Genetic Diversity of Miscanthus spp. in Photosynthesis

저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.

A DISSERTATION FOR THE DEGREE OF MASTER OF SCIENCE

Genetic Diversity of Miscanthus spp. in Photosynthesis

BY

JASTIN EDRIAN REVILLEZA

FEBRUARY, 2013

MAJOR IN CROP SCIENCE AND BIOTECHNOLOGY DEPARTMENT OF PLANT SCIENCE

Genetic Diversity of Miscanthus spp. in Photosynthesis

UNDER THE DIRECTION OF PROFESSOR DO-SOON KIM SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF SEOUL NATIONAL UNIVERSITY

BY

JASTIN EDRIAN REVILLEZA

MAJOR IN CROP SCIENCE AND BIOTECHNOLOGY DEPARTMENT OF PLANT SCIENCE

NOVEMBER, 2012

APPROVED AS A QUALIFIED DISSERTATION OF JASTIN EDRIAN REVILLEZA

FOR THE DEGREE OF MASTER OF SCIENCE BY THE COMMITTEE MEMBERS

FEBRUARY, 2013

C H A I R M A N

Prof. BYUN-WOO LEE, PhD

V I C E

-C H A I R M A N _{Prof. DO-SOON KIM, PhD}

M E M B E R

ABSTRACT

Genetic Diversity of Miscanthus spp.

in Photosynthesis

Jastin Edrian Revilleza

Department of Plant Science

The Graduate School

Seoul National University

Miscanthus, a weed thriving in South-eastern Asia and some parts of

Africa, is a well-known potential biomass grass due to its C

photosynthesis and high biomass potential in temperate region. Its

photosynthetic ability under low temperature and shaded condition when

canopy closed is considered as an important trait in determining biomass

yield potential. Thus, the objectives of this study were to investigate the

photosynthetic ability under low temperature and shaded conditions and

the photosynthetic diversity of different Miscanthus accessions with

relation to its biomass yield. Three factors were examined: temperature

effects when the plants were exposed to 25/20

C, 20/15

C, and 15/10

C;

light intensity effects when the plants are exposed to varying light

intensities of 100%, 75%, 45%, 25%, and 5%; and genotypic variation in

photosynthetic ability and biomass yield using 53 three-year stands

accessions measured in the field condition. Results show that lower

temperature and shaded condition tend to limit the photosynthetic ability

of the plant but the trends of declining with decreasing temperature and

light intensity depend on species and accessions. Miscanthus

lutarioriparius was most sensitive to low temperature in photosynthesis

and biomass yield responses, while M. sacchariflorus was the second

least sensitive in photosynthesis and M. sinensis in biomass yield

response after M. x giganteus, which was most insensitive to low

temperature in photosynthesis and biomass yield responses. Miscanthus

sacchariflorus was most insensitive to low light intensity in

photosynthesis, while M. sinensis was most insensitive in biomass yield.

Therefore, it can be concluded that under low temperature and low light

intensity, M. sacchariflorus can maintain stable photosynthesis and M.

sinensis can maintain more stable biomass yield. The field study with 53

accessions showed diversity in physiological parameters including

photosynthesis and biomass yield but no significant correlation between

photosynthesis rate and biomass yield, suggesting that biomass yield

may not solely explained by photosynthetic rate measured on a specific

spot of a leaf but by whole plant photosynthetic ability comprised of

whole leaf area and photosynthesis rate. Miscanthus lutarioriparius and

some M. sinensis accessions, M-96, M-107 and M-177, showed high

biomass yield even with low photosynthesis. They may be a good source

for breeding a new variety as a biomass crop and for further study to

understand the relationship between photosynthesis and biomass yield.

Keywords: Bioenergy, biomass, genetic diversity, light intensity,

CONTENTS

ABSTRACT...i

CONTENTS...iii

LIST OF FIGURES...v

LIST OF TABLES...viii

1. INTRODUCTION...1

2. LITERATURE REVIEW...2

2.1 Miscanthus as a potential bioenergy crop...2

2.2 Photosynthetic ability of Miscanthus in relation with biomass

production...3

2.3. Effects of low temperature on photosynthesis and biomass

yield...4

2.4 Developing a new Miscanthus variety for biomass production

in temperate regions...5

3. MATERIALS AND METHODS...7

3.1 Plant material ...7

3.2 Photosynthetic responses under low temperature condition...7

3.4 Diversity of Miscanthus spp. in photosynthetic ability...9

3.5 Statistical analysis...10

4. RESULTS AND DISCUSSIONS...13

4.1 Effect of low temperature on photosynthetic responses of

Miscanthus spp………...13

4.2 Effect of low light intensity on photosynthetic responses of

Miscanthus spp...26

4.3 Diversity of Miscanthus spp. genotypes in photosynthetic

abilities...39

5. CONCLUSION...45

6. REFERENCES...47

ABSTRACT IN KOREAN...53

LIST OF FIGURES

Figure 1. Photosynthetically active radiation (PAR) values of 100%

(●), 75% (■), 45%(▲), 25% (▼), and 5% () light intensities….11

Figure 2. Plant height of M. x giganteus (●), M. lutarioriparius (■)

M. sinensis (▲), and M. sacchariflorus ( ▼) accessions grown at

different temperatures. Measurements were made at 14 (A) and 28

DAT (B)……….…….14

Figure 3. Leaf area of M. x giganteus (●), M. lutarioriparius (■)

M. sinensis (▲), and M. sacchariflorus ( ▼) accessions grown at

different temperatures. Measurements were made at 14 (A) and 28

DAT (B)………...16

Figure 4. Photosynthesis rate of M. x giganteus ( ● ), M.

lutarioriparius (■), M. sinensis (▲), and M. sacchariflorus (▼)

accessions grown at different temperatures. Measurements were made

at 14 (A) and 28 (B) DAT………...18

Figure 5. Biomass yield of M. x giganteus (●), M. lutarioriparius

(■), M. sinensis (▲), and M. sacchariflorus (▼)accessions grown at

different temperatures. Measurements were made at 35 DAT……...20

Figure 6. Relationship between photosynthesis rate measured at 14

(A) and 28 DAT (B) and biomass yield measured at 35 DAT of

various Miscanthus spp. accessions grown at varying temperatures

25/20

°C

(●) and 20/15

°C

(■), and 15/10

°C

(▲)………..24

Figure 7. Plant height of M. x giganteus (●), M. lutarioriparius (■),

M. sinensis (▲), and M. sacchariflorus ( ▼) accessions grown at

various light intensities. Measurements were made at 21 (A), 42 (B),

and 56 (C) DAT………..…26

Figure 8. Number of tillers of M. x giganteus (●), M. lutarioriparius

(■), M. sinensis (▲) and M. sacchariflorus (▼) accessions grown at

various light intensities. Measurements were made at 21 (A), 42 (B),

and 56 (C) DAT...28

Figure 9. Leaf area of M. x giganteus (●), M. lutarioriparius (■),

M. sinensis (▲), and M. sacchariflorus (▼)accessions grown at low

light intensity at 42 (A) and 70 DAT (B)………...….30

Figure 10. Photosynthesis rate of M. x giganteus ( ● ), M.

lutarioriparius (■), M. sinensis (▲), and M. sacchariflorus (▼)

accessions grown at various light intensities. Measurements were

made at 21 (A), 42 (B), and 56 (C) DAT………....32

Figure 11. Biomass yield of M. x giganteus (●), M. lutarioriparius

(■), M. sinensis (▲), and M. sacchariflorus (▼) accessions grown at

various light intensities. Measurements were made at 21(A), 42 (B),

and 56 (C) DAT….………...34

Figure 12. Relationship between relative photosynthesis rate and

biomass yield of various Miscanthus spp. accessions grown various

light intensities 100% (●), 75% (■), 45% (▲), 25% (▼), and 5%

() at 21 (A) 42 (B), and 56 (C) DAT………...37

Figure 13. Relative photosynthesis rate of various Miscanthus spp.

accessions relative to M. x giganteus in June (●) August (■), and

September (▲) 2012………...40

Figure 14. Relationship between photosynthesis rate and biomass

yield of M. x giganteus (●) M. lutarioriparius (■), M. sinensis (▲),

and M. sacchariflorus (▼) accessions in June (A), August (B), and

September (C) 2012………...….42

LIST OF TABLES

Table 1. Accessions of Miscanthus spp. used in this study…………12

Table 2. Slopes of Miscanthus spp. responses to varying temperature

in their plant growth, photosynthesis and biomass yield. Plant growth

and photosynthesis were measured at 14 DAT and 28 DAT, and

biomass yield at 35 DAT. Numbers in parentheses are standard

error……….21

Table 3. Slopes of Miscanthus spp. responses to varying light

intensity in their plant growth, photosynthesis and biomass yield.

Plant growth and photosynthesis were measured at 21 DAT, 42 DAT

and 56 DAT, and biomass yield at 70 DAT The numbers in

parentheses are standard errors………...………....35

Table 4. Correlation of photosynthesis to SPAD reading, chlorophyll

fluorescence (PI), and biomass yield (g plant

) of various Miscanthus

spp. accessions...43

Table 5. Miscanthus spp. showing high biomass yield with different

photosynthetic abilities. The numbers in parentheses are standard

deviations………....…44

1. INTRODUCTION

Miscanthus x giganteus is exceptional among C4 species for its high productivity

in temperate climates (Dohleman and Long 2009). It is native to the East Asian region including Korea, China and Japan (Chung and Kim 2012). However, the study of C4 photosynthesis on different environmental conditions is limited. First,

studies focused mainly on the difference between Maize and Miscanthus. Naidu et

al. (2003) measured the amount of PPDK and Rubisco at low temperatures

between M. x giganteus in contrast with Maize, with results showing that the maintenance of large amounts of PPDK and Rubisco in M. x giganteus is critical in maintaining high rates of C4 photosynthesis at low temperature. Wu and Wedding

(1987) studied temperature effects on PEPC from a CAM and C4 plant using maize

as a model plant. Secondly, most studies are limited to Miscanthus species grown only in the fields of America and Europe. Little has been studied on the eastern parts of Asia especially in South Korea. Beale et al. (1996) did a study to determine the seasonal variation in photosynthetic of M. x giganteus grown in southern England. Results showed that under the cool temperate conditions of southern England, M. x giganteus, unlike all C4 species examined, is able to realize

the photosynthetic potential of the C4 process without suffering any apparent low

temperature impairment. Third, there are only a few reports on the influence of light and temperature on C4 plants (Grammatikopoulos and Manetas 1990;

Selionoti et al. 1986). Miscanthus x giganteus is the only Miscanthus species being commercially cultivated for biomass production due to its high biomass yield (Chung and Kim 2012). This study aims to (1) investigate the effects of low temperature and low light intensity on the growth and photosynthesis rate of various Miscanthus spp. accessions and (2) to compare the biomass yield among the different accessions with relation to their photosynthetic ability.

2. LITERATURE REVIEW

2.1Miscanthus as a potential bioenergy crop

Miscanthus has been studied as a potential bioenergy crop in Europe since the

1980s. Chung and Kim (2012) reported several factors on how Miscanthus is an ideal alternative bioenergy source. Due to its high biomass yield potential, it is estimated that M. x giganteus would require 63 and 35% of land to produce the same amount of ethanol compared with corn and switchgrass, respectively (Anderson et al. 2011; Heaton et al. 2008). Not only does Miscanthus x giganteus is a potential bioenergy crop. Jezowski et al. (2011) suggested that apart from M. x

giganteus particularly hybrids of M. sinensis and M. sacchariflorus should be taken

into consideration in genetic and breeding studies on the improvement of its yield from energy grasses. He also reported that Miscanthus requires few years to reach its peak yields. In general substantial increase in yield is noticed from first year of cultivation to second year (Clifton-Brown et al. 2001). Peak yields can be reached in two years but it may take up to five years for some sites (Lewandowski et al. 2000). Miscanthus x giganteus is the only Miscanthus species being commercially cultivated for biomass production due to its high biomass yield. Field trials in Europe showed that biomass yield potential of M. x giganteus was over 20 t dry matter (DM) ha-1 year-1 in central Europe and 30-40 DM ha-1 year-1 in southern Europe (Dantalos et al. 2007; Schwarz et al. 1994). Species which are of interest for biomass production are Miscanthus sacchariflorus (Diploid in China, tetraploid in Japan), Miscanthus sinensis (diploid), Miscanthus floridulus (diploid), and

Miscanthus x giganteus (triploid) which are all native to the East Asian Region

(Korea, Japan, and China). The other species are known form the Himalayas and Southern Africa (Chung and Kim 2012; Hodkinson et al. 2002). The full establishment of M. x giganteus takes 3-5 years (Lewandowski and Kahnt 1993) during which time the yield increases in each successive year. Miscanthus x

giganteus grow up to 3-4 m tall by the end of the growing season after full

establishment, at which typical autumn biomass yields range from 10 to 30 t DM ha-1 yr-1 depending on local agronomic conditions (Clifton-Brown et al. 2001).

Nonetheless, Miscanthus has the greatest yield potential for biomass production in temperature regions as compared with other major bioenergy crops. With senescence, Miscanthus recycles N and other nutrients to the roots and rhizomes, and the stems may be harvested at maximum dry matter, allowing the primary harvest of carbon and hydrogen and sustained growth from the rhizome biomass for future years (Beale et al. 1999). In addition, energy-use efficiency and carbon fixation capacity of Miscanthus was better than other crops (Bullard and Metcalfe 2001).

2.2 Photosynthetic ability of Miscanthus in relation with

biomass production

Miscanthus belongs to the family Poaceae. It is a member of the grass tribe

Andropogoneae, which includes maize, sorghum and sugarcane (Daniels and Roach 1987). Like these crop species, M. x giganteus uses the NADP-malic enzyme pathway of C4 photosynthesis (Naidu et al. 2003; Wang et al. 2008). It’s

distribution is largely tropical or sub-tropical, widely ranging from south-eastern Asia, China, Japan and few species occurring in Africa (Scally et al. 2001). Molecular and morphological systematics places Miscanthus closest to sugarcane followed by sorghum. Diploid and tetraploid forms of Miscanthus are the most common. Hybrids between 2x and 4x forms have occurred naturally, producing vigorous triploids such as M. x giganteus. Cytological studies confirmed M x

giganteus as an interspecific hybrid between M. sinensis and M. sacchariflorus

(Linde-Larsen 1993). The wide distribution of Miscanthus spp. provides an opportunity to study adaptation of C4 plants to changes in environmental factors

associated with changing altitudes (Kao et al. 1998). Chou et al. (1991) reported that the response of isozymes to temperature was different in two Miscanthus populations from altitudes of 780 and 2700 m. In addition, Miscanthus plants growing at 780 m could not survive when transplanted to a habitat of 2700 m, whereas those from 2700 m survived well when transplanted to 780 m (Chou and

Chang 1988). M. sinensis is tolerant to low pH and other metals. This enables it to dominate plant communities on naturally harsh soil environments (Murakoshi et al. 1998; Shimoda and Oikawa 2006) and contaminated sites (Nishizono et al. 1987).

2.3 Effects of low temperature on photosynthesis and biomass

yield

Although the majority of C4 species are of tropical and subtropical origin and

unsuited to the cool climate, there are a small number of C4 species native to cool

climates, which appear to show increased tolerance to low temperatures (Long et

al. 1975). Several C4 tropical grasses show low temperature compensation points

between 6.5 and 10°C (Ludlow and Wilson 1971) and many are chilling sensitive, showing impaired photosynthesis and Chlorophyll breakdown after relatively brief exposure to temperatures to 10°C (McWilliam and Naylor 1967; Taylor and Rowley 1971). Long et al. (1975) studied C4 plants from cool temperate regions

with particular reference to Spartina townsendii. They found out that this is a C4

species adapted and limited in distribution to cool temperate climatic regions. One feature of this adaptation is the ability of the species to maintain significant rates of photosynthesis at relatively low temperatures, which contrasts with observations on other C4 grasses. Indeed, there is no fundamental reason why C4 pathway

photosynthesis should exclude a species from cool regions (Bjorkman 1975). Pittermann and Sage (2001) tested the effects on long- and short-term on C4

photosynthesis by using Muhlenbergia montana, a high altitude C4 grass. Results

demonstrated that C4 plants from climates with low temperature during the

growing season can fully acclimate to cold stress given sufficient time. This acclimation appears to involve reversal of injury to the C4 cycle following initial

exposure to low temperature. By contrast, carbon gain at low temperatures generally appears to be constrained by the carboxylation capacity of Rubisco, regardless of acclimation time. The inability to overcome the Rubisco limitation at low temperature may be an inherent limitation restricting C4 photosynthetic

performance in cooler climates. Beale (1995) examined two perennial C4

rhizomatous grasses of cool temperate origin, Miscanthus x giganteus and Spartina

cynosuroides for two years in fertilized, irrigated plots in southeastern England.

The study provided the evidence that the superior potential light conversion efficiencies associated with C4 photosynthesis can be realized under cool temperate

conditions and that such climatic conditions do not inherently impair the C4

process. Farage et al. (2006) compared the suboptimal growth temperature between the quantum efficiencies of photosynthetic electron transport through photosystem II (PSII) and CO2 assimilation in leaves of two chilling-tolerant C4 species,

Miscathus x giganteus and Cyeprus longus. Results showed that there are

differences in the growth of two chilling tolerant species where Miscanthus x

giganteus grown at 10°C, further protection of PSII was effected by a 20-fold

increase in zeaxanthin content in dark-adapted leaves, which was associated with much higher levels of non-photochemical quenching of excitation energy, compared to that observed in leaves grown at 14 and 25°C which may explain the remarkable productivity of this C4 plant in cool climates, even in comparison to

other C4 species such as Cyperus longus which also occur naturally in such

climates.

2.4 Developing a new Miscanthus variety for biomass

production in temperate regions

Several research groups have started breeding a new Miscanthus varieties for biomass production in Europe (Clifton-Brown et al., 2008) and USA (Long et al., 2007). Miscanthus x giganteus of which rhizome was first collected in Japan and transferred to Denmark by Olsen (Heaton et al. 2010), has been cultivated for biomass production in Europe. However, as it was originated from a single clone, it has a high potential risk of vulnerability to outbreaks of new pests or diseases due to its lack of genetic diversity. Recently, some research groups in East Asia have also started collecting their own Miscanthus germplasm and began breeding a new

Miscanthus variety as a bioenergy crop in Korea (Chung and Kim 2012), Japan

(Wang, et al. 2011) and China (Jiang et al. 2012). Developing a new Miscanthus variety having tolerant low temperature, hence able to overwinter, and drought stress may allow growing Miscanthus in this region. Under physiological responses of Miscanthus to various locations imposed in temperate regions, particularly low temperature may help us breed a new variety of Miscanthus having high biomass yield with abiotic stress tolerance.

3. MATERIALS AND METHODS

Pot and field experiments were conducted in 2011 and 2012. Pot experiments under controlled environment of the growth chamber and outdoor environment were conducted to investigate the effects of low temperature and light intensity, respectively. A field experiment was also conducted at the Experimental Farm Station of Seoul National University, Suwon, Korea, to investigate genetic diversity of Miscanthus genotypes originated from various locations in their photosynthetic ability.

3.1 Plant materials

Various accessions belonging to Miscanthus x giganteus, M. lutarioriparius, M.

sinensis, and M. sacchariflorus were selected from 300 genotypes collected from

various locations in Korea and neighboring East Asian countries such as China, Japan, and Russia (Table 1).

3.2 Photosynthetic responses under low temperature

condition

To investigate the effect of low temperature on the photosynthesis and the growth of Miscanthus spp. ten different accessions of Miscanthus spp. were used for this experiment (Table 1). Plants grown up to 10 cm tall from the seeds except

M. x giganteus, which was grown from the rhizome, were transferred to the

glasshouse maintained at approximately 30°C (day) /20°C (night). When the plants reached a plant height of 25 cm, they were transferred to three different growth chambers (Hanbaek Sci. Ltd., Korea) maintained at 25/20°C (C1), 20/15°C (C2)

and 15/10°C (C3). The plants were placed in the growth chamber in a completely randomized design with three replications.

For the morphological assessments, plant height, leaf number, tiller number, and leaf area were measured from each of the accessions. Plant height was measured from the base to the highest point of the plant. At the same time, tiller number and leaves were counted.

Various field instruments were used to measure the physiological parameters. SPAD (Konica Minolta, Japan) was used to measure leaf chlorophyll content. Handy PEA (Hansatech Instruments, UK) was used to measure the leaf chlorophyll fluorescence. This was done by placing the plants into a dark room for 30 min dark adaptation before measurement. To measure the photosynthetic rate, LI-6400XT (LI-COR, USA) was used with a setting of 400 umol-2s-1 photosynthetic photon flux density, 20°C chamber temperature and 10% blue light for all the treatments. It was measured between 9 am to 4 pm where light intensity is at its maximum peak. Measuring gas exchange and chlorophyll content repeatedly on the same leaves in field may provide useful information on the relationship between these parameters (Schaper and Chako 1991). Plant biomass yield was measured during the end of the observation period.

Assessments of photosynthesis and plant growth were made at 0, 7, 14, 21, 28, and 35 days after treatment (DAT). The leaf and the stem dry weight were measured after 48 h drying in an oven (Hanbaek Sci. Ltd., Korea) at under 80°C for 48 hours.

3.3 Photosynthetic responses under low light intensity

To investigate the effect of light intensity on the photosynthesis and the growth of Miscanthus spp., eight different accessions of Miscanthus spp. were used for this experiment (Table 1). Plants derived from rhizomes were grown in the glasshouse maintained at approximately 30°C (day)/20°C (night) for 60. The plants were placed under five different light conditions, 100% (control, T1), 75% (T2), 45% (T3), 25% (T4), and 5% (T5), by placing different layers of net over the plants. The

photosynthetically active radiation (PAR) was measured using an AccuPAR LP-80 (Decagon Devices, USA) during the period of this light treatment (Figure 1). The actual light intensities measured were 85.18 ± 9.19 for 75%, 33.01 ± 8.25 for 45%, 22.48 ± 8.63 for 25%, and 5.70 ± 1.24 for 5%.

Morphological and physiological parameters were done with the same procedure of the difference in low temperature experiment (See section 3.2), with some modifications of measuring leaf chlorophyll fluorescence where it was made during night time for maximum dark adaptation of the plant.

Assessments of photosynthesis and plant growth were made at 0, 7, 14, 21, 42, 49, 56, 63, and 70 DAT. During the last week of observation, plants were pre-weighted for leaf blade, leaf sheath and stem fresh weight and their dry weight were measured after drying for 48 hours under 80°C using a drying oven (Hanbaek Sci. Ltd., Korea)

3.4 Diversity of Miscanthus spp. in photosynthetic ability

To assess the photosynthesis rate of various Miscanthus spp. accessions in the field condition, three-year stands of 53 accessions of Miscanthus spp. planted on the germplasm field in the Experimental Farm Station were selected (Table 1). Three leaves from different tillers of the same accession were used in measuring the different parameters in June, August and September of 2012. In November during plant senescence, the biomass yield was measured by oven-drying of selected five plants for each accession. Assessments of physiological parameters were done with the same procedures of different in temperature experiment (See section 3.2), with some modifications in measuring leaf fluorescence where a leaf clip was attached to the leaf for 30 min as a dark adaptation before measurement. The biomass yield was measured in November by oven dry at 80°C for 48 hours.

3.5 Statistical analysis

All measurements were initially subjected to analysis of variance (ANOVA). Linear regression analyses were also conducted to investigate the relationship between temperature or light intensity and photosynthesis of Miscanthus accessions. All statistical analyses were conducted using SAS 9.3 (SAS Institute Inc., 2010).

Days After Transplanting

PA

R (

µ

molm

s

)

Figure 1. Photosynthetically active radiation (PAR) values of 100% (●), 75% (■), 45% (▲

Table 1. Accessions of Miscanthus spp. used in this study.

Miscanthus spp. Code Country Geographic Information Location

Latitude Altitude

M.xgiganteus 57T,L _{UK N 35° 25' 30.5" 10 Rhotmasted}

78 USA N 35° 25' 30.5" 10 HantaekBotanical garden

M. lutarioriparius 165T,L _{Korea N 35° 08′ 17.10″ 450 Jeonnam}

271 Korea N 35° 00' 42.8" 31 Jeonnam

M sacchariflorus 1 Korea N 37° 06′ 59.3″ 26 Gyeonggi 4 Korea N 36° 56′ 03.9″ 17 Chungnam 7 Korea N 36° 31′ 30.5″ 74 Chungbuk 9 Korea N 36° 55′ 49.1″ 159 Chungbuk 40 Korea N 37° 49′ 45.9″ 84 Gangwon 41 Korea N 35° 02′ 32.8″ 34 Jeonnam 46 Korea N 37° 31′ 01.5″ 5 Incheon 66 Korea N 37° 45′ 38.2″ 89 Gyeonggi 90T,L _{Korea N 37° 11′ 20.5″ 261 Gangwon} 93 Korea N 37° 12′ 02.8″ 380 Gangwon 108 Korea N 35° 30′ 16.8″ 26 Gyeongnam 113 Korea N 35° 33′ 14.5″ 9 Gyeongnam 115T,L* _Korea N 35° 33′ 05.4″ 6 Gyeongnam 128 Russia N 43° 28′ 37.1″ 22 Primorsky 157 Korea N 36° 11′ 38.9″ 2 Chungnam 160 Korea N 35° 04′ 05.1″ 15 Jeonnam 175 Korea N 37° 33′ 56.9″ 102 Gyeonggi 180 Korea N 37° 33′ 56.9″ 102 Gyeonggi 188 Korea N 36° 07′ 41.0″ 355 Gyeongbuk 200 Korea N 34° 33′ 12.9″ 5 Jeonnam 223 China N 42° 48′ 08.4″ 238 Jilin 243 Japan N 33° 22' 46.3" 7 Fukuoka 265T,L _{Korea N 36° 03' 26.3" 10 Jeonbuk} 289 Korea N 34° 55' 32.4" 10 Jeonnam

M. sinensis 5 Korea N 36° 34′ 40.0″ 44 Chungnam 14 Korea N 37° 33′ 54.6″ 106 Jeju 26 Korea N 38° 04′ 40.5″ 565 Gyeonggi 33T,L,* _Korea N 33° 19′ 10.8″ 359 Jeju 37 Korea N 33° 26′ 25.3″ 430 Jeju 45T,L _{Korea N 37° 31′ 01.5″ 5 Incheon} 50 UK - - Purchased 80T,L,* _Korea N 37° 16′ 13.8″ 1004 Gangwon 88 Korea N 37° 15′ 59.2″ 1082 Gangwon 96 Korea N 37° 00′ 25.3″ 231 Chungbuk 104 Korea N 35° 32′ 34.3″ 647 Geongnam 107T _{Korea N 35° 31′ 20.3″ 86 Gyeongnam,} 116 Korea N 35° 33′ 05.7″ 6 Gyeongnam 120 Korea N 35° 23′ 47.2″ 393 Jeonbuk 131T _{Russia N 42° 42′ 47.9″ 17 Primorsky} 135 Russia N 42° 42′ 41.6″ 20 Primorsky 138 Russia N 43° 41′ 03.3″ 134 Primorsky 161 Korea N 34° 58′ 31.9″ 18 Jeonnam 162 Korea N 35° 32′ 03.7″ 355 Jeonbuk, 177 Korea N 37° 33′ 57.1″ 97 Jeju 187 Korea N 36° 35′ 55.6″ 155 Gyeongbuk 194 Korea N 34° 30′ 52.8″ 20 Jeonam 199 Korea N 34° 33′ 20.1″ 5 Jeonnam 209L Japan N 32° 52′ 51″ 1323 Kumamoto

227 China N 42° 37′ 52.3″ 34 Jilin, Hunchun 231 China N 43° 01′ 33.1″ 123 Jilin, Domun 257 Japan N 32° 52' 49.3" 1339 Kumamoto 255 Japan N 32° 52' 28.2" 509 Kumamoto T - accessions used for temperature experiment

L - accessions used for light intensity experiment

4. RESULTS AND DISCUSSION

4.1 Effect of low temperature on photosynthetic responses of

Miscanthus spp.

Temperature significantly affected the growth and photosynthesis of Miscanthus spp. observed at 14 and 28 DAT, and biomass measured at 35 DAT with significant difference among species and accessions.

The growth response to varying temperature in plant height varied between species and accessions (Figure 2). Plant growth was fastest at 25/20°C and slowest at 15/10°C as expected. Among species, M. lutarioriparius showed the greatest slope in response of plant growth to decreasing temperature (Figure 2, Table 2), suggesting that M. lutarioriparius is most sensitive to low temperature. Least slope was observed at M. x giganteus, suggesting that this specie is the least sensitive to low temperature. The slope between 14 and 28 DAT was decreasing for all species except M. sacchariflorus with response of plant growth to decreasing temperature. Among accessions, M. lutarioriparius and M. x giganteus were still the most and least sensitive, respectively with response to decreasing temperature (Figure 2, Table 2). However, difference in slope between 14 and 28 DAT was decreasing for all accessions except M. sacchariflorus 90 and 265, indicating that plant growth with response to different temperatures was varying between days. Using M. x

giganteus as the reference accession, M. lutarioriparius has the greater slope for

both 14 and 28 DAT and M. sinensis 80 and 107 has the lesser slope for 14 and 28 DAT, respectively with response to decreasing temperature.

Plant Heig ht ( cm) 60 80 100 120 Temperature (oC) 25/20 20/15 15/10 60 80 100 120 Mxg M165 M33 M45 M80 M107 M131 M90 M115 M265 60 80 100 120 Mxg M165 M33 M45 M80 M107 M131 M90 M115 M265

Figure 2. Plant height of M. x giganteus (●), M. lutarioriparius (■), M. sinensis

( ▲ ), and M. sacchariflorus ( ▼ ) accessions grown at different temperatures. Measurements were made at 14 (A) and 28 (B) DAT.

B

A

Leaf area growth also showed the similar response to plant growth with response to varying temperature, where it varied among species and accessions. Leaf area growth was fastest at 25/20°C and slowest at 15/10°C as expected. Among species,

M. lutarioriparius showed the steepest slope in response of growth in leaf area to

decreasing temperature (Figure 3, Table 2). Least slope was observed at M. x

giganteus, suggesting that this specie is more tolerable to low temperature. The

slope with response to decreasing temperature between 14 and 28 DAT was decreasing for all species. Among accessions, M. lutarioriparius and M. sinensis 107 showed the greatest and least slope, respectively, with response to decreasing temperature. Difference in slope between 14 and 28 DAT was decreasing for all accessions where majority of the accessions of M. sacchariflorus showed the greatest difference. The photosynthesis rate per leaf area (computed by multiplying the leaf area and photosynthesis rate) of all the accessions also showed a decreasing trend with response to decreasing temperature. Using M. x giganteus as the reference accession, M. lutarioriparius and M. sinensis 131 has the greater and lesser slope for both 14 and 28 DAT, respectively, with response to decreasing temperature.

100 150 200 250 300 350 400 450 500 Mxg M165 M33 M45 M80 M107 M131M90 M115 M265 Lea f Ar ea (cm 2 ) 100 150 200 250 300 350 400 450 500 Mxg M165 M33 M45 M80 M107 M131M90 M115 M265 Temperature (oC) 25/20 20/15 15/10 100 150 200 250 300 350 400 450 500 Mxg M165 M265 M115 M90 M33 M45 M80 M107 M131

Figure 3. Leaf area of M. x giganteus (●), M. lutarioriparius (■), M. sinensis (▲

), and M. sacchariflorus ( ▼ ) accessions grown at different temperatures. Measurements were made at 14 (A) and 28 (B) DAT.

A

Photosynthesis also decreased with lowering the temperature but the extent of decrease depends on the species and accessions (Figure 4). Among species, M.

lutarioriparius showed the greatest slope in response of the photosynthesis rate to

decreasing temperature (Figure 4, Table 2), suggesting that M. lutarioriparius is most sensitive to low temperature. Least slope was observed at M. x giganteus suggesting that this specie is the least sensitive to low temperature. The slope between 14 and 28 DAT was increasing for all species, indicating that the difference in photosynthesis was more prominent in prolonged exposure to lower temperature. Among accessions, M. lutarioriparius and M. x giganteus were still the most and least sensitive, respectively, with response to decreasing temperature (Figure 4, Table 2). The slope with response to decreasing temperature between 14 and 28 DAT was increasing for all accessions except M. sinensis 45 and 90. Using

M. x giganteus as the reference accession, M. lutarioriparius has the greater slope

for both 14 and 28 DAT and M. sinensis 107 and M. sacchariflorus 265 has the lesser slope for 14 and 28 DAT, respectively, with response to decreasing temperature.

25/20 20/15 15/10 Ca rbon Dioxide As similation ( µ CO 2 m -2 s -1 ) 0 2 4 6 8 10 12 Mxg M165 M33 M45 M80 M107 M131 M90 M115 M265 0 2 4 6 8 10 12 Mxg M165 M33 M45 M80 M107 M131 M90 M115 M265 Temperature (oC) 25/20 20/15 15/10 0 2 4 6 8 10 12 Mxg M165 M265 M115 M90 M33 M45 M80 M107 M131

Figure 4. Photosynthesis rate of M. x giganteus (●), M. lutarioriparius (■), M.

sinensis ( ▲ ), and M. sacchariflorus ( ▼ ) accessions grown at different

temperatures. Measurements were made at 14 (A) and 28 (B) DAT.

A

There was no significant correlation of photosynthesis rate to chlorophyll content (SPAD reading) and leaf chlorophyll fluorescence (PI, performance of index) for all the Miscanthus accessions. In addition, Green et al. (1998) reported about the relationship between chlorophyll fluorescence and photosynthesis is complex between higher plants.

The biomass yield showed that accessions placed on 15/10°C have the lowest biomass yield, which indicated that lower temperature tends to decrease such parameter (Figure 5). Among species, M. lutarioriparius showed the greatest slope in response of biomass yield to decreasing temperature (Figure 5, Table 2), suggesting that M. lutarioriparius is most sensitive to low temperature. Least slope was observed at M. x giganteus, suggesting that this specie is the least sensitive to low temperature. However, among accessions, M. lutarioriparius still had the greatest slope in response to decreasing temperature but M. sinensis 90 showed the least slope (Figure 5, Table 2). In addition, M. sinensis 107 has the highest yield among the accessions.

Temperature (

C)

Biomass Yield (g

/pl

ant)

Figure 5. Biomass yield of M. x giganteus ( ● ), M. lutarioriparius( ■ ), M.

sinensis( ▲ ), and M. sacchariflorus ( ▼ ) accessions grown at different

21

Table 2. Slopes of Miscanthus spp. responses to varying temperature in their plant growth, photosynthesis and biomass yield. Plant growth

and photosynthesis were measured at14 DAT and 28 DAT, and biomass yield at 35 DAT. Numbers in parentheses are standard error.

Genotype Code

Plant height Leaf area Photosynthesis rate

Biomass yield 14 28 14 28 14 28 M. x giganteus 57 -0.833 (0.017) -0.666 (0.013) -8.378 (0.168) -6.597 (0.132) -0.089 (0.002) -0.130 (0.003) -0.362 (0.007) M. lutarioriparius 165 -22.830 (0.457) -19.417 (0.388) -101.660 (2.033) -100.430 (2.049) -3.600 (0.072) -3.953 (0.079) -1.145 (0.023) M. sinensis 33 -5.333 (0.107) -4.500 (0.090) -44.257 (0.865) -43.718 (0.874) -2.793 (0.056) -2.863 (0.057) -0.453 (0.009) 45 -3.667 (0.073) -3.167 (0.063) -67.159 (1.343) -63.658 (1.273) -0.745 (0.015) -0.723 (0.012) -0.292 (0.006) 90 -2.333 (0.047) -1.583 (0.032) -49.382 (0.988) -45.027 (1.021) -0.811 (0.017) -0.691 (0.017) -0.118 (0.002) 107 -2.500 (0.050) -1.667 (0.033) -6.645 (0.113) -4.891 (0.098) -0.138 (0.003) -0.747 (0.015) -1.060 (0.021) 131 -7.334 (0.147) -6.667 (0.133) -10.942 (0.219) -7.469 (0.149) -0.302 (0.006) -0.409 (0.008) -0.365 (0.007) M. sacchariflorus 90 -4.917 (0.098) -7.167 (0.143) -11.918 (0.238) -10.327 (0.287) -0.811 (0.016) -0.961 (0.019) -0.717 (0.014) 115 -3.667 (0.073) -3.428 (0.069) -11.541 (0.231) -9.638 (0.253) -0.701 (0.014) -0.730 (0.015) -0.737 (0.015) 265 -2.917 (0.058) -4.000 (0.080) -12.035 (0.241) -11.764 (0.255) -0.667 (0.013) -0.925 (0.019) -0.508 (0.010) Mean Value M. x giganteus -0.833 -0.666 -8.378 -6.597 -0.089 -0.130 -0.362 M. lutarioriparius -22.830 -19.417 -101.660 -100.430 -3.600 -3.953 -1.145 M. sinensis -4.233 -3.517 -35.677 -32.953 -0.966 -1.099 -0.458

21

There was no significant correlation between the relative photosynthesis rate and biomass yield (Figure 6). However, it is clear that different accessions have different photosynthesis rates and biomass yields. In general, accessions of M.

sacchariflorus have the lowest biomass yield. In addition, the lowest temperature

(indicated by the triangle shape) tends to show the lowest biomass yield as expected. An accession of M. sinensis 107 showed a high biomass yield and high photosynthesis at the same time. Its high biomass yield could be attributed at its early presence of tillers. Accessions from M. Sinensis showed the earliest tiller emergence among the other accessions. Although M. x giganteus was well known for its high biomass yield, the reason for such low value could be attributed at the growth stage of the plant. All of the accessions are exposed to different temperatures without reaching the flowering stage, which is why it did not reach its maximum growing capacity. As for the other accessions (M. lutarioriparius, M.

sinensis, and M. sacchariflorus) most of them showed tillers at these early stages,

however for M. x giganteus, no tillers were visible during the course of the experiment.

Relative Carbon Dioxide Assimilation (%) 60 80 100 120 140 160 180 0 2 4 6 8 10 Mxg m165 m33 m45 m80 m107 m131 m90 m115 m265 Mxg m165 m33 m45 m80 m107 m131 m90 m115 m265 Mxg m165 m33 m45 m80 m107 m131 m90 m115 m265 0 2 4 6 8 10 Mxg m165 m33 m45 m80 m107 m131 m90 m115 m265 Mxg m165 m33 m45 m80 m107 m131 m90 m115 m265 Mxg m165 m33 m45 m80 m107 m131 m90 m115 m265 Temperature (oC) 25/20 20/15 15/10 0 2 4 6 8 10 Mxg M165 M265 M115 M90 M33 M45 M80 M107 M131 Biomass Yield (g /pl ant)

Figure 6. Relationship between relative photosynthesis rate measured at 14 (A)

and 28 DAT (B) and biomass yield measured at 35 DAT of various Miscanthus spp grown at varying temperatures 25/20°C (●), 20/15°C (■), and 15/10°C (▲).

A

Similarly, other C4 plants demonstrate the same photosynthetic response to low

temperatures. Du (1999) reported on this study using different Saccharum spp. in origin and cold sensitivity suggested that the C4 enzymes pyruvate

orthosphosphatedikinase (PPDK) and NADP-malate dehydrogenase (NADP-MDH) are the key enzymes that may determine the cold sensitivity in photosynthesis. Caldwell et al. (1977) reported substantial decrease of photosynthetic capacity of Atriplex species between 4 and 10°C. Naidu and Long (2004) reported that another potential mechanism for the maintenance of high photosynthesis rates in M. x giganteus at low temperature could also be attributed to its high levels of leaf absorption during growth at low temperature. Miscanthus well-established over several years can survive even much lower temperatures than the first year. Overwintering survival and tolerance depend on Miscanthus species and genotypes. Miscanthus sacchariflorus showed a greater survival rate after overwintering than M. sinensis (Kang et al. 2010) which may be related to the soil position of buds from new rhizomes.

In general, plant growth, leaf area, photosynthesis rate and biomass yield were greatly affected in M. lutarioriparius and least affected in M. x giganteus among species. Comparing among accessions, still M. lutarioriparius was greatly affected but the least affected varied among accessions. Most of the accessions showed a decreasing slope with response of plant growth, leaf area to decreasing temperature and an increasing slope with response of photosynthesis rate to decreasing temperature between 14 and 28 DAT. There was no correlation between photosynthesis rate and biomass yield for all species and accessions.

4.2 Effect of low light intensity on photosynthetic responses

of Miscanthus spp.

Light intensity significantly affected plant growth in plant height, no. of tillers, leaf area, and photosynthesis rate observed at 21, 42, and 56 DAT, and biomass yield measured at 70 DAT. These responses to light intensity showed statistically significant differences among species and accessions.

The plant height of Miscanthus spp. exposed to different light intensities varies with species and accession (Figure 7). Plants are growing despite of being in different light intensity. All accessions showed that plants are taller at light intensities of between 45% and 25% except M. sinensis where the plant growth is highest at 100% light intensity. With the absence of trend on decreasing light intensity, such figure has no slope on this study. M. x giganteus showed the fastest growth among species and accession. Tiller emergence was also lower at 5% light intensity for all the accessions (Figure 8) with the slope (Table 3) showed a decreasing trend. Among species, M. lutarioriparius showed the greatest slope in response of the number of tillers to decreasing light intensity (Figure 8, Table 3). Least slope was observed at M. x giganteus, suggesting that this species is the least sensitive to low light intensity. The slope was increasing for all species from 21 to 56 DAT with response of the number of tillers to decreasing light intensity. Among accessions, M. lutarioriparius, M. sinensis 209, and M. sacchariflorus 115 have the greatest slope with response of the tiller number to decreasing light intensity at 21, 42 and 56 DAT, respectively. In addition, M. x giganteus at 21 DAT and M. sinensis 33 at 42 and 56 DAT has the least slope with response of the tiller number to decreasing light intensity. The slope was increasing for all species except M. sinensis 33 from 21 to 56 DAT with response of the number of tillers to decreasing light intensity. Using M. x

giganteus as the reference accession, there was no clear trend seen on which

accession has the greater and lesser slope because it varied among 21, 42, and 56 DAT.

Plant Heig ht ( cm) 60 80 100 120 Light Intensity 100% 75% 45% 25% 5% 40 60 80 100 120 140 160 180 Mxg M165 M33 M45 M209 M90 M115 M265 40 60 80 100 120 140 160 180 Mxg M165 M33 M45 M209 M90 M115 M265 40 60 80 100 120 140 160 180 Mxg M165 M33 M45 M209 M90 M115 M265

Figure 7. Plant height of M. x giganteus (●), M. lutarioriparius (■), M. sinensis

(▲), and M. sacchariflorus (▼) accessions grown at various light intensities.

A

B

Light Intensity 100% 75% 45% 25% 5% 0 2 4 6 8 10 12 14 Mxg M165 M33 M45 M209 M90 M115 M265 Ti ll er Number ( no ./ pl ant) 0 2 4 6 8 10 12 14 Mxg M165 M33 M45 M209 M90 M115 M265 0 2 4 6 8 10 12 14 Mxg M165 M33 M45 M209 M90 M115 M265

Figure 8. Number of tillers of M. x giganteus (●), M. lutarioriparius (■), M.

sinensis( ▲ ), and M. sacchariflorus ( ▼ ) accessions grown at various light

intensities. Measurements were made at 21 (A), 42 (B), and 56 (C) DAT.

A

B

Leaf area growth also showed the similar response to low light intensity, tending to decrease the growth of the leaf of the plant (Figure 9) and varies among species and accessions. Among species, M. sacchariflorus showed the steepest slope in response of growth in leaf area to decreasing light intensity (Figure 9, Table 3). Least slope was observed at M. lutarioriparius, suggesting that this specie is more tolerable to low light intensity. The slope with response to decreasing light intensity between 42 and 70 DAT was decreasing for all species except for M. sacchariflorus. Among accessions, M. sacchariflorus 115 and M.

sinensis 45 showed the greatest and least slope respectively with response to

decreasing light intensity. Difference in slope between 42 and 70 DAT was decreasing for all accessions except for M. sacchariflorus 115 and 265. The photosynthesis rate per leaf area of all the accessions also showed a decreasing trend with response to decreasing light intensity. Using M. x giganteus as the reference accession, M. sacchariflorus 115 and M. sinensis 209 showed the greater and lesser slope for both 42 and 70 DAT, respectively, with response to decreasing light intensity.

100% 75% 45% 25% 5% Lea f Ar ea (cm 2 ) 100 200 300 400 500 Mxg M165 M33 M45 M209 M90 M115 M265 Light Intensity 100% 75% 45% 25% 5% 100 200 300 400 500 600 MxgM165 M33 M45 M209 M90 M115 M265 100 200 300 400 500 MxgM165 M33 M45 M209 M90 M115 M265

Figure 9. Leaf area of M. x giganteus (●), M. lutarioriparius (■), M. sinensis

(▲), and M. sacchariflorus (▼) accessions grown at various light intensities. Measurements were made at 42 (A) and 70 (B) DAT.

A

Photosynthesis also decreased with lower light intensity but the extent of decrease depends on accessions (Figure 10). The slope (Table 3) is negative, meaning it was decreasing together with lower light intensity as expected. Among accessions, M. x giganteus showed the greatest slope on 42 and 56 DAT. Least slope was observed at M. sacchariflorus at 21, 42 and 56 DAT, suggesting that this species is the least sensitive to low light intensity (Figure 10, Table 3).The slope of photosynthesis with response to low temperature between 21 and 42 DAT decreased and increased between 42 and 56 DAT for all species, indicating that the difference in photosynthesis was more prominent during longer periods at different light intensities. Among accessions, M x giganteus has the greatest slope of photosynthesis with response to low light intensity at 42 and 56 DAT (Figure 10, Table 3). The least slope of photosynthesis rate with response to low light intensity was observed at accessions of M. sacchariflorus 90 at 21 DAT and 265 at 42 and 56 DAT. The slope of photosynthesis rate with response to low light intensity was decreasing between 21 and 42 DAT and increasing between 42 and 56 DAT for all accessions except M. sacchariflorus 90 and 115 where it was increasing from 21 to 56 DAT. Using M x giganteus as the reference accession,

M. sacchariflorus 265 and M. sacchariflorus 115 has the greater slope and lesser

slope respectively for both 42 and 56 DAT with response to decreasing light intensity.

In C4 plants, reductions in photorespiration are achieved through the spatial

separation of dark and light reactions, as found in single-cell C4 species. In

single-cell C4, intracellular compartmentalization of enzymatic activities enables a

two-step carbon fixation process where the initial carboxylation reaction occurs at one end of a cell and decarboxylation and refixation of CO2 by RuBisCO occur at the

Light Intensity 100% 75% 45% 25% 5% 0 2 4 6 8 10 12 Mxg M165 M33 M45 M209 M90 M115 M265 Carbon Di oxid e Ass imi lati on (µ CO 2 m -2 s -1 ) 0 2 4 6 8 10 12 Mxg M165 M33 M45 M209 M90 M115 M265 0 2 4 6 8 10 12 14 Mxg M165 M33 M45 M209 M90 M115 M265

Figure 10. Photosynthesis rate of M. x giganteus (●), M. lutarioriparius (■),

M. sinensis (▲), and M. sacchariflorus (▼) accessions grown at various light

intensities. Measurements were made at 21 (A), 42 (B), and 56 (C) DAT.

A

B

Difference in light intensity also showed that there was no significant correlation of photosynthesis rate to chlorophyll content and leaf chlorophyll fluorescence for all the Miscanthus accessions. Chiba et al. (2003) reported that the photosynthetic capacity of a leaf changes with age and its position in the canopy. These changes in photosynthetic capacity are related to the degradation of chlorophyll and the breakdown of Rubisco and other proteins into amino acids that are exhorted as a source of nitrogen to growing organs. In addition, lowered chlorophyll content in the leaves appeared to protect the plants somewhat from the pronounced stress as evidenced by their higher photosynthetic activity (Taylor and Rowley 1971).

The biomass yield measured at 70 DAT showed that accessions exposed at 5% light intensity has the lowest biomass yield which indicated that lower light intensity tends to decrease such. This was the case for all accessions. Among species, M. sacchariflorus showed the greatest slope in response of biomass yield to decreasing temperature (Figure 11, Table 3), suggesting that this specie is most sensitive to lower light intensity. Least slope was observed at M. sinensis suggesting that this specie is the least sensitive to lower light intensity. This was also the case among accessions where the greatest where M. sacchariflorus115 and M. sinensis45 showed the greatest and least slope, respectively in response of biomass yield to decreasing temperature (Figure 11, Table 3). In addition, M.

Light Intensity

Biomas

s y

ield (g

/pl

ant)

Figure 11. Biomass yield of M. x giganteus (●), M. lutarioriparius (■), M.

sinensis ( ▲ ), and M. sacchariflorus ( ▼ ) accessions grown at various light

3

5

Table 3. Slopes of Miscanthus spp. responses to varying light intensity in their plant growth, photosynthesis and biomass yield. Plant growth

and photosynthesis were measured at 21 DAT, 42 DAT and 56 DAT, and biomass yield at 70 DAT. The numbers in parentheses are standard errors.

Genotype ID

Tiller number Leaf area Photosynthesis rate

Biomass yield 21 42 56 42 70 21 42 56 M. x giganteus 57 -0.200 (0.004) -0.800 (0.016) -0.900 (0.018) -23.525 (0.471) -20.137 (0.403) -6.976 (0.140) -6.141 (0.123) -8.388 (0.168) -2.212 (0.044) M. lutarioriparius 165 -0.667 (0.013) -1.033 (0.021) -1.100 (0.022) -11.996 (0.240) -10.713 (0.254) -8.043 (0.161) -6.006 (0.120) -7.898 (0.158) -3.284 (0.066) M. sinensis 33 -0.550 (0.011) -0.327 (0.007) -0.425 (0.009) -22.933 (0.459) -20.108 (0.482) -9.146 (0.183) -5.238 (0.105) -7.463 (0.109) -1.532 (0.031) 45 -0.360 (0.007) -0.617 (0.012) -0.690 (0.014) -11.306 (0.226) -9.5911 (0.192) -7.389 (0.148) -5.180 (0.104) -7.094 (0.122) -1.261 (0.025) 209 -0.233 (0.005) -1.400 (0.028) -1.500 (0.030) -30.207 (0.604) -22.41 (0.448) -9.967 (0.199) -5.517 (0.110) -8.299 (0.166) -1.322 (0.026) M. sacchariflorus 90 0.200 (0.004) -1.100 (0.022) -1.137 (0.023) -12.289 (0.246) -25.545 (0.511) -5.059 (0.101) -5.235 (0.105) -8.261 (0.165) -2.279 (0.046) 115 -0.600 (0.012) -1.340 (0.027) -1.627 (0.033) -45.334 (0.907) -67.147 (1.343) -5.910 (0.118) -6.127 (0.123) -7.250 (0.145) -4.455 (0.089) 265 -0.233 (0.005) -0.460 (0.009) -0.470 (0.009) -16.599 (0.332) -17.018 (0.340) -5.574 (0.111) -3.334 (0.067) -4.504 (0.090) -3.397 (0.068) Mean Value M. x giganteus -0.200 -0.800 -0.900 -23.525 -20.137 -6.976 -6.141 -8.388 -2.212 M. lutarioriparius -0.667 -1.033 -1.100 -11.996 -10.713 -8.043 -6.006 -7.898 -3.284 M. sinensis -0.381 -0.781 -0.872 -21.482 -17.370 -8.834 -5.312 -7.619 -1.372 M. sacchariflorus -0.211 -0.967 -1.078 -24.741 -36.570 -5.514 -4.899 -6.672 -3.377

There was no significant correlation between the relative photosynthesis rate and biomass yield (Figure 12). However, it is clear that different accessions have different photosynthesis rates and biomass yields. In general, accessions from M.

sinensis have the lowest biomass yield and accessions from M. sacchariflorus

have the highest biomass yields. In addition, the decreasing light intensity tends to show the lowest biomass yield as expected. An accession of M. sacchariflorus 115 showed a high biomass yield and high photosynthesis at the same time. Its high biomass yield could be attributed at its early presence of tillers. Lower light intensity tends to delay the flowering and tiller emergence of the plant. Therefore, the biomass yield is low.

Relative Carbon Dioxide Assimilation (%) 0 50 100 150 200 250 300 0 5 10 15 20 25 30 35 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 Bio mas s Yiel d (g /p lant ) 0 5 10 15 20 25 30 35 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 0 5 10 15 20 25 30 35 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45 m209 m90 m115 m265 mxg m165 m33 m45m209 m90 m115 m265

Figure 12. Relationship between relative photosynthesis rate and biomass yield of

various Miscanthus spp. accessions grown at various light intensities 100% (●), 75% (■), 45% (▲), 25% (▼), and 5% () at 21 (A), 42 (B), and 56 (C) DAT.

A

B

It was generally stated that leaves of shade plants are thinner and their chloroplasts are larger and richer in chlorophyll than the leaves of sun plants. However a striking factor of shade plant chloroplasts is their large grana stacks which may contain as many as 100 thylakoids per granum. Leaves grown at low light intensities have more chlorophyll per unit weight or unit volume of leaf, but the chlorophyll content per unit area of leaf surface is very often lower than that of leaves grown at higher light intensities (Boardman 1977). In addition, solarization (Holman 1930) and the photo inactivation (Jones 1966) of isolated chloroplasts are some of the reported damaging effects of high light intensity.

In general, plant growth was the highest between 45% and 25%. Furthermore there was no specific accession or specie that plant growth was greatly affected on the response of different species and accessions at decreasing light intensities: M.

lutarioriparius on increase in tiller number and M. sacchariflorus on the growth

of leaf area. The most prominent trend of the slope of increase in tiller number with response to decreasing light intensity was increasing from 21 to 56 DAT while the slope of leaf area growth was decreasing from 21 to 56 DAT. This was also the same case of the accessions. The photosynthesis rate of M. x giganteus was greatly affected and M. sacchariflorus was least affected with response to decreasing light intensity. The most prominent trend of the slope of photosynthesis with response to decreasing light intensity was decreasing between 21 and 42 DAT and increasing between 42 and 56 DAT. Biomass yield of M.

sacchariflorus was greatly affected and M. sinensis was least affected with

response to decreasing light intensity. There was no correlation between photosynthesis rate and biomass yield for all species and accessions

4.3 Diversity of Miscanthus spp. genotypes in photosynthetic

abilities

To further investigate the photosynthetic diversity, 53 accessions from 300 three-year stand accessions of different Miscanthus species collected from various locations were selected and measured for the photosynthesis rate during the month of June, August and September in 2012.

Figure 13 shows the relative photosynthesis rate of M. lutarioriparius, M.

sinensis, and M. sacchariflorus to M. x giganteus (reference accession) at

different months. First prominent result was that the photosynthesis rate was low at June, the highest during the month of August and decreased on September. This could be attributed for the high light intensity (data not shown). The measured photosynthetically active radiation (PAR) was the highest during this month. Relative to M. x giganteus, majority of the accessions from M. sacchariflorus showed a higher rate (01, 07, 09, 40, 41, 46, 93, 113, 200, 289, and 289) followed by M. sinensis (37, 96, 135, 161, 177, 231, and 257). Table 4 shows that the correlation of photosynthesis rate with SPAD and PI are low. Generally, SPAD is negatively correlated while PI is positively correlated. In addition, the correlation was decreasing from June to September. Senescence of Miscanthus spp. monitored over 3 years in a trial of 244 genotypes in the UK revealed that on the average, M. sinensis genotypes remained greener for longer than M.

Miscanthus spp. Accession ID M-5 7 M-7 8 M-1 6 5 M-2 7 1 M-0 1 M-0 4 M-0 7 M-0 9 M-4 0 M-4 1 M-4 6 M-6 6 M-9 0 M-9 3 M-1 0 8 M-1 1 3 M-1 2 8 M-1 5 7 M-1 6 0 M-1 7 5 M-1 8 0 M-1 8 8 M-2 0 0 M-2 2 3 M-2 4 3 M-2 6 5 M-2 8 9 M-0 5 M-1 4 M-2 6 M-3 7 M-4 5 M-5 0 M-8 8 M-9 6 M-1 0 4 M-1 0 7 M-1 1 6 M-1 2 0 M-1 3 1 M-1 3 5 M-1 3 8 M-1 6 1 M-1 6 2 M-1 7 7 M-1 8 7 M-1 9 4 M-1 9 9 M-2 0 9 M-2 2 7 M-2 3 1 M-2 5 7 M-2 5 5 Relative Carbo n Dio xid e Assimilatio n (% ) 0 20 40 60 80 100 120 140 160 180 M. sacchariflorus M. sinensis M. x giganteus M. lutarioriparius

Figure 13. Relative photosynthesis rate of various Miscanthus spp.

accessions relative to M. x giganteus in June (●), August (■), and

September (▲) 2012.