Effects of Fully Open-Air [CO2] Elevation on Leaf Photosynthesis and Ultrastructure of Isatis indigotica Fort

Traditional Chinese medicine relies heavily on herbs, yet there is no information on how these herb plants would respond to climate change. In order to gain insight into such response, we studied the effect of elevated [CO2] on Isatis indigotica Fort, one of the most popular Chinese herb plants. The changes in leaf photosynthesis, chlorophyll fluorescence, leaf ultrastructure and biomass yield in response to elevated [CO2] (550±19 µmol mol–1) were determined at the Free-Air Carbon dioxide Enrichment (FACE) experimental facility in North China. Photosynthetic ability of I. indigotica was improved under elevated [CO2]. Elevated [CO2] increased net photosynthetic rate (P N), water use efficiency (WUE) and maximum rate of electron transport (J max) of upper most fully-expended leaves, but not stomatal conductance (gs), transpiration ratio (Tr) and maximum velocity of carboxylation (V c,max). Elevated [CO2] significantly increased leaf intrinsic efficiency of PSII (Fv’/Fm’) and quantum yield of PSII(ΦPS II), but decreased leaf non-photochemical quenching (NPQ), and did not affect leaf proportion of open PSII reaction centers (qP) and maximum quantum efficiency of PSII (Fv/Fm). The structural chloroplast membrane, grana layer and stroma thylakoid membranes were intact under elevated [CO2], though more starch grains were accumulated within the chloroplasts than that of under ambient [CO2]. While the yield of I. indigotica was higher due to the improved photosynthesis under elevated [CO2], the content of adenosine, one of the functional ingredients in indigowoad root was not affected.


Introduction
Global atmospheric CO 2 concentration ([CO 2 ]) is predicted to reach 550 mmol mol -1 by the middle of this century [1]. The increase in [CO 2 ] may improve the photosynthetic efficiency of plants, thereby increasing the supply of photoassimilates, dry mass and yield [2][3][4]. Higher [CO 2 ] increases the carboxylation rate of Rubisco but inhibits the oxygenation of Ribulose-1, 5-bisphosphate (RubP) [3]. These [CO 2 ] effects vary with crops, cultivars and plant development stages.
Chinese medicinal herbs are among the oldest alternative and complementary medicines. Their ever-increasing use indicates public interest in such medicines and their important roles. Chinese herbal medicines constitute multi-billion-dollar industries worldwide and 1500 herbals are sold as dietary supplements or ethnic traditional medicine. It is expected that there would be a greater boost in the use of Chinese herbal medicine [5]. Consumers spending on herbal products in the United States was estimated to be more than $5 billion per year, mainly from self-prescription of over-the-counter products [6]. The research on traditional Chinese medicine has been taken more and more seriously [7,8].
Isatis indigotica Fort, grown and used in most regions of China, belongs to the Brassicaceae family. The dried root and leaf of I. indigotica, or indigowoad root and indigowoad leaf, can be used as medicine according to Chinese Pharmacopeia [9]. I. indigotica is biennial, with the leaf and root from the first but not the second year being used as medicine. Seeds are harvested in the second year. I. indigotica has multiple pharmacological properties such as anti-viral, anti-cancer, anti-bacterial and immune enhancement [10,11]. It can reduce fever, detoxify and benefit the pharynx [12]. Adenosine is one of the main medical ingredients of indigowoad root [13,14], and is an indicator of the quality of indigowoad root [14]. Adenosine can be used as cardioprotective and therapeutic agent for chronic heart failure [13,14]. It also shows antiinflammatory efficacy [13,15]. The most popular medicine form of the herb is indigowoad root infusion, which is used for treating flu. The current annual consumption of indigowoad root was about 2 million kg in China [16].

Leaf Mesophyll Cells
Elevated [CO 2 ] increased the number of starch grains per chloroplast profile and the area per starch grain by 150.0% and 144.3%, respectively (Table 3 and Fig.1 A, 2 ], respectively. The total weight per plant was significantly increased by 8.8% (P,0.05) (Fig.2). The mean adenosine contents in root were 0.44 mg/g and 0.59 mg/g under ambient [CO 2 ] and elevated [CO 2 ], respectively, and was not significantly affected by elevated [CO 2 ] (Fig.3).

Discussion and Conclusion
The enhancement of photosysnthesis by elevated [CO 2 ] diminishes over time (days to years), termed photosynthetic acclimation [4]. Photosynthetic acclimation has been well documented in C 3 plants e.g. rice [24,25], soybean [23,26] and wheat [27], and the response varied with plant species, cultivars, developmental stages and environmental conditions [2,23]. Quantum yield of PSII(WPSII) may serve as a quantitative indicator of electron transport through PSII, which is related to the photochemical efficiency of PSII [28]. Non-photochemical quenching (NPQ) indicates plants dissipate energy by the means of thermal energy rather than linear electron transport. Nonphotochemical quenching increases dramatically when sink is limited (no development of new sinks) [29]. In our study, increases in P N and J max under elevated [CO 2 ] coincided with increases in WPSII, the efficiency of light harvesting and energy transduction of open PSII centres (Fv'/Fm'). V c,max , the maximum quantum efficiency of PSII(Fv/Fm) and qP (an estimate of open PSII centres) showed no significant change under elevated [CO 2 ], but NPQ was decreased. These results are partially in agreement with the study by Tausz-Posch et al [28], which showed increases in WPSII, Fv'/ Fm' and qP of wheat grown under elevated [CO 2 ] and mild or moderate stress conditions. During the first year of cultivation, I. indigotica was only in its vegetative growth stage, plant growth might require large amount of photosynthates, hence no limitation in sink. Therefor NPQ was decreased and more energy was used in linear electron transport (WPSII) under elevated [CO 2 ].
The increase in the number and size of starch grain in chloroplast indicated that more photosynthates were produced through increased photosynthesis under elevated [CO 2 ]. More photosynthates would restrain photosynthesis if no new sink is available to accommodate superfluous assimilation [23,30,31], resulting in photosynthetic acclimation. Photosynthetic acclimation occurs in rice flag leaves under FACE because N content in the leaves was reduced under a high N demand for reproductive development, but not in the eighth leaf stage when vegetative growth occured [32]. Photosynthetic acclimation occurred at the seedling stage (R5) in soybean cultivar Zhonghuang 13, but not in the other soybean cultivar Zhonghuang 35. Acclimation did not occur for Zhonghuang 35, because extra C sink was developed and the photosynthesis was not restricted under elevated [CO 2 ] [23]. Acclimation to high [CO 2 ] was not apparent when the ratio of sink to source activity was increased, but it was observed when the ratio was reduced [30]. Photosynthetic acclimation did not occur in I. indigotica under elevated [CO 2 ] in the first year when new C sinks developed.
The [CO 2 ]-induced increased photosynthesis increased biomass by 8.8% in our experiment. Similar [CO 2 ] effects were also observed for other species of the Brassicaceae family such as  Climate, soil type and fertilizer application influence the growth of herbal medicine plant, and its active ingredients [33]. Global temperature may increase with [CO 2 ] in future, which could lead to climate change with perturbations like more severe and frequent drought events [28]     oilseed rape [36]. Increased temperature, drought and elevated [O 3 ] would reduce the fertilization effect of elevated [CO 2 ] [37,38]. The effect of growth, metabolism, effective compositions of I. indigotica with climate change including elevated [CO 2 ], increased temperature, drought and elevated [O 3 ] would be a new area to explore in future research.
The decrease in g s under elevated [CO 2 ] is associated with increased water use efficiency [39]. There was no significant reduction in g s of I. indigotica under elevated [CO 2 ], but the water use efficiency increased by 17.5% because of the increase in P N . These indicate that the herbal plant may be tolerant to some extent of the drought conditions potentially accompanied with elevated [CO 2 ].
In addition, the structure of grana layer and stroma thylakoid membranes were intact, but much tighter than those under ambient [CO 2 ]. A correlation might exist between the conformation of stroma thylakoids and starch grain accumulation, because the chloroplast was occupied by an excess of large starch grains [21]. The grana layer and the membrane structure under elevated [CO 2 ] were not intact in the leaf, which contributed to photosynthetic acclimation for soybean [23]. P N , J max , Fv'/Fm' and WPSII under elevated [CO 2 ] were improved, and photosynthetic acclimation was not encountered for I. indigotica,which is in accordance with the intact structural of grana layer and stroma thylakoid membranes. In

Site Description
The study was conducted at the Mini-FACE facility developed by IEDA (Institute of Environment and Sustainable Development in Agriculture) located at an experimental station of the Chinese Academy of Agricultural Sciences at Changping, Beijing, China (40.13uN, 116.14uE). The operational procedures of the facility were as described in a previous experiment [23]. The long term average rainfall and temperature during the I. indigotica growing season were 475 mm and 21.3uC, respectively.

I. indigotica Cultivation and Irrigation
I. indigotica were sown on 26 June 2011 in 40 cm pots (25 cm depth). Three plots were with ambient [CO 2 ] of 411615 mmol mol -1 , and another three plots with elevated [CO 2 ] of 550619 mmol mol -1 , simulating the expected environment in 2050. Three plants were grown in each pot and four pots were included in every plot. The soil was a clay loam with a pH (1:5 soil:water) of 8.6 and contained 1.21% organic carbon (C) and 0.11% total N. Fertilizers were applied at sowing at the rates of 3.68 g N per plot and 4.08 g P per pot. Irrigation equivalent to 10-20 mm of rainfall was applied every 2-3 days after the seedling emerged from soil.

Transmission Electron Microscopy
For anatomical and ultra-structural investigations, samples from the upper most fully-expanded leaves facing the sunlight were collected (three plants per plot) on 19 August 2011 (between 10:00 and 12:00, at 11-leaf stage). Leaves were observed using an electron microscope (JEOL JEM-2100F) operating at 80 kV as described previously [23].

Gas Exchange Measurements
Measurements of P N (Net photosynthetic rate) vs C i (intercellular CO 2 concentration) were conducted 36, 53 and 84 days after sowing. One of the upper most fully-expanded leaves was randomly selected. The number of leaves on plants was 7, 11 and 18 correspondingly. Gas exchange measurements were conducted using portable gas exchange systems (LI-COR 6400; LI-COR, Lincoln, Neb, USA). The [CO 2 ] in the leaf chamber was controlled by the LI-COR CO 2 injection system, and an irradiance of 1,400 mmol photons m -2 s -1 was supplied using an built-in LED lamp (red/blue). Temperature in the 26362.5 cm 3 leaf chamber was set at 25uC, and the actual temperature ranged from 25 to 28uC. The vapour pressure deficit (VPD) on the leaf surface was between 1.9 and 2.1 kPa. The [CO 2 ] surrounding the leaf for all control and treatment leaves was controlled across the series of 550, 400, 300, 200,100, 50, 400, 550, 600, 700, 800, 1000, 1200 and 550 mmol mol -1 , and measurements were recorded after equilibrium was reached. Measurements were made between 9:00 and 14:00 local time. Each individual curve took approximately 35 min to complete. Values for P N and C i were used to calculate V c,max (Maximum velocity of carboxylation) and J max (Maximum rate of electron transport) values using the model and software provided by Sharkey et al [40]. P N , Tr (transpiration ratio), WUE (water use efficiency, WUE = P N /Tr) and g s (stomatal conductance) were also measured at the same irradiance, temperature and vapour pressure when the measurements of P N vs C i were conducted. [CO 2 ] in the leaf chamber was set to 400 mmol mol -1 for current [CO 2 ] treatment and 550 mmol mol -1 for elevated [CO 2 ] treatment, and one upper most fullyexpanded leaf was measured per pot.

Chlorophyll fluorescence
The photosynthetic performance of upper most fully-expanded leaves was assessed in terms of the chlorophyll a fluorescence parameter Fv/Fm (Maximum quantum efficiency of PSII), Fv'/Fm' (Intrinsic efficiency of PSII), WPSII (Quantum yield of PSII), qP (Proportion of open PSII reaction centers ), NPQ (Non-photochemical quenching) using a miniaturized pulse-amplitude modulated fluorescence analyzer (Mini-PAM, Walz, Effeltrich, Germany) with a leaf clip holder as described by Bilger et al [41]. The fluorescence parameter was measured at incident PPFD between 9:00 and 15:00 h over 3 consecutive days, for a total of individual leaves in each plot. F09 and Fm9 of darkness-adapted leaves were measured between 23:00 and 01:00 h on the same day. The high light flash used to measure saturated fluorescence had a PPFD (Photosynthetic Photon Flux Density) of 4,000 mmol m 22 s 21 and a duration of 800 ms. All chlorophyll fluorescence parameters were calculated as described by Rascher et al [42].
Harvesting I. indigotica plants were harvested on 8 October 2011 (102 days after sowing). All the plants were separated into above ground parts and roots, air dried and weighed.

The Content of Adenosine
Standard of adenosine were purchased from National institutes for food and drug control (Beijing, China). All the roots were dried at 50uC until constant weight is reached. Samples were pulverized to 80 meshes. 1.0 g of pulverized powder was ultrasonically extracted with 25 ml of methanol for 30 minutes. The supernatant was filtered and cooled to ambient temperature. The obtained solution extracted from the root and standards solution were filtered through a syringe filter (0.45 mm) and aliquots (10 ml) were subjected to HPLC analysis by Agilent 1100 series HPLC-DAD system (Agilent, Palo Alto, CA, USA). Agilent Zorbax Eclipse XDB-C 18 column (4.66250 mm, 5 mm, USA) was used. The mobile phase was a mixture of methanol/water (8:92, v/v). Elution was performed at a solvent flow rate of 1 ml/min. Detection was performed with a variable-wavelength UV detector (L-4250) at 260 nm [13].

Statistical Analysis
All experimental data presented were analyzed by analysis of variance at 0.05 probability level using SAS System 8.1.