, 73cm in breadth and 175 cm in height). There are 10 cuttings in each pot. Potted plants were grown in the conditions: day/night air temperature, 28/22^°C; photoperiod, 12h; relative humidity, 65-85%. One Chamber was kept at a CO₂ concentration (average ± SD) of 370 ± 15 μmol/mol (control), while another chamber was treated with an elevated CO₂ concentration of 1500 ± 50 μmol/mol (hereafter denoted as treatment). In order to study the effect of CO₂ for every variety of poplar, we choose the seventh day after treatment to JIP-test in this study.We repeated the experiment three times with 10 plants in total, 10 for controls and 10 for elevated treatment.

2.2.              Chlorophyll a Fluorescence Transient Measurement and the JIP-Test

JIP - test is a method to analyze the transient fluorescence, also known as fluorescence fast dynamic research method. Measuring the curve of rising fluorspar light (FLR) (rapid fluorescence kinetics) needed very strict about equipment, and the light source for high strength of saturated excitation light (strong degrees in 3000 ~ 10000 μmol photons/(m^-2 s)), starting fast, and can quickly capture the fluorescence signal.

JIP - test is an important method of analysis of fluorescence kinetics curve, the purpose of this method is to turn the parameters of the measured values into a biological significance that can direct energy intensity produced by PSII. In general, there are four important inflection point on the fluorescence fast dynamic curve, namely the O - J - I - P turning point, respectively is: 1) O point shows that the system (PSI) release amount of the fluorescent light, which can be understood as the light of the PSI and efficiency, the fluorescence intensity is at 0.02 ms (F₀). 2) Fluorescence intensity at 2ms, called F_j, J inflection point reflect the reaction in the accumulation of the electron acceptor Q_A^- to Q_B^-, the oxidation of the Q. 3) Fluorescence intensity at 30ms calledF_i.4）Maximum fluorescence intensity is F_m.

Leaves were dark adapted for 30 min to ensure that all PSII RCs were in the dark adapted state with open RCs. Then chlorophyll-a fluorescence transients were recorded and digitized with the portable Handy PEA (Hansatech Instruments, Ltd., King’s Lynn Norfolk, UK) with a 12-bit resolution from 10 s to 1 s and a time resolution of 10 s for the first 200 data points (Strasser et al., 1995). All measured and derived parameters (Table 1) are based on Strasser et al. [24,25] and Tsimilli-Michael[26].

Poplar leaves can completely cover the fluorescent clip test hole, it can measure directly. The excitation light was red light whose intensity was 3000 µmol/m²s saturated light intensity with peak wavelength of 650 nm. Fluorescent signal recording time of 10s, each group of plant test repeat 5 times.

2.3.              Date Analysis

Each experiment was conducted at least five times independently. All data presented were mean values of each treatment. Standard Errors (SE) for the values obtained were calculated. All the above data analyses were processed using SPSS17.0 Software.

3.                  Results

3.1.              Changes in Photosystem II (PS II) Under CO₂ Stress

In Figure 1, four typical O-J-I-P chlorophyll fluorescence transient in leaves of untreated and treated seedlings of four Populus species are shown. To analyze changes in the shape of the transients among all Populus genotypes, All O-J-I-P transients were normalized at the O- and P-step. In four genotypes the response of treated seedlings was not similar.

In PopulusX, at the 7th day, relative fluorescence intensity was decreased (Figure 1A). Thus high CO₂ conditions after long time inhibited the fluorescence intensity, especially from I- to P- step of Populus X. In Populusdeltoides×cathayana, at 7th day, relative fluorescence intensity was increased. While, relative fluorescence intensity was increased from J- to I- step (Figure 1B), which showed different changes from these genotypes in relative fluorescence intensity. In Populus alba ‘Berdinensis’ L, relative fluorescence intensity was similar between control and treatment from J- to I- step (Figure 1C). InPopuluseuramericana′N3016′ ×Populusussuriensis, an increase in relative fluorescence intensity was observed from I- to P- step (Figure 1D).

3.2.              Changes of Fluorescence Parameters Under CO₂Stress

In Table 2, no significant high CO₂-induced changes were observed in the minimum fluorescence (F_o), fluorescence at the J-step (F_j) and fluorescence at the I-step (F_i). In contrast, drastic decreases inF maximum fluorescence (F_m)values of fourPopulusspecies were recorded after 7 days of CO₂ treatment (Table 2). In parallel, all Populus species showed significant decrease in maximum quantum yield of PSII primary photochemistry (φ_Po), the efficiency with which the energy of a trapped exciton is converted into electron transport beyond Q_A^- (ψ_Eo), the quantum yield of electron transport beyond Q_A(φ_Eo) and total performance index per absorption basis (PI_total) after high CO₂ treatment. Especially, PI_total value sharply decreased after treatment. On the other hand, the values of relative variable fluorescence at the J-step (V_j) and relative variable fluorescence at the I-step (V_i) of treated-plants were higher than those of controls. There is higher value in ABS/RC (measure of the average total absorbance per active PSII RC) of treated plants compared to those of control plants. However, an exception for the Populusdeltoides × cathayanaafter treatment had no difference between CO₂-treated and control plants (Table 2).

4.                  Discussion

In this study, the shapes of chlorophyll a fluorescence transient (O-J-I-P) were markedly different in the four high CO₂-treated leaves, however the values of F_v/F_m showed similar decreasing tendency (Table 2). Thus, there is the heterogeneous behavior of PSII in Populusleaves as similar F_v/F_m. Generally, the level of photochemical reaction can be evaluated according to the chlorophyll fluorescence intensity. And F_v/F_m was used to describe the trapping efficiency of the absorbed light, which can reduce primary quinone electron acceptor of PSII (Q_A) [27]. Therefore, the different fluorescence transients of leaves under high CO₂ levels indicated that the photochemical reactions were different in differ Populusspecies, although there were similar decreasing F_v/F_m(reflecting trapping efficiencies of the absorbed light). In contrast, previous studies showed the F_v/F_mratio reflects the photochemical efficiency of PSII [28]. Various environmental stress can lead to inhibition of photosynthetic efficiency, accordingly, affecting state of the photosynthetic apparatus.

For O-J-I-P, point O represents the fluorescence of PS Ⅱ action center when all of the electron acceptor (Q_A, A_B, PQ, etc.) are fully open in the maximum oxidation state. The fluorescence intensity of point O is connected with the content of the antenna pigment and the activity of action center. Point J reflects the rate of reduction of Q_A, which is connected with reaction centre pigments, light-harvesting pigment and the state of Q_A and Q_B. If the electrons transfer from Q_A to Q_B is restricted, the value of point J will rise [29]. Here, higher J value in the leaves. In addition, point I reflects the heterogeneity of PQ, the electron acceptor state in point I mainly is related to Q_A^- and Q_B^2-. Furthermore, the structure and function of PSⅡ complexes and the size of the PQ library is attributed to appearing time of point P. In this study, high CO₂ level caused a significant decrease in the P-step level of the fluorescence transients OJIP curves of four Populus species after 7 days of treatment with high CO₂. So a fast decrease of the P-step level indicated there was main change to fluorescence transients. Generally, the “P” level was connected with the process of the electron transportation from Q_Bto PQ, and it can mark concentrations of both Q_A⁻ Q_B²⁻ and PQH[30, 31].

No significant changes in F_o, F_j and F_iof different Populusspecies were observed when   subjected to high CO₂ stress. However, high CO₂stress result F_m in decrease. F_m reflects maximum fluorescence intensity at the P-step[32]. Therefore, this result suggested that the F_mmay be a good marker of PSII vitality under CO₂ stress.

The PSII switches from the process of converting light energy into biochemical energy storage to the energy conversion process that transforms absorbed light energy into heat dissipation[33].One of parameters used to analyze the response of the plant’s PSII is PI_total. PI_totalis sensitive to changes in either antenna properties, trapping efficiency or electron transport beyond Q_A[34]. In this study, we found that PSII performance was represented using this index (PI_total). In general, the observed PI_totalis influenced by changes in antenna, RC, electron transport and end-acceptor reduction dependent parameters. Thus, PI_totalintegrates the response of RC/ABS, TRo/ABS(=F_v/F_m), ETo/TRo(= [1 −V_j]) and REo/ETo(=[1 − V_i]/[1 −V_j]).

In summary, inhibition of PSII under CO₂stress involved in changes of F_m and PI_total. In other words, these two parameters can be used in indicate the changes of PSII. These results indicated that the main reasons why PSII were affected by CO₂were degradation of antenna pigment and inhibition of the electron transport at the acceptor side of PSII.

5.                  Acknowledgement

This study was supported by the Fundamental Research Funds for the Central Universities (No. 2572015DA03 and No. 2572016EAJ4).

6.                  Conflict of Interest

The authors declare that there are no conflicts of interest.

Figures 1(A-D):The relative fluorescence transients in fully dark-adapted PopulusX leaves (A), Populusdeltoides×cathayanaleaves (B), Populus alba ′Berdinensis′ L leaves (C) and Populuseuramericana′N3016′× Populusussuriensisleaves (D) under CO₂stress and the control condition at 7 days. 

1.                   IPCC (2007) Intergovernmental panel on climate change, Climatic change 2007: the physical science basis, Geneva.

2.                   Amthor JS (2010) Terrestrial higher-plant response to increasing atmospheric [CO2] in relation to the global carbon cycle. Global Change Biology 1:243-274.

3.                   Griffin DW, Garrison VH, Herman JR, Shinn EA (2001) African desert dust in the Caribbean atmosphere: Microbiology and public health[J]. Aerobiologia 17:203-213.

4.                   Long SP, Ainsworth EA, Rogers A, Ort DR (2004) RISING ATMOSPHERIC CARBON DIOXIDE: Plants FACE the Future*[M]//. Annual review of plant biology 55:591-628.

5.                   Peters GP, Hertwich EG (2008) CO₂ embodied in international trade with implications for global climate policy. Environmental Science & Technology 42:1401-1407.

6.                   Cui C, Huang C, Yu G, Cui Y,Zhao J (1999) Research of Nursery Growth Rule of Populusxiaohei Planting by Cutting. Journal Ofence of Teachers College & University.

7.                   Jiang ZH, Wang XQ, Fei BH, Ren HQ, Liu XE (2007) Effect of stand and tree attributes on growth and wood quality characteristics from a spacing trial with Populusxiaohei.Annals of Forest Science 64:807-814.

8.                   Kostiainen K, Kaakinen S, Warsta E, Kubiske ME, Nelson ND (2008) Wood properties of trembling aspen and paper birch after 5 years of exposure to elevated concentrations of CO₂ and O₃. Tree Physiology 28:805-813.

9.                   Verhoeven AS, Adams WW, Demmigadams B, Croce R,Bassi R (1999) Xanthophyll Cycle Pigment Localization and Dynamics during Exposure to Low Temperatures and Light Stress inVinca major. Plant Physiology 120:727.

10.                Jiang CD, Gao HY, Zou Q(2002) Characteristics of Photosynthetic Apparatus in Mn-Starved Maize Leaves. Photosynthetica 40:209-213.

11.                Force L, Critchley C, Rensen JJSV(2003) New fluorescence parameters for monitoring photosynthesis in plants. Photosynthesis Research 78:17-33.

12.                Jiang CD, Gao HY, Zou Q(2003) Changes of Donor and Acceptor Side in Photosystem 2 Complex Induced by Iron Deficiency in Attached Soybean and Maize Leaves. Photosynthetica 41:267-271.

13.                Heerden PDRV, Strasser RJ, Krüger GHJ(2004) Reduction of dark chilling stress in N₂ -fixing soybean by nitrate as indicated by chlorophyll a, fluorescence kinetics. Physiologia Plantarum 121:239.

14.                Albert KR, Mikkelsen TN, Ro-Poulsen H(2005) Effects of ambient versus reduced UV-B radiation on high arctic Salix arctica, assessed by measurements and calculations of chlorophyll a, fluorescence parameters from fluorescence transients. Physiologia Plantarum 124: 208-226.

15.                Schansker G, Kissimon J, Kovács L (2005) Biophysical studies of photosystem II-related recovery processes after a heat pulse in barley seedlings (Hordeum vulgare L.). J Plant Physiol162:181.

16.                Tóth SZ, Schansker G, Garab G, Strasser RJ (2007) Photosynthetic electron transport activity in heat-treated barley leaves: the role of internal alternative electron donors to photosystem II. Biochimica Et Biophysica Acta 1767:295-305.

17.                Ilík P, Schansker G, KotabováE, Váczi P, Strasser RJ, et al. (2006) A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants. Biochimica et biophysica acta 1757: 12.

18.                An L (2006) The polyphasic chlorophyll a fluorescence rise measured under high intensity of exciting light. Functional Plant Biology 33:9-30.

19.                Strauss AJ, Ghj K, Strasser RJ, van Heerden PD (2007) The role of low soil temperature in the inhibition of growth and PSII function during dark chilling in soybean genotypes of contrasting tolerance. Physiologia Plantarum 131: 89-105.

20.                Chen LS, Li P, Cheng L (2008) Effects of high temperature coupled with high light on the balance between photooxidation and photoprotection in the sun-exposed peel of apple. Planta 228:745-756.

21.                Yordanov I, Goltsev V, Stefanov D, Chernev P, Zaharieva I, et al. (2008) Preservation of photosynthetic electron transport from senescence-induced inactivation in primary leaves after decapitation and defoliation of bean plants. Journal of Plant Physiology 165: 1954.

22.                Strasserf R J, Srivastava A, Govindjee G(1995) Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. PhotochemPhotobiol. Photochemistry & Photobiology 61:32-42.

23.                Šesták Z (2001) Probing Photosynthesis. Mechanisms, Regulation and Adaptation. Photosynthetica 39: 10.

24.                Strasser RJ, Tsimillimichael M, Srivastava A(2004) Analysis of the Chlorophyll a Fluorescence Transient[M]// Chlorophyll a Fluorescence. Springer Netherlands Pg No: 321-362.

25.                Strasser R J, Tsimilli-Michael M, Qiang S, Goltsev V (2010) Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant Haberlea rhodopensis. BiochimBiophys Acta 1797: 1313-1326.

26.                Tsimilli-Michael M, Strasser RJ (2008) In vivo Assessment of Stress Impact on Plant’s Vitality: Applications in Detecting and Evaluating the Beneficial Role of Mycorrhization on Host Plants[M]// Mycorrhiza. Springer Berlin Heidelberg Pg No: 679-703.

27.                GHK, Weis E (2003) Chlorophyll Fluorescence and Photosynthesis: The Basics. Annual Review of Plant Physiology 42: 313-349.

28.                Rachoski M, Gazquez A, Calzadilla P, Bezus R, Rodriguez A, et al. (2015) Chlorophyll fluorescence and lipid peroxidation changes in rice somaclonal lines subjected to salt stress. Acta Physiologiae Plantarum 37: 1-12.

29.                Ainsworth EA, Long SP (2005) What Have We Learned from 15 Years of Free-Air CO₂ Enrichment (FACE)? A Meta-Analytic Review of the Responses of Photosynthesis, Canopy Properties and Plant Production to Rising CO₂. New Phytologist 165: 351-371.

30.                Hill R, Larkum AW, Frankart C, Kühl M, Ralph PJ (2004) Loss of Functional Photosystem II Reaction Centres in Zooxanthellae of Corals Exposed to Bleaching Conditions: Using Fluorescence Rise Kinetics. Photosynthesis Research 82: 59-72.

31.                Stirbet A, Strasser B J, Strasser RJ, Govindjee (1998) ChlorophyllaFluorescence Induction in Higher Plants: Modelling and Numerical Simulation. Journal of Theoretical Biology 193: 131-151.

32.                Li PM, Gao HY, Strasser RJ (2005) [Application of the fast chlorophyll fluorescence induction dynamics analysis in photosynthesis study]. Acta PhotophysiologicaSinica 31: 559-566.

33.                Thach le B, Shapcott A, Schmidt S, Critchley C (2007) The OJIP fast fluorescence rise characterizes Graptophyllumspecies and their stress responses. Photosynthesis Research 94: 423-436.

34.                Oukarroum A, Madidi SE, Schansker G, Strasser RJ (2007) Probing the responses of barley cultivars( Hordeum vulgare, L.) by chlorophyll a, fluorescence OLKJIP under drought stress and re-watering. Environmental & Experimental Botany  60: 438-446.