A Comparative Study on Photosynthetic Characteristics of Dryopteris fragrans and Associated Plants in Wudalianchi City, Heilongjiang Province, China

Introduction

Dryopteris fragrans (L.) Schott, a deciduous perennial herb used in China for the treatment of skin diseases (Shen et al., 2006), exhibits antibacterial,antioxidant, analgesic, antitumor and immunomodulatory activities.Multiple substances, such as flavonoids,sterols and other medicinal components, have been isolated and characterized from this fern (Fan et al.,2012).Notably, D.fragrans has a very narrow geographic distribution and is limited to Asia, Europe and North America.In China, it is found exclusively in Wudalianchi City, Heilongjiang Province and thrives only in areas within volcanic geological landforms.There it grows in association with several other plant species, mainly Sambucus williamsii, Artemisia sacrorum, Chelidonium majus, Sorbaria sorbifolia,Woodsia ilvensis, Potentilla asperrima and Urtica angustifolia.The unique, but limited, geographic distribution of D.fragrans has probably played an important role in shaping its physiological and ecological characteristics.

Fern occupies an important place in plant phyletic evolution and uniquely exhibits independent gametophyte and sporophyte life stages.Moreover,D.fragrans is a unique type of fern that grows in extreme environment.Therefore, due to its medicinal,nutritional and ornamental value, achieving optimal D.fragrans growth is currently an important cha-llenge.Because photosynthesis is a very complex process, photosynthetic physiological ecology can systematically address this complexity through experimental indoor control methods, field sampling methods, isotopic techniques and other methods(Knight and Mitchell, 1989; Aguilar et al., 2015).Such studies can elucidate the relationships between ecological factors and multiple plant physiological phenomena.For example, on the one hand, photosynthetic efficiency of plants is influenced by external factors, such as light intensity, temperature and relative atmospheric humidity (Freeland, 1952;Arzari et al., 2005).On the other hand, internal factors, such as leaf size, leaf maturity, chlorophyll content and nitrate-reductase activity also play a role (Spoeher and Mcgee, 1924) (Osterhout et al.,1919).In spite of this complexity, researchers have successfully employed several endangered plants.For example, researches on Trillium tschonoskii found that this endangered plant photosynthesis(Liao et al., 2006) and cannot adapt to humid environments (Macedo et al., 2011; Hang et al., 2008).In this study, various photosynthetic characteristics of D.fragrans and its associated plants were measured and compared, including net photosynthesis rate,chlorophyll content, nitrate reductase activity, light compensation point (LCP) and light saturation point(LSP).The results indicated that coordination exists between D.fragrans photosynthetic characteristics and its growth environment.Moreover, these results also served to identify factors underlying the narrow geographic distribution of D.fragrans and provided a theoretical foundation to justify protection of wild resources and facilitate artificial cultivation of D.fragrans.

Materials and Methods

Natural conditions

The experimental site was located within the main D.fragrans natural habitat regions (Wudalianchi City,China).This area has a temperate continental monsoon climate, with average temperature of –0.5℃, average annual precipitation of 476 mm and average relative humidity of 69.2%.The frost period typically lasted from early October to early May, with an average annual frost-free period of 121 days.

高等学校应将原账会计科目余额表中 “非流动资产基金”“事业基金”“财政补助结转”“财政补助结余”“非财政补助结转”“经营结余”的期末余额合计记入新账“累计盈余”贷方期初余额。

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Fig.5 showed that changes in chlorophyll a,chlorophyll b and chlorophyll a+b values exhibited similar trends.However, these values were much higher for A.sacrorum and C.majus, with large variations among different months.In contrast, for P.asperrima, S.williamsii, D.fragrans, S.sorbifolia,W.ilvensis and C.majus, these values were lower,with only minimal variation.In D.fragrans, these values were only a little higher than for C.majus and W.ilvensis.It was well known that chlorophyll a mainly absorbed red light, while chlorophyll b absorbs blue light.Red light absorption for D.fragrans was initially higher than for W.ilvensis and C.majus, while blue light absorption by D.fragrans was still higher than that of W.ilvensis but lower than that of C.majus later, from August to October.To summarize, plant chlorophyll content had a direct effect on photosynthetic efficiency.Thus, in this D.fragrans community,when W.ilvensis exhibited higher photosynthetic efficiency, C.majus photosynthetic efficiency was noticeably lower, while D.fragrans efficiency fell among values for these species.

σ(4)，……, σ(n))，根据序列数目确定可容覆盖范围，并判断极比序列数据范围。当极比序列都落在可容覆盖范围时，可以进行GM(1, 1)建模。

Plant materials

（4）A:We’ve decided to enlarge the production as there is a strongdemand fromoverseas.

Healthy representative plants of D.fragrans and its main associated plants, including Sambucus williamsii,Artemisia sacrorum, Chelidonium majus, Sorbaria sorbifolia, Woodsia ilvensis, Potentilla asperrima and Urtica angustifolia were chosen for sampling.Leaves, which carried out plants' major photosynthetic function, were typically sampled using two or three leaves per plant (13 cm long, 3.5 cm wide).Leaves with similar spatial orientation and angle were chosen(Cai et al., 2008) with westward posture and 30º dip angle with respect to the ground (Yang et al., 2010; Li,2005).

Diurnal variations of photosynthetic rate (Pn)

On sunny days in mid-July, a LCi portable photosynthesis measurement system (ADC BioScientific,Ltd., UK) was used to measure net photosynthesis rate [Pn, μmol · (m2 · s-1)-1] each hour from 6: 00 a.m.to 6: 00 p.m (Jin, 2002).Each measurement was repeated 3 times.

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电子信息系统控制要求运用电子信息技术手段建立控制系统，减少和消除内部人为控制的影响，确保内部控制的有效实施，同时要加强对电子信息系统开发与维护、数据输入与输出、文件储存与保管、网络安全等方面的控制。

Measurement of light compensation point and light saturation point

CO2 concentration was set to 450 µmol · mol-1 and the relative humidity to 80% as described previously(Zhang et al., 2010).The saturating light intensity was determined by varying the light intensity until it was no longer a factor limiting the photosynthesis rate.Light compensation point (LCP) was determined using photosynthetically active radiation-net photo-synthetic rate response curves.

Measurement of chlorophyll content

A soaking extraction method was applied to extract chlorophyll using a mixed ethanol-acetone solution.Chlorophyll content was determined using spectrophotometry (Shu et al., 2010).Three individual D.fragrans plants growing at the same location were used to measure Pn and were chosen to determine chlorophyll a and chlorophyll b contents using the Beer-Lambert law.

Measurement of nitrate reductase activity

The activity of nitrate reductase was measured as previously described (Fresneau et al., 2007; Giaimo et al., 2002).NaNO2 was used to generate a standard curve and the activity of nitrate reductase was determined from the curve.

Results

Chlorophyll content changes followed a consistent pattern, with a gradual decrease from May to July,followed by an increase from July to September.In October, C.majus, S.sorbifolia and U.angustifolia plants lost their leaves.The chlorophyll a/b ratio changes were quite stable across the entire growing season, with the most values remaining between 1.5 and 3.0 and exhibiting common trends.However, early in the growing season, chlorophyll a/b values were the highest, followed by a slow decline until they reached their lowest values in July and August.Therefore,the chlorophyll content was maximal, the chlorophyll a/b value was the lowest.With leaf aging, the ratio gradually rose again, during spring and autumn, the chlorophyll a/b ratio was larger, favoring absorption of longer light wavelengths.In summer, chlorophyll a/b value was relatively lower, favoring absorption of shorter wavelengths.Therefore, chlorophyll a,chlorophyll b, chlorophyll a+b and chlorophyll a/b each varied significantly for different months.

The diurnal variations in leaf net photosynthetic rate(Pn) for D.fragrans and its main associated plants are shown in Fig.1, showing dramatic changes for all the plants studied.Moreover, the diurnal Pn profiles of D.fragrans, W.ilvensis and U.angustifolia exhibited unimodal change pro files, while C.majus and A.gmelinii exhibited bimodal rate change pro files.The maximal photosynthesis rate (Pmax) for S.williamsii and P.asperrima were the highest and were mainly observed at noon or 1: 00 p.m.Only C.majus and P.asperrima exhibited an additional Pmax peak around 11: 00 a.m.Pmax values were the lowest in D.fragrans and A.gmelinii (Fig.2).

The changing trend of nitrate reductase activity for all the plants during the growing season is illustrated in Fig.7.Nitrate reductase activity rose gradually with leaf growth for all the plants, reaching peak activity in July.After July, with leaf aging, leaf physiological function and photosynthetic capacity decreased concurrently with declining nitrate reductase activity.

The results demonstrated that leaf chlorophyll content directly correlated with photosynthetic capacity for all the plants grown under similar conditions within a certain range (Figs.3-5).During the growth period from April to October, the chlorophyll was tested content monthly for all the plants, except for a gap in some data for April (Fig.4), when the average temperature was only –11.2℃.Due to the cold temperatures, S.williamsii, S.sorbifolia and U.angustifolia leaves were not completely grown by this time point and data were not collected.

Fig.1　Diurnal variations of Pn

Diurnal variations of Pn in D.fragrans and its main associated plants

Fig.2　Average diurnal Pmax of D.fragrans and associated plants

1, D.fragrans; 2, S.williamsii; 3, A.gmelinii; 4, C.majus; 5, S.sorbifolia; 6, W.ilvensis; 7, P.asperrima; 8, U.angustifolia

Fig.3　Chlorophyll a content of D.fragrans and main associated plants

Fig.4　Chlorophyll b content of D.fragrans and main associated plants

社会意识形态、现代信息技术的不断发展是形成并传播当代大学生流行语的基本条件。当代社会生活节奏、文化交流速度不断加快，政府政策、生活方式不断转变，节奏、交流、政策、方式等方面的变化在大学生流行语中留下了相应的痕迹。大学生流行语的改变体现着现代社会发展的同时，也折射出当代国民社会文化价值观念，透露出社会各个层次的生活心态。可见，当代大学生流行语与现代社会之间相互依赖并相互依存。

Fig.5　Chlorophyll a+b comparisons for D.fragrans and associated plants for each month

Variation in activity of nitrate reductase in D.fragrans and associated plants

Fig.6　Comparison of chlorophyll a/b for each plant species averaged over all the months

1, D.fragrans; 2, S.williamsii; 3, A.gmelinii; 4, C.majus; 5, S.sorbifolia; 6, W.ilvensis; 7, P.asperrima; 8, U.angustifolia

Low chlorophyll a/b values for smaller plants have established that they utilize blue-purple wavelengths more efficiently.Fig.6 showed the highest maximum chlorophyll a/b value for D.fragrans followed by A.gmelinii, W.ilvensis, C.majus, U.angustifolia,P.asperrima, S.sorbifolia, S.williamsii.These results showed that D.fragrans was poorly adapted to its environment relative to its associated plants.

Comparison of monthly variations of chlorophyll content in D.fragrans and its main associated plants

As shown in Fig.8, the maximum nitrate reductase activity varied.The highest maximum nitrate reductase activity value was observed for S.williamsii, while the minimum was for A.gmelinii.Notably,the average nitrate reductase activity in D.fragrans was higher than only that of A.gmelinii.In addition,because Pn showed a positive correlation with enzyme activity, the relatively low D.fragrans nitrate reductase activity reflected its relatively lower photosynthesis rate and weaker photo-synthesis capacity than its associated plants.

Fig.7　Monthly variation in nitrate reductase activity for all the studied plants

Fig.8　Comparison of maximum nitrate reductase activity in D.fragrans and associated plants

1, D.fragrans; 2, S.williamsii; 3, A.gmelinii; 4, C.majus; 5, S.sorbifolia; 6, W.ilvensis; 7, P.asperrima; 8, U.angustifolia

Variation of LCP and LSP in D.fragrans and its main associated plants

A common pattern in LCP seasonal dynamic variation was observed overall (Figs.9 and 10).With the attainment of leaf maturity and increase in chlorophyll content, LCP appeared to decline generally, reaching a minimum in July.Subsequently, with leaf aging and reduction of chlorophyll content, LCP steadily and consistently increased, reaching a peak in September.A low LCP, small canopy density and strong light intensity in the rocky environment of D.fragrans community gave rise to excessive photosynthesis,which greatly impacted growth.Therefore, in these eight species, the chlorophyll content and associated leaf growth both peaked in July, followed by a steady decrease with leaf aging.Generally, LSP could be utilized to measure plant photosynthetic capacity,as a higher LSP correlates with a larger Pn value.Compared with its associated plants, D.fragrans exhibited a relatively low LSP, suggesting a narrower ecological amplitude to light adaptation.

Fig.9　LCP variation D.fragrans and main associated plants

Fig.10　LSP variation in D.fragrans and associated plants

Discussion

The net photosynthetic rate (Pn) in different habitats was a single peak pattern.In the summer morning, leaf photosynthetic rate of D.fragrans and other associated plants increased gradually.With the increased of the height of the sun, maximum value was at 1: 00 p.m.This was due to the high temperature and light in the northeast in summer, but in the morning the temperature was low, with the increased of PAR and temperature Pn was also rising to and peak at the same time.Then the temperature was higher,the leaf water content was reduced, and the stomata were partially closed, which resulted in the decrease of Ci concentration and the decreased of Pn.Plant net photosynthetic rate determines the level of accumulation of plant photosynthetic products, which can further affect the speed of plant growth (Zhang et al., 2014).D.fragrans niche similarity and niche overlap of this plant were higher, which showed that their niches were more similar (Huang et al.,2013).Previous findings had shown that the growth of D.fragrans responded to specifically defined environment factors.Here, measurements of photosynthetic rate and other photosynthetic physiological indices demonstrated that these values were not higher for D.fragrans, but were lower among most of its associated plants.For example, a lower Pn value reflected a weak photosynthetic capacity for D.fragrans relative to other plants in this community.In addition, the low Pn changed at noon coupled with a higher light energy utilization rate both suggested that D.fragrans had a certain resistance to strong light.

Strong light environment was not conducive to the synthesis of chlorophyll and chloroplast development.Chlorophyll content and chlorophyll a/b had a direct effect on the photosynthetic rate.Chlorophyll a/b were small, meant the higher the use of blue violet, the higher ability to adapt to less light environment (Li et al., 2011).When chlorophyll a and chlorophyll b decreased, photosynthetic activity of plants increased.Compared with associated plants, the total chlorophyll content and chlorophyll a to chlorophyll b content ratio in D.fragrans remained consistently at a middle level, demonstrating that D.fragrans might adapt to light, but had weak competitive ability.At the same time, the study found that D.fragrans nitrate reductase activity varied significantly in different seasons, reaching the maximum in July before declining.

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The heliophytes had high LCP and LSP; however,the shade plants had low LCP and LSP (Li et al., 2011).In our study, low LCP and LSP values for D.fragrans suggested it had a stronger ability to utilize weak light than its associated plants.Overall, the results of this study linked the narrow geographic distribution of D.fragrans to its growth disadvantage relative to its associated plants.

Conclusions

Photosynthesis is one of the most significant physiological processes underlying plant growth and greatly impacts subsequent plant size and development.D.fragrans is mainly distributed in rocks, an inhospitable environment that is neither warm nor damp enough for most plants to thrive.Therefore, during competition within a mixed plant community, the success of D.fragrans partly depends on its growth speed.Because Pn determined the rate of plant growth to a certain extent, this factor should play a role.Moreover, because previous researches indicated that D.fragrans growth characteristics helped it to adapt to environmental factors, photosynthetic physiological indices and the photosynthetic rate of D.fragrans and its main associated plants were analyzed.The study showed that D.fragrans were not dominant and exhibit even lower values than for the associated plants.By comparing these photosynthetic characteristics, a potential coordination between D.fragrans and the growing environment were observed that partly explained the reason behind the narrow geographic distribution of D.fragrans.Moreover, the information obtained from the analyses should provide a theoretical basis for further resource protection, exploitation and artificial cultivation of D.fragrans.

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