Rekha R. Warrier ? C. Buvaneswaran ? P. Priyadharshini R.S.C. Jayaraj

Introduction

Human activities are causing a steady rise in carbon dioxide concentration (CO2) in the atmosphere (IPCC 2001). CO2elevation can lead to changes in physiological and growth activities of plants, and consequently, changes in the biosphere (Eichelmann et al. 2004). Considerable attention has been devoted to plant physiological and growth responses to elevated CO2(Rey and Jarvis 1998; Roberntz and Stockfors 1998; Rogers and Humphries 2000; Jach and Ceulemans 2000; Zhang and Dang 2005;Karnosky et al. 2005; King et al. 2005; Cao et al. 2007; Kubiske et al. 2007). Growth rates usually accelerate when terrestrial plants are grown in CO2enriched atmospheres. The net photosynthetic rate of trees generally increases in response to CO2elevation if there are no other limiting environmental factors(Karnosky et al. 2005). Transient effects of elevated CO2on plant growth have been correlated with changes of net photosynthesis and attributed to altered levels of chlorophyll (a+b), Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) and various other photosynthetic proteins (Sage 2002). However, this growth stimulation typically subsides within a few days or weeks as plants acclimate to the elevated CO2treatment (Sage 1994).The acclimation of growth and photosynthesis to enhanced CO2is usually less pronounced in seedlings than in larger, older plants (Sage 2002; Geiger et al. 1998). Therefore, seedlings and developing tissues are important tools for studying plant responses to CO2enrichment. This study investigated the responses of seedling traits and primary metabolites in three tropical plantation species under the current ambient and doubled CO2. It was hypothesized that different species respond differently to elevated CO2levels initially but adapt to the changed environment over a period of time.

Materials and methods

Materials

The experiment was carried out at the Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore, Tamil Nadu, India.Seedlings of each of the selected tree species namely Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia were directly sown in hycopots filled with soil : sand : farmyard manure at 2:1:1 and placed inside polytunnels made of polythene sheets of 500 microns thickness. CO2enrichment was done to elevate the concentration (to 640 μL·L-1) using CO2cylinders.Seedlings placed in polytunnels under ambient (365 μL·L-1) CO2served as controls. The enrichment was done on daily basis and CO2levels were monitored using a PorTablePhotosynthesis System of CID, Inc., USA. Seedlings in polytunnels without CO2enrichment served as control. The experiment lasted 180 days.Observations were recorded on the 90thand 180thdays following treatment.

Plant growth

The experiments were carried out in five replicates of each treatment. The seedlings were selected randomly from each treatment. Each seedling was separated into shoot and root. The harvested above- and below-ground plant materials were weighed. Root traits including total root length, root fresh weight and root-collar diameter, and shoot traits including height of plant and shoot fresh weight were measured. The seedlings were then oven-dried at 70°C for 48 h. Seedling Quality Index was quantified using the method of Dickson et al. (1960) and root to shoot allometric coefficient was calculated from paired measurements of root and shoot biomass. The amount of biomass accumulated during the six-month experiment was used as a measure of growth.

Biochemical analysis

Five seedlings were sampled from each set. Sample extraction was carried out with different types of solvents for various analyses (phosphate buffer, hydrolyzed 2.5 N HCl and 80% acetone)centrifuged and then supernatant was taken for the estimation.Leaf tissue was analysed for total carbohydrates by use of the Yemm and Fokes (1954) method. A 100-mg sample was digested in 2.5 N HCl and the resulting green color was read at 630 nm in a Systronics UV-visible spectrophotometer. Total soluble proteins were measured in the leaves using the method of Lowry et al. (1951). Chlorophylls were extracted into solution with 80%acetone, and absorbance measured at 645, 652 and 663 nm to determine the total chlorophyll, chlorophylls a and b contents(Yoshida et al. 1976). Carbonic anhydrase activity was measured following Wilbur and Anderson (1948).

Data analysis

Data were analyzed using analysis of variance (ANOVA) with SPSS statistics package. Means were compared using DMRT where ANOVA showed a significant effect. Replications were also considered as variates since the seedlings did not exhibit homogeneity in growth performance due to segregation. The percentage variation of each parameter due to increased CO2at different growth stages was calculated over ambient level values and the maximum responsive growth stage to different levels of CO2was identified.

Results

Root characteristics

The results are presented here as the response of the different species in growth and chemical composition to elevated levels of CO2, i.e. 640 μL·L-1at different time intervals namely three and six months (90 and 180 days following treatment).

Tectona grandis: Root length increased throughout the growth period studied. Root length after 180 days reached a maximum of 33.36 cm. Root length of treated samples was greater than that of controls but the difference was not significant (Tables 1 and 5).The increment in root length following exposure to elevated CO2was 12% with 640 μL·L-1, over the control recorded at 180 days of growth (Fig.1 and Table5). Ailanthus excelsa: Though root length increased throughout the growth period (Tables 2 and 4),the increment following exposure to elevated CO2 was only 0.77% over the control (Fig.1 and Table5). The root length after 180 days reached a maximum of 15.13 cm (Table4). Under elevated CO2, mean root length exceeded that of controls but not significantly (Tables 2 and 5). Compared to the root length at 90 days, the root length doubled at 180 days (Table4). Casuarina equisetifolia: Elevated CO2did not significantly influence root length (Tables 3 and 5). Furthermore, mean root length in Casuarina equisetifolia following exposure to elevated CO2was 16%below the control (Fig.1).Chlorophyll a, CHLB: Chlorophyll b, TCHL: Total Chlorophyll, CA: Carbonic Anhydrase, RL: Root length, SL: Shoot length, FRW: Fresh Root weight, FSW: Fresh Shoot weight, DRW: Dry Root weight, DSW: Dry Shoot weight, SQI: Seedling Quality Index, PTN: Proteins, SUG: Sugars, RSRATIO:Root Shoot Ratio.

Fig.1 Percent variation in root characteristics ratio of Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia under elevated CO2 levels and ambient conditions

Table1. ANOVA for various growth, biomass and chemical characteristics in Tectona grandis seedlings over 180 days and their response to elevated CO2 levels (640 μL·L-1) and ambient (365 μL·L-1)conditions

Table2. ANOVA for various growth, biomass and chemical characteristics in Ailanthus excelsa seedlings over 180 days and their response to elevated CO2 levels (640 μL·L-1) and ambient (365 μL·L-1)conditions

Table3. ANOVA for various growth, biomass and chemical characteristics in Casuarina equisetifolia seedlings over 180 days and their response to elevated CO2 levels (640 μL·L-1) and ambient (365 μL·L-1)conditions

Root fresh and dry weights of Tectona grandis: Both root fresh and dry weights followed the same trend as root length.The percentage increase over control was about 9.74% in root fresh weight and 21.78 % in root dry weight (Fig.1; Table5).

Ailanthus excelsa: Elevated CO2did not significantly influence the fresh and dry weights, but percentage increase over control was about 11.49% in root fresh weight and 4.33 % in root dry weight (Fig.1; Table5). Casuarina equisetifolia: The trend observed in Tectona grandis and Ailanthus excelsa was observed in this species. The percent increment over the control was recorded as 38.13 and 29.30 for fresh and dry weights, respectively (Fig.1; Table5).

Shoot characteristics

Shoot length of Tectona grandis: Shoot length was shorter in elevated conditions of CO2when compared with controls, the difference being 5% less than controls after 180 days of growth(Fig.2; Table5). Ailanthus excelsa: CO2significantly increased the shoot length of Ailanthus excelsa (Table2). The combined effects of growth period and CO2did not influence the plant height significantly (Table2). The increment was 22.94%greater than for controls. This was the highest recorded among the three species studied. Casuarina equisetifolia: Similar to

Ailanthus excelsa, elevated CO2significantly influenced the shoot length in Casuarina equisetifolias (Tables 3 and 5). Theincrement in shoot length in Casuarina equisetifolia following exposure to elevated CO2was 21.74 percent.

Table4. Various growth, biomass and chemical characteristics in Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia seedlings over 90 and 180 days in closed chambers.

Table5. Various growth, biomass and chemical characteristics in Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia seedlings over 180 days and their response to elevated CO2 levels (640 μL·L-1) and ambient (365 μL·L-1) conditions

Shoot fresh and dry weights of Tectona grandis: Tectona grandis showed increase in shoot fresh and dry weights over growth periods (Tables 1 and 4). CO2did not influence the fresh and dry weights significantly (Tables 1 and 5) but increments over the controls were 8.17% and 17.62%, respectively). Ailanthus excelsa: Similar to shoot length, CO2had significant influence on shoot fresh and dry weights (Table2). The combined effect of CO2and growth period had no significant influence on the fresh and dry weights. The percentage increase over control was about 40.27% in shoot fresh weight and 52.91% in shoot dry weight under 640 μL·L-1. (Fig.2; Table5). Casuarina equisetifolia: The trend observed for Ailanthus excelsa was also observed for Casuarina equisetifolias (Table3). The percentage increase over control in shoot fresh weight was 60.03% and 54.10% in shootdry weight under 640 μL·L-1(Fig.2; Table5)..

Fig.2 Percent variation in shoot characteristics of Tectona grandis,Ailanthus excelsa and Casuarina equisetifolia under elevated CO2 levels and ambient conditions

Total biomass

Elevated CO2levels enhanced the total biomass at higher levels of CO2. The increment in total biomass was 19.58% in Tectona grandis, 44.51% in Ailanthus excelsa and 49.89% in Casuarina equisetifolia over their respective controls. Casuarina equisetifolia showed the greatest biomass increment in response to elevated CO2levels (Fig.3).

Root / shoot (R/S) ratio

Tectona grandis seedlings showed significant variation in root:shoot ratio (R/S) with respect to CO2level, growth period and interaction between the two main factors (Table1), while Casuarina equisetifolia showed significant increase over periods of time. Ailanthus excelsa showed significant variation with respect to combined effects of CO2elevations and growth periods but not individually. The increments observed in root:shoot ratio in the species were 69.3, 6.4 and 27.2 (Fig.3) for Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia respectively.

Fig.3 Percent variation in Total biomass, seedling quality index and root/shoot ratio of Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia under elevated CO2 levels and ambient conditions

Seedling quality index (SQI)

A trend similar to root-shoot ratio was observed in all three species. The SQI increment was highest in Tectona grandis (48.4 %;p ＜5%; Table1) followed by Casuarina equisetifolia (22.39%; p＞5%; Table2) and Ailanthus excelsa (10.10 %; p ＞5%; Table3;Fig.3). The increase in Tectona grandis could be attributed to the increase in the leaf size as a result of elevated CO2levels. This result is in agreement with the R/S ratio.

Biochemicals

Chlorophylls of Tectona grandis: Chlorophylls a, b and total showed significant increase with elevated CO2levels and with the combined effects of growth period and elevated CO2levels(Tables 1 and 5). The increment in chlorophylls was 64.5%,95.84% and 49.06%, respectively for chlorophylls a, b and total recorded at 180 days of growth (Fig.4; Table5). Ailanthus excelsa: CO2had a negative influence on the chlorophylls in Ailanthus excelsa (Fig.4; Table5). The decline in chlorophylls was highest in chlorophyll b (18.52 per cent), followed by total chlorophyll (11.24 per cent). Chlorophyll a levels were similar in treated and control seedlings but slightly lower for controls (Fig.4; Table5). The combined effects of growth period and CO2did not in any way influence the chlorophylls (Table2). Casuarina equisetifolia: Elevated CO2and time period significantly influenced the chlorophylls in Casuarina equisetifolia (Table3). Increments of chlorophylls a, b, and total were 56.66, 122.7 and 103.9, respectively (Fig.4, Table5). Replications showed significant increase in the chlorophyll b and total levels suggesting a need to screen genotypes to understand their varied responses.Combined effects of CO2and growth period showed significant responses for chlorophyll b and total chlorophyll (Table3).

Fig.4 Percent variation in biochemicals namely chlorophylls, CA activity, total sugars and proteins of Tectona grandis, Ailanthus excelsa and Casuarina equisetifolia under elevated CO2 levels and ambient conditions

Carbonic anhydrase: CA increased significantly in Casuarina equisetifolia, Tectona grandis and Ailanthus excelsa in response to elevated CO2(Tables 1 to 3). The increments were16.02%, 61.74% and 21.38%, respectively for Tectona grandis,Ailanthus excelsa and Casuarina equisetifolia (Fig.4; Table5).

Sugars and proteins: Sugars did not show significant responses to increased levels of CO2though treatment means were higher for all three species, the increments being 10.0, 12.95 and 13.12, respectively. However, protein levels varied significantly in Casuarina equisetifolia and Ailanthus excelsa, with recorded increments of 31.7 and 24.8 (Fig.4; Table5).

Discussion

Elevated levels of atmospheric CO2can increase production in greenhouses (Mortensen, 1987). Effects on gas exchange, respiration, growth, and development have been documented for a variety of plant species (Curtis and Wang 1998). Use of elevated CO2in the greenhouse, therefore, can be used to modify the physiology, size, or morphology of plants in order to meet specific objectives relevant to the desired end use (e.g. afforestation,agroforestry, reclamation). We subjected three tropical species to increased levels of CO2at the nursery stage. The most responsive was Casuarina equisetifolia, a nitrogen fixing species, preferred by tree farmers due to its multiple uses and short rotation.This was followed by Ailanthus excelsa, an indigenous fast growing tree species in use by match industries. Tectona grandis,a long rotation species, showed less pronounced responses than the fast-growing species. The performance of Tectona grandis was poor at the end of three months, indicating adverse effect of CO2enrichment on morphological traits of this species (Varadarajan et al. 2010) however at the end of six months, Tectona grandis showed higher root:shoot ratio and seedling quality index indicating better adaptability to elevated CO2levels. Since stump planting is the preferred propagule for planting Tectona grandis, higher SQI indicates that the species would be able to perform better under varied climatic conditions.

A second significant feature of the data sets is varying root:shoot ratios of the three species. Allometrics is a useful tool to evaluate biomass allocation among different plant organs. It is based on the logarithmic relationships between biomass partitioned to the two plant organs (root to shoot) (Nicklas 2005). In nature, plants are believed to develop a root to shoot ratio that is partly genetically inherited and partly determined by the environment. Plants sense the environment and respond to fluctuations in the resources availability by applying morphological and physiological controls that alter, among other processes, the carbon allocation pattern.

The root:shoot ratio is one measure that helps assess the overall health of plants. An increase in root:shoot ratio can indicate a healthier plant provided the increase is from greater root size and not from lower shoot weight. In the present study, elevated CO2levels maintained better root:shoot ratios than did controls. Surprisingly, Tectona grandis had the highest increment for root:shoot ratio with higher root biomass but low shoot biomass increment. The high root:shoot ratio and SQI suggests that Tectona grandis, although it did not show significant morphological responses during the study period, could adapt better to elevated CO2levels over longer periods of exposure. However, in Casuarina equisetifolia and Ailanthus excelsa, increases in root:shoot ratio were more attribuTableto increases in shoot characteristics than the root though both showed increments in their overall growth performance.

Morphological and growth responses to elevated CO2include increased biomass yield, height, total leaf area, total leaf weight and size, leaf weight per unit area, and dry matter allocation to roots (Radoglou and Jarvis, 1990a, b; Ceulemans and Mousseau,1994; Curtis and Wang, 1998). In our study, there was an increase in all shoot characteristics except in Tectona grandis where shoot length did not show positive response. Field observations revealed larger leaf area for the species under elevated CO2suggesting resource allocation for expansion of leaves rather than to height. Root characteristics also showed positive response, except in root length in Casuarina equisetifolia, but field observations showed increased secondary and tertiary roots, this observation is in agreement with the root biomass increment. In all the three species, the biomass increased over the ambient grown species.

Aspen and poplars grown in elevated CO2accumulated 55%more dry mass than trees grown in ambient CO2conditions. This increase resulted from dry mass increase of all the tree components, i.e. stem, branches, buds, needles and roots. In work with woody species at ambient and elevated CO2, Tischler et al.(2004) observed significant effects of elevated CO2on total biomass for mesquite (Prosopis glandulosa) at day 3, and for parkinsonia (Parkinsonia aculeata L.), honey locust (Gleditsia triacanthos L.), and huisache (Acacia farnesiana (L.) Willd.) at eighth day. Similar increases in dry mass in response to CO2enrichment have been observed for a range of tree species grown under field conditions (Norby et al. 1999). Elevated CO2has been found to cause greater allocation to root biomass (Dickson et al. 1998) and in our study, this was observed for all three species. An increase of 15% in stem height and 30%?45% in biomass under elevated CO2has been reported in tree species(Ceulemans et al. 1996; Curtis and Wang 1998; Wang et al. 2000;Zak et al. 2000). An increase in height of 8% and biomass(15%?30%) was observed in hybrid poplar under elevated CO2(Tupker et al. 2003).

Accumulation of foliar carbohydrates is one of the most pronounced and universal changes observed in the leaves of C3 plants grown at elevated CO2concentration. In our study, the levels of total carbohydrates / sugars did not show significant variation in response to elevated CO2levels. Rogers and Ainsworth (2006) report that trees have large sinks for photosynthates and may be expected to avoid foliar carbohydrate accumulation in the presence of elevated CO2. Developing loblolly pines experiencing a step change in CO2at the Duke Forest FACE experiment (Hendrey et al. 1999) did not show an accumulation of carbohydrates when measured at multiple stages during the first season of CO2exposure (Myers et al. 1999). Rogers and Ellsworth (2002) did report foliar carbohydrate accumulation. Herrick and Thomas (2001) did not report carbohydrate accumulation in sun or shade leaves of Liquidambar styraciflua (sweetgum) growing at elevated CO2in the understory at the DukeForest FACE site (Herrick and Thomas 2001). However, Tissue et al. (2002) did report carbohydrate accumulation at elevated CO2in the same species at the Oak Ridge National Laboratory FACE site (Norby et al. 2001). Singaas et al. (2000) reported carbohydrate accumulation in Acer rubrum, Ceris canadensis and L. styraciflua at the Duke site. So it could be inferred that our understanding of the mechanisms underlying the response of foliar carbohydrates to elevated CO2in these tree species needs to be increased.

Further, if high-CO2grown plants invest relatively more in cell walls and/or secondary compounds, this may increase their leaf longevity, but slow their growth, whereas an additional investment in proteins (for example in photosynthetic machinery)may accelerate growth (Poorter and Bergkotte 1992). Here in our experiments, we observed an increase in the levels of proteins in Casuarina equisetifolia a major nitrogen source of the plant suggesting that the total non structural carbohydrate (TNC) levels could have increased, with an addition to the photosynthetic machinery as is evidenced by higher levels of chlorophylls.

Conclusion

Elevated CO2is a tool that can be used to modify growth and resource allocation in tropical tree species during nursery production prior to large-scale use. Overall, the three species responded positively to elevated CO2with increased growth and allocation to roots. Modification of morphology through control of atmospheric CO2can be combined with selection of clones to produce planting stock appropriate to the end purpose. For agroforestry/afforestation, both early establishment and maximum growth are of interest; in reclamation of ecologically degraded sites, large root systems that help ensure survival and rapid uptake of water and minerals are important. The latter would be useful even in bioremediation by absorption of contaminants.

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